scripta scientifica medica -...

Medical UniversityProf. Dr. Paraskev Stoyanov

VARNA, Bulgaria

ScriptaScientifica

Medica

Vol. 44 (1), 2012Supplement 1

SCRIPTA SCIENTIFICA MEDICAAn official publication of Medical University "Prof. Dr. Paraskev Stoyanov", Varna

Editor-in-Chief:

Prof. Anelia Klissarova, MD, PhD, DSc

Rector of Medical University of Varna

e-mail: [email protected]

Co-Editor-in-Chief:

Prof. Rossen Madjov, MD, PhD, DSc

Vice Rector of Medical University of Varna


Editorial Board:

Assoc Prof. Peter Genev, MD, PhD Assoc. Prof. Boriana Varbanova, MD, PhD

Department of Pathoanatomy Department of Pediatrics and Medical Genetics

E-mail: [email protected] e-mail: [email protected]

Assoc. Prof. Minko Minkov, MD, PhD Assoc. Prof. Zhaneta Georgieva, MD, PhD

Head, Department of Anatomy, Histology and Vice Rector University Hospital Coordination and

Embryology Postgraduate Education

e-mail: [email protected] e-mail: [email protected]

Prof. Krasimir Ivanov, MD, PhD, DSc Assoc. Prof. Svetoslav Georgiev, MD, PhD

Head, Department of Surgery Department of Internal Medicine


Prof. Iskren Kotsev, MD, PhD, DSc Assoc. Prof. Negrin Negrev, MD, PhD

Department of Hepato - Gastroenterology Vice Rector for Students Affair


Assoc. Prof. Marinka Peneva, MD, PhD Assoc. Prof. Stoyanka Popova, MD, PhD

Dean, Faculty of Medicine Dean, Faculty of Public Health


Assoc. Prof. Vasil Svechtarov, DMD, PhD Assoc. Prof. Violeta Tacheva, PhD

Dean, Faculty of Dental Medicine Head, Department of Language Teaching,

e-mail: [email protected] Communications and Sports


Assoc. Prof. Diana Ivanova, MD, PhD Assoc. Prof. Valentina Madjova, MD, PhD

Head, Department of biochemistry molecular medicine Department of Family Medicine

and nutragenomics e-mail: [email protected]


Assoc. Prof. Emanuela Mutafova, PhD Assoc. Prof. Radoslav Radev, MD, PhD

Head, Department of Economics Head, Department of Surgery

and Healthcare Management e-mail: [email protected]


Secretary:

Nikola Kolev, MD, PhD Emilia Jordanova

Department of Surgery IT Center

University Hospital "St. Marina" e-mail: [email protected]

MEDICAL UNIVERSITY “PROF. DR. PARASKEVSTOYANOV” VARNA

BULGARIAN SOCIETY FOR PHYSIOLOGICAL SCIENCES

PROCEEDINGS

OF THE X NATIONAL CONGRESS

OF BULGARIAN SOCIETY FOR PHYSIOLOGICAL SCIENCES

6 - 9 OCTOBER 2011

V A R N A

UNDER THE AUSPICES OF

Prof. Anelia Klissarova, MD, PhD, DSc Rector, Medical University “Prof. Dr. Paraskev Stoyanov”

Varna

and

Prof. Alexander Stoynev, MD, PhD, DSc President, Bulgarian Society for Physiological Sciences

Volume 44(1) Supplement 1, 2012

CONTENTS

Kossev A. - SENSORIMOTOR INTEGRATION: TMS STUDIES OF PROCESSING

OF PROPRIOCEPTIVE INFORMATION IN HEALTH AND DISEASE . . . . . . . . . . . . . . . . . . . . 5

Genova B., N. Bocheva, M. Stefanova, S. Stefanov - EFFECTS OF ADAPTATION

IN VISUAL PERCEPTION OF GLOBAL SPEED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Dushanova J., D. Mitov - VISUAL EVENT-RELATED POTENTIALS BY

AN IDENTIFICATION OF GRATING ORIENTATION DIFFERENCE . . . . . . . . . . . . . . . . . . . . 15

Tancheva L., E. Encheva, M. Novoselski, V. Petkov, R. Klisurov -

SEX-DEPENDENT EFFECT OF A NEW PEPTIDOMIMETIC ON COGNITIVE FUNCTION

OF ISOLATED RATS AFTER MATERNAL DEPRIVATION . . . . . . . . . . . . . . . . . . . . . . . . . 19

Popova E., P. Kupenova, I. Ivanov - EFFECTS OF D1 RECEPTOR BLOCKADE

ON THE INTENSITY-RESPONSE FUNCTION OF FROG DARK ADAPTED ELECTRORETINOGRAM . . 23

Pavlova E., G. Uzunova, P. Somlev - DYNAMICS OF HEART RATE RECOVERY

AFTER CYCLE ERGOmetric PWC170 TEST IN SOCCER PLAYERS. . . . . . . . . . . . . . . . . . . . . 27

Bekyarova G., M. Hristova - URIC ACID, THIOLS AND BURN-INDUCED OXIDATIVE

INJURY OF GASTRIC MUCOSA IN RATS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Bekyarova G., M. Hristova, M. Tsaneva - CYTOPROTECTIVE EFFECT

OF MELATONIN AND ANTIOXIDANT DEFENSE AFTER THERMAL SKIN INJURY . . . . . . . . . . . 35

Sarov G. - UNDERSTANDING PATHOGENESIS OF RISKY BEHAVIOR: A HOLISTIC APPROACH . . 39

Sarov G. - COMPARISON OF FUNCTIONAL AND STRUCTURAL CLASSIFICATION IN

PATHOPHYSIOLOGICAL THEORY AND TEACHING . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Tchekalarova J., D. Pechlivanova, Zl. Petkova, Al. Stoynev - ÅFFECT OF CHRONIC

TREATMENT WITH MELATONIN ON DIURNAL VARIATIONS OF SPONATENOUS MOTOR

SEIZURES AND BEHAVIOR OF WISTAR AND SPONTANEOUSLY HYPERTENSIVE RATS

IN KAINATE MODEL OF TEMPORAL LOBE EPILEPSY . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Georgiev K., E. Miladinova, T. Pajpanova - DESIGN, SYNTHESIS AND ANALYSIS OF NOVEL

ENDOMORPHIN-2 AND MORPHICEPTIN ANALOGUES. . . . . . . . . . . . . . . . . . . . . . . . . . 51

Petrov L., P. Atanasov, N. Zaekov, A. Alexandrova, Z. Zsheliaskova-Koynova,

I. Achkakanov - PHYSIOLOGICAL AND NON-INVASIVE BIOCHEMICAL INDEXES

IN A MODEL OF EMOTIONAL STRESS IN SHOOTERS . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Ivanova M., S. Belcheva, I. Belcheva, N. Negrev, R. Tashev - OLFACTORY BULBECTOMY

INDUCES SHIFTS IN LATERALIZATION OF LOCOMOTOR RESPONSES TO VASOACTIVE

INTESTINAL PEPTIDE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Danovska M., M. Alexandrova - LOW GRADE INFLAMMATION IN DIABETIC

HYPERTENSIVE INDIVIDUALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Mihaylova M. , I. Hristov, K. Racheva, Ts. Totev, D. Mitov - EARLY VEP WAVES

ARE AFFECTED STRONGER BY GRATING LENGTH THAN WIDTH . . . . . . . . . . . . . . . . . . . 67

Stefanova M., N. Bocheva, O. Georgieva - EVALUATION OF INDIVIDUAL, GENDER

AND AGE DIFFERENCES IN VISUAL MOTION PERCEPTION . . . . . . . . . . . . . . . . . . . . . . . 73

Kolev N., L. Halacheva - SEX DIFFERENCES IN FUNCTIONAL ORGANIZATION OF BIMANUAL

SHOT-LIKE ISOMETRIC HANDGRIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Angelova P., N. Boyadjiev, A. Ivanova, S. Muletarov - CHANGES IN THE PHYSICAL

WORKING CAPACITY, BODY COMPOSITION AND FAT METABOLISM OF WOMEN ON A

THREE-MONTH SPECIALIZED TRAINING AND DIETARY PROGRAM. . . . . . . . . . . . . . . . . . 81

Somlev P. - THE EFFECTS OF PACED BREATHING ON SPECTRAL PARAMETERS

OF HEART RATE VARIABILITY IN ATHLETES AND UNTRAINED CONTROLS . . . . . . . . . . . . . 85

Somlev P., G. Uzunova, E. Pavlova - HEART RATE VARIABILITY AT REST IN ELITE AND FORMER

SOCCER PLAYERS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

3

Georgiev Ts., P. Hadzhibozheva, A. Tolekova - CONTRACTILE RESPONSES OF THE RAT

UTERINE SMOOTH MUSCLE TO INFLUENCES WITH ANGIOTENSIN II AND VASOPRESSIN . . . . . 93

Kolarova R. - CARDIOVASCULAR STRESS AND MYOCARDIAL PROTECTION (I) . . . . . . . . . . 97

Kolarova R., Z. Gendzhev - ANTIOXIDANT PREVENTION IN EXPERIMENTAL

HYPERTENSION: HISTOMORPHOLOGICAL CHANGES (II) . . . . . . . . . . . . . . . . . . . . . . . 101

Kolarova R. - EXPERIMENTAL HEMODINAMIC STRESS AND ITS INFLUENCE FROM

PHYSICAL EXERCISE, HYPOKINESIA AND ANTIOXIDANTS . . . . . . . . . . . . . . . . . . . . . . 105

Stefanov S., K. Alexiev, N. Bocheva, B. Genova - AGE-RELATED EFFECTS

ON THE SENSITIVITY TO GLOBAL MOTION DIRECTION DETERMINED BY THE METHOD

OF CLASSIFICATION IMAGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Tzvetkov S. - SPECIFICITY IN BREATHING REGULATION DURING PHYSICAL EXERCISE

IN TAEKWONDO NATIONAL ATHLETES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Krustev S. M., N. Negrev, D. I. Stephanova - THE STRENGTH-DURATION PROPERTIES

IN SIMULATED DEMYELINATING NEUROPATHIES DEPEND ON THE MYELIN SHEATH

AQUEOUS LAYERS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Minchev Zl., P. Gatev - PSYCHOPHYSIOLOGICAL EVALUATION OF EMOTIONS DUE

TO THE COMMUNICATION IN SOCIAL NETWORKS. . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Decheva L., I. Pashalieva, E. Stancheva, Y. Nyagolov, N. Negrev - MELATONIN

AND THE FIBRINOLYTIC SYSTEM IN THE RAT: EFFECTS ON THROMBIN ACTIVATABLE

FIBRILOLYSIS INHIBITOR (TAFI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Nikolova J., M. Orbezova, P. Atanasova, P. Nikolov, F. Nikolov, P. Hrischev -

PREHYPERTENSION IN WOMEN WITH METABOLIC SYNDROME . . . . . . . . . . . . . . . . . . . 133

Nyagolov Y., I. Pashaliewa, N. Doncheva, N. Negrev - SOMATOTROPIN, SOMATOSTATIN

AND PROLACTIN EFFECTS ON VITAMIN K-DEPENDENT PLASMA CLOTTING FACTOR

(FII, FVII, FIX, FX) ANTIGEN CONCENTRATIONS IN RATS . . . . . . . . . . . . . . . . . . . . . . . 137

Hristov K. - RESEARCH ON VARIATIONS IN SOME ELECTROPHYSIOLOGICAL

CHARECTERISTIC DATA OF ACUPUNCTURE POINTS IN EXPERIMENTAL ANIMALS PUT

UNDER DIFFERENT ATMOSPHERE PRESSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Daskalov Ì. - LONG-TERM SURVIVAL OF A SERIES OF CANCER PATIENTS WITH PURULENT

SEPTIC POSTOPERATIVE COMPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

AUTOR'S INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

PERMUTERM SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

INSTRUCTIONS TO AUTHORS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

4

KRASI

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KRASI

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KRASI

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KRASI

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SENSORIMOTOR INTEGRATION: TMS STUDIES OF PROCESSING

OF PROPRIOCEPTIVE INFORMATION IN HEALTH AND DISEASE

Kossev A.

ABSTRACT

Sensorimotor integration was investigated by transcranial magnetic stimulation (TMS) and muscle vibration

(MV) in healthy subjects and patients with cervical dystonia (CD), idiopathic Parkinson’s disease (IPD), clini-

cally probable Parkinsonian multiple system atrophy (MSA-P) and amyotrophic lateral sclerosis (ALS). MV

(80 Hz, 0.5 mm) was applied to the right extensor carpi radialis muscle (ECR). Single and paired TMS pulses

were applied 3 s after onset of the MV. Motor evoked potentials (MEPs) were recorded from the vibrated ECR

and its antagonist, the flexor carpi radialis muscle using conventional surface electromyographic techniques.

In healthy subjects a vibratory related facilitation of the motor cortex was described. In patients with CD MV

induced facilitation was significantly reduced. During MV the level of intracortical inhibition (ICI) was also

reduced in CD patients in contrast to increased level of intracortical facilitation. The impaired sensorimotor

integration in CD patients is probably due to the alterations of basal ganglial function. We found that process-

ing of proprioceptive information is differently affected in IPD and MSA-P patients. The impaired ICI and

missing MV induced facilitation of MEP in IPD p-atients can be partially normalized by the Levodopa. Our

results suggested an impairment of ICI and processing of proprioception in ALS patients. Inhibition of excit-

atory neurotransmitters by riluzol might explain the recovery of ICI in ALS patients.

Key words: transcranial magnetic stimulation (TMS), muscle vibration (MV), motor evoked potential (MEP),

intracortical inhibition (ICI), intracortical facilitation (ICF), silent period (SP)

INTRODUCTION

Sensorimotor integration is the process whereby sensory in-

put is integrated by the central nervous system and used for

assisting motor program execution. Motor evoked potentials

(MEPs) in response to transcranial magnetic stimulation

(TMS) are subject to various influences. A powerful means

to modify MEPs is muscle vibration (MV) (4). The vibration

stimuli mainly excite the muscle spindle afferents and the

motor cortex is primarily responsible for the processing of

muscle spindle signals and limb movement perception.

METHODS

We have studied healthy volunteers and patients with cer-

vical dystonia (CD), idiopathic Parkinson”s disease (IPD),

clinically probable Parkinsonian multiple system atrophy

(MSA-P) and amyotrophic lateral sclerosis (ALS). The re-

sults from patient’s groups were compared to those of

age-matched control subjects. The study was approved by

the local Ethics Committee and subjects and patients were

studied after giving written informed consent. For the clini-

cal details see the original papers (5,16,18,19).

Subjects and patients were seated with the right arm gently

fixed in slight abduction from the trunk (20o) and flexion in

the elbow (110 o). MV was used with a frequency of 80 Hz

and an amplitude of 0.5 mm which avoided generation of the

tonic vibration reflex (TVR). MV was applied to the right

extensor carpi radialis muscle (ECR) by means of an electro-

magnetic mechanical stimulator (Ling Dynamic Systems,

Model V100) with a disk surface (2 cmin diameter). The du-

ration of the MV trains was 4 s with random intertrial inter-

vals between 12 and 22 s. In a group of healthy subjects we

used different MV frequencies – 80, 120 and 160 Hz.

TMSwasappliedassingleorpairedpulsesusing twoMagStim

200 stimulators (Magstim Company, Ltd., UK) connected to a

stimulating coil through a BiStim module. The circular coil

(mean diameter 9 cm; current flow anticlockwise when viewed

from above) was adjusted over the vertex to evoke optimal

muscle responses in the muscles of the right side.

Motor evoked potentials were recorded from the vibrated

ECR and its antagonist, the flexor carpi radialis muscle

(FCR) using conventional surface electromyographic

(EMG) techniques. Signals were amplified (band pass 10

Hz - 5 kHz) and digitized (sampling rate 10 kHz). Periods

of 400 ms duration (100 ms prior to the stimulus and 300

ms after it) were stored on the disk. The EMG activity was

continuously monitored, and patients and subjects had au-

dio-visual feedback to ensure the absence of voluntary

background activity as well as of TVR. In the group of

healthy subjects we recorded also MEPs of the muscles

contralateral to the vibrated arm.

Motor threshold (MT) was determined at rest as the lowest

stimulus intensity eliciting three responses of at least 50 µV

peak to peak amplitude in four stimuli.

5

Scripta Scientifica Medica, 2012; vol. 44 (1), Supplement 1 Copyright © Medical University, Varna

Address for correspondence:

A. Kossev, Bulgarian Academy of Sciences, Sofia 1113,Acad. G.

Bontchev Str., Bl.21.,


Single pulse TMS (s-TMS) was applied using an intensity

of 120 % of MT. Paired stimuli (p-TMS) were performed

with intensities of 70% of MT for the conditioning first

pulse and 120 % of MT for the following test pulse at

interstimulus intervals (ISI) of 3 ms to evaluate intracortical

inhibition (ICI) and 13 ms for intracortical facilitation (ICF)

as originally described by Kujirai et al. (10,15).

TMS was applied first without MV. Five single stimuli were

followed by 10 paired stimuli (five of each ISI presented in

random order) with random intertrial intervals. Thereafter,

the procedure was repeated with MV of the ECR. Single and

paired TMS pulses were applied 3 s after the onset of MV.

In the group of healthy subjects we used both TMS and

transcranial electrical stimulation (TES), which allows for a

differentiation between the cortical and spinal actions. TES

was carried out using a Digitimer (Model D180, Digitimer

Ltd., Welwyn Garden City, UK) as the cathode placed over

the vertex, and the anode - about 7 cm lateral to the left.

Cortical silent period (SP) was investigated at 30 % of maxi-

mal isometric voluntary contraction force (MVIC) of ECR. A

single TMSpulse (intensity120 % of MT) was applied during

voluntary contraction of the ECR. Five trials were obtained

and the procedure was repeated with vibration of the ECR.

The MEP parameters of interest was MEP size as assessed by

the total voltage time integral (area) or peak-to- peak amplitude

and MEP latency. In each subject, MEP values were normal-

ized to themeanvaluesobtainedwithsingleTMSwithoutMV.

The resulting values were pooled for the groups and were ex-

pressed as mean ± standard deviation.

Paired t-test was used to assess the effects of paired pulse TMS.

Differences between controls and patients and the effect of MV

were assessed using analysis of variance (ANOVA). Probabil-

ity (p) values of < 0.05 were considered significant.

RESULTS AND DISCUSSION

MV in healthy subjects.

In healthy subjects MEPs in the vibrated muscle are aug-

mented when TMS is applied more than 120 ms after MV

onset (Fig.1). Simultaneously MEPs recorded from the an-

tagonist of the vibrated muscle are suppressed (20). The

augmentation of the MEPs in the vibrated muscle was ob-

served at MV frequencies 80 and 120 Hz but not at 160 Hz

and was increasing with ongoing MV. In contrast, the inhi-

bition in the antagonist revealed a reverse time course and

was also induced by vibration frequency 160 Hz.

When TES was used, MEPs were not augmented in the vi-

brated muscles while those in the antagonist were de-

creased (9) (Fig.2). This dissociation between the effects of

TMS and TES as well as the different frequency dependen-

cies and different time courses suggest involvement of dif-

ferent neuronal circuits and possibly different receptor in-

puts in the MEP augmentation. It seems that MEP augmen-

tation in the vibrated muscle is caused by cortical activation

while MEP depression in the antagonist seems to involve

both spinal and cortical circuits and probably other dy-

namic mechanoreceptors (8).

MV induced facilitation was stronger at 80 Hz than at

higher frequency stimulation. Using lower vibration fre-

quency Steyvers et al. (21) confirmed that the optimal fre-

quency is in the range around 75 Hz, which is the optimal

for stimulation of the primary endings (14). All this find-

ings suggest that the primary muscle spindle messages (Ia

fibers) are the major sensory input underlying the vibratory

related facilitation of the motor cortex.

During MV the homonymous contralateral muscle revealed

a slight non-significant augmentation of MEPs but its antag-

onist showed a gradual depression of MEPs with ongoing

MV reaching significant levels (7). These findings suggest

transcallosal MEP modulation which involve the Ia

afferents.

MV in patients with cervical dystonia.

Eleven patients with cervical dystonia and 11 age-matched

healthy control subjects were enrolled in the study (5,19).

Motor thresholds (MT) as well as MEP areas after single

TMS without MV in ECR and FCR for CD patients were

fairly similar to those in the control subjects. With condi-

tioning TMS, ICI with short ISIs (3 msec) and ICF with

6

Sensorimotor integration: TMS studies of processing of ...

Fig. 1: Area of motorevoked potentials (MEPs)

recorded from vibrated extensor carpi radialis muscle

(ECR) and its antagonist flexor carpi radialis (FCR).

The parameters are normalized to the mean control

values and pooled for all subjects (mean ± standard

error; n = 10). Motor evoked potentials were recorded

without muscle vibration - MV (control – c.), 0.5 s and

3 s after onset of MV, and 1 s after offset of MV (pv.).

Three different vibration frequencies were used: 80 Hz,

120 Hz, and 160 Hz. Asterisks indicate significant

differences to control values.

long ISIs (13 msec) were significantly pronounced simi-

larly to the control subjects (see Fig.3).

With single-pulse TMS there was a significant MV induced

MEP augmentation in the vibrated ECR, although it was

much less pronounced than in control subjects (see Fig. 3).

Simultaneously, the MEPs recorded from the antagonist

showed a tendency to be reduced compared with single

TMS without MV. Comparison of the two groups (control

subjects and patients) revealed significantly smaller MEP ar-

eas recorded from both muscles during MV in the patients.

The MV-induced changes of the conditioned MEPs were

also different from those in the control subjects. The MEP

reduction at ISIs of 3 ms was smaller than in control sub-

jects and did not reach significant level. In contrast to con-

trol subjects, there was a significant ICF with ISIs of 13 ms.

The MV-induced augmentation in patients was suppressed

significantly, resembling previous findings in patients with

musician’s cramp (Rosenkranz et al., 2000). The essential

difference between the two studies was that the vibrated

forearm flexors were localized in the affected body segment

in musician’s cramp, whereas this was clearly not the case in

cervical dystonia. Therefore, this similarity suggests that the

changes of cortical function in focal dystonia are more gen-

eralized and extend beyond the projection areas of the clini-

cally involved muscle groups. The comparison of MV-in-

duced effects on MEPs in response to TMS and TES

strongly suggest that MV-induced augmentation results from

the cortical rather than spinal activation (9), supporting the

hypothesis that abnormal MEP augmentation in our patients

is caused by an impairment of the cortical mechanisms.

The mechanisms underlying abnormal MV induced effects

in patients, however, are not clear. Because the MEPs in the

patients were abnormal only during MV, it seems most

likely that the sensorimotor integration is impaired. Proba-

bly, the gain of proprioceptive input to the cortex is

changed, or focusing to the target sensorimotor areas is im-

paired as a consequence of the alterations of the basal

ganglial function in dystonia (22). In fact, several studies

have suggested an abnormal central processing of the sen-

sory information in dystonia (1).

7

Kossev A.

Fig. 3: Unconditioned (single TMS) recorded during

MV and conditioned (paired TMS) MEP areas (mean ±

standard error; n = 11) recorded from ECR and FCR

without MV and 3 s after onset of MV applied on the

ECR. The unconditioned values during MV are

normalized to the corresponding mean value of MEP

areas recorded without MV. The conditioned MEP

areas at interstimulus intervals of 3 msec and 13 msec

are normalized to the corresponding mean value of

MEP areas recorded in response to single

(unconditioned) TMS. Open bars - healthy control

subjects; shaded bars - patients with cervical dystonia.

Asterixes indicate a significant effect of MV-induced

augmentation in the vibrated muscle, significant effect

of intracortical inhibition (ISI 3-msec) and intracortical

facilitation (3-msec ISI) and significant differences

between patients and the control group.

Fig. 2: MEPs in response to transcranial electric (TES)

(A) and transcranial magnetic stimulation (TMS) (B)

(both with intensity 120% of motor threshold). MEPs

were recorded from vibrated ECR muscle and its

antagonist FCR without MV and 3 s after the onset of

MV. The responses on the figure are average of 5

stimulations.

MV in patients with idiopathic

Parkinson´s disease (IPD) and

Parkinsonian multiple system atrophy

(MSA-P).

Ten patients with IPD, 10 patients with clinically probable

MSA-P criteria and 10 healthy control subjects were studied.

All patients were examined in a defined off-state, i.e. at least

12 hours after last intake of anti-Parkinson medication (16).

Motor thresholds at rest did not differ significantly between

healthy controls, IPD and MSA-P. Without MV MEP sizes

in both patient groups compare to controls were smaller

(not significant), ICI was missing in ECR in IPD patients

and missing in both muscles in MSA patients (Fig.4).

During MV MEPs of the vibrated ECR in response to sin-

gle and paired TMS were significantly augmented. The

MEPs recorded from the non-vibrated antagonist muscle

(FCR) were only slightly, but not significantly increased

(see Fig.4). In IPD, MEPs were not augmented by MV. In

MSA, however, MV induced significant MEP augmenta-

tion in both muscles, i.e. in the vibrated ECR and in the

non-vibrated FCR.

The duration of SP without MV did not differ significantly

between controls and both patient groups although it

tended to be shorter in IPD (p < 0.08) (Fig.5). The MV did

not change SP in controls and in IPD while it caused signif-

icant SP prolongation in MSA.

Without MV ICI in resting forearm muscles is reduced in

IPD in the off-state (13,2). It was proposed that this deficit

may reflect a reduction of excitability of GABAA depend-

ent inhibitory pathways (23) as a consequence of changed

basal ganglia output (13). In MSA we found the same defi-

cit and the impaired ICI seems to be a common

neurophysiological feature of IPD and MSA-P.

Concerning MV, it has been shown in IPD that the vibra-

tory input has less effect on the accuracy in positioning

tasks than in the healthy subjects (12,6). Thus we had ex-

pected that MV would have a less facilitatory effect on

MEPs which was basically confirmed by our results. The

MV induced MEP augmentation in IPD was completely

absent. We agree with several other authors that the

changed modulation of cortical excitability by sensory in-

formation in IPD is caused by the impaired sensory pro-

cessing. Although the level of these changes within the

CNS is not clear it is generally assumed that the dysfunc-

tion of the basal ganglia plays an important role (11).

In contrast to IPD, our MSA-P patients showed a signifi-

cant MEP augmentation which was, however, weaker than

in controls. Additionally this augmentation was not focal

and was similar in the vibrated muscle and in the non-vi-

brated antagonist.

The duration of cortical SP without MV was not signifi-

cantly different between healthy subjects and both groups

of patients with a tendency to be shorter in IPD. These re-

sults confirm previous findings in IPD and MSA (3). The

MV did not change SP significantly in control group or in

IPD patients while it prolonged the duration of SP signifi-

cantly in the MSA patients. The reason for this change and

8


Fig. 5: Cortical silent period (SP) (mean + standard

error, n = 10) of healthy controls, patients with

idiopathic Parkinson’s disease (IPD) and multiple

system atrophy (MSA) patients without muscle vibration

(MV) (white bars) and during MV (grey bars). The

asterisk indicates a significant prolongation of SP

Fig. 4: MEP areas (mean + standard error, n = 10)

normalized to unconditioned MEPs recorded in

response to single pulse TMS (s-TMS, S) without muscle

vibration (MV) (white bars) and during MV (grey bars).

The responses to s-TMS are marked by “S” and

responses to paired pulse TMS with 3 and 13 ms

interstimulus interval (ISI) interval are marked by “3

ms” and “13 ms”, respectively. The significance (p <

0.05) of inhibition due to ICI is marked over the white

bars (without MV) by asterisks. The asterisks between

the grey and the white bars indicate the significance of

facilitation due to MV comparing the area of the MEP

its functional significance is not clear, but obviously MSA

shows more widespread MV evoked changes. Differences

in the influence on inhibitory intracortical circuits between

IPD and MSA-P may reflect the different underlying pa-

thology.

Additionally, the processing of proprioceptive information

in IPD patients group was studied also in the on state (1

hour after the take of Levodopa) (17). The impaired ICI and

missing MV induced facilitation of MEP can be partially

normalized by Levodopa (Fig.6)

MV in patients with amyotrophic lateral

sclerosis (ALS).

Thirteen ALS patients (7 with Riluzol, 6 without Riluzol)

and 10 age-matched healthy controls were enrolled into this

study (18). The MV facilitated MEPs significantly in

healthy subjects, but not among ALS patients (Fig.7).

ALS patients without Riluzol showed an impairment of

ICI, while those on riluzole presented with normal inhibi-

tion of MEPs (Fig.8). In contrast to healthy controls the

ICF in ALS patients was significant neither in patients on

Riluzole nor in those without Riluzole although treated pa-

tients revealed a tendency to ICF in both muscles (p=0.08

and p=0.09). The results suggest an impairment of ICI and

processing of proprioception in ALS. The inhibition of ex-

citatory neurotransmitters by Riluzole might explain the re-

covery of ICI in ALS patients.

9

Kossev A.

Fig. 7: MEP areas (mean + standard error) (patients

with amyotrophic lateral sclerosis) in response to single

pulse TMS applied 3 s after mV onset, normalized to the

MEPs recorded in response to single pulse TMS without

muscle vibration (MV). The asterisks indicate

significant effect of MV.

Fig. 8: MEP areas (mean + standard error) (patients

with amyotrophic lateral sclerosis) in response to

paired pulse TMS (ISI - 3 and 13 ms) without MV,

normalized to the corresponding MEP in response to

single pulse TMS. The asterisks indicate significant

effect of conditioned stimulation.

Fig. 6: MEP areas (mean + standard error, n = 10)

normalized to unconditioned MEPs recorded in

response to single pulse TMS without muscle vibration

(MV). White bars – without MV and grey bars 3 s after

MV onset. The responses to s-TMS are marked by “S”

and responses to paired pulse TMS with 3 and 13 ms

ISI interval are marked by “3 ms” and “13 ms”,

respectively. IPD–off and IPD–on - patients with

idiopathic Parkinson’s disease studied in off state and

on state (1 hour after the take of Levodopa). The

asterisks between the grey and the white bars indicate

the significance of facilitation due to MV comparing the

area of the MEP during MV to the corresponding data

without MV.

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Buccol ier i , e t al . Abnormalities of sensorimotor

integration in focal dystonia: a transcranial magnetic

stimulation study.- Brain, 2001, 124, 537–545.

2. Bares , M., P. Kanovsky, H. Klajblova, I .

Rektor . Intracortical inhibition and facilitation are

impaired in patients with early Parkinson’s disease: a

paired TMS study.- Eur. J. Neurol., 2003, 10,

385-389.

3. Cantel lo , R. , R. Tarlet t i , C. Civardi .

Transcranial magnetic stimulation and Parkinson’s

disease.- Brain Res. Rev., 2002, 38, 309-327.

4. Claus, D. , K. R. Mil ls , N. M. F. Murray. Fa-

cilitation of muscle responses to magnetic brain stim-

ulation by mechanical stimuli in man.- Exp. Brain

Res., 1988, 71, 273–278.

5. Dengler , R. , S. Siggelkow, J. D. Rollnik, J .

Duper , C. Moll , A. Kossev. Changes of motor

cortical function in dystonia.- In: Sensorimotor Con-

trol. R. Dengler, A. Kossev, eds. NATO Science Se-

ries, Series 1.- Life and Behavioural Sciences, 2001,

326, 150-158.

6. Khudados, E., F. W. Cody, D. J. O’Boyle.

Proprioceptive regulation of voluntary ankle move-

ments, demonstrated using muscle vibration, is im-

paired by Parkinson’s disease.- J. Neurol. Neurosurg.

Psychiatry, 1999, 67, 504-510.

7. Kossev, A., S. Siggelkow, H.-H. Kappels, R. Dengler,

J. D. Rollnik. Crossed effects of muscle vibration on

motor-evoked potentials.- Clin. Neurophysiol., 2001,

112, 453-456.

8. Kossev, A., S. Siggelkow, J. D. Rollnik, J. Däuper, R.

Dengler. Modulation of corticospinal excitability and

intracortical mechanisms during muscle vibration: a

study using transcranial magnetic stimulation.- In:

Sensorimotor Control. R. Dengler, A. Kossev, eds.

NATO Science Series, Series 1.- Life and Behav-

ioural Sciences, 2001, 326, 19-28.

9. Kossev, A., S. Siggelkow, M. Schubert, K.

Wohlfarth, R. Dengler. Muscle vibraation: different

effects on transcranial magnetic and electrical stimu-

lation.- Muscle Nerve, 1999, 22, 946-948.

10. Kujirai, T., M. D. Caramia, J. C. Rothwell, et al.

Corticocortical inhibition in human motor cortex.- J.

Physiol. (Lond.), 1993, 471, 501–519.

11. Maschke,M., C. M. Gomez, P. J. Tuite, J. Konczak.

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lum, impairs kinaesthesia.- Brain, 2003, 126,

2312-2322.

12. Rickards, C., F. W. Cody. Proprioceptive control of

wrist movements in Parkinson’s disease. Reduced

muscle vibration-induced errors.- Brain, 1997, 120,

977-990.

13. Ridding, M. C., R. Inzelberg, J. C. Rothwell. Changes

in excitability of motor cortical circuitry in patients

with Parkinson’s disease.- Ann. Neurol., 1995, 37,

181-188.

14. Roll, J. P., J. P. Vedel, E. Ribot. Alteration of

proprioceptive messages induced by tendon vibration

in man: a microneurographic study.- Exp. Brain Res.,

1989, 76, 213–222.

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Moll , A. R. Kossev, R. Dengler . Die

transkranielle magnetische Doppelstimulation zur

Beurteilung kortikokortikaler Inhibition und

Fazilitierung.- Klin. Neurophysiol., 2001, 32: 26-29.

16. Schrader, C., T. Peschel, J. Däuper, J. D. Rollnik, R.

Dengler, A. Kossev. () Changes in processing of

proprioceptiv information in Parkinson’s disease and

Multiple System Atrophy. Clin. Neurophysiol., 2008,

119, 1139-1146.

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A. R. Kossev. Impaired proprioception in

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Neurophysiol., 2008, 39, 262-266.

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sensorimotor integration in cervical dystonia - a study

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Kappels, W. Wolf, R. Dengler. Modulation of motor

evoked potentials by muscle vibration: the role of vi-

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873-881.

10


EFFECTS OF ADAPTATION IN VISUAL PERCEPTION

OF GLOBAL SPEED

Genova B., N. Bocheva, M. Stefanova, S. Stefanov

Department of Sensory Neurobiology, Institute of Neurobiology, BAS, Sofia

Our aim was to investigate how adaptation to the mean speed of motion affects global speed perception. The

stimuli were composed of 50-frame sequences of moving elements presented in two circular apertures to the

left and to the right of a fixation point. Each element moved in random direction and with speed taken from

uniform distribution with different range. We presented two different standard speeds and several speed

ranges. Each block of trials began with an adaptation phase with duration of 32 s. Top-up stimuli with dura-

tion of 2 s were shown 500 ms before each stimulus pair. The standard stimulus was presented always at the

position of the adaptor. The task of the observer was to determine which of two stimuli in the pair, left or right,

was faster. Our data show that the bias and the sensitivity to differences in global speed perception are differ-

ently affected by the adapting speed and the speed range. The results are discussed in relation to tuning char-

acteristics of MT area neurons.

Key words: Vision, Perception, Motion, Speed, Adaptation, Sensitivity

INTRODUCTION

Visual system adapts to the recently viewed stimuli. After

prolonged exposure to moving stimuli sensitivity to speed

differences is increased (2) but absolute speed sensitivity is

reduced and the stimulus may appear to move at a different

speed (10). Adaptation is a process that occurs at different

levels in the visual pathway, and thus tracing its effects

would significantly clarify the organization of the visual

system. Furthermore, motion perception is also affected by

different parameters of stimulation, such as spatial fre-

quency, contrast, etc. For example, the sensitivity to speed

differences is impaired by the presence of noise (1). Motion

perception also declines during normal aging (e.g. (7)).

Our aim is to investigate the effects of adaptation to the

mean speed of motion on sensitivity to global speed differ-

ences and to evaluate whether these effects vary with age.

To achieve this goal we evaluated the changes in the pa-

rameters of the psychometric function with and without ad-

aptation to the mean motion speed.

MATERIAL AND METHODS

Ten younger subjects (mean age 20 yrs., range 17–24 yrs.,

five male) and 10 older subjects (mean age 75 yrs., range

65–85 yrs, four male) took part in the experiments. All ob-

servers were naive to the purposes of the experiment and

reported having normal or corrected to normal vision. The

older observers passed a routine ophthalmologic examina-

tion that excluded ocular diseases, particularly lens opaci-

ties, glaucoma, macular degeneration.

The motion stimuli consisted of movie sequences of 50

dots moving in directions randomly selected in the range of

0o-360o. The stimuli were generated and presented with

Dell computer running MATLAB with the help of

PsychToolbox. The subject sat at a distance of 114 cm from

the computer screen. The monitor operated in a 1280x1024

resolution mode with a refresh rate of 60 Hz.

Two experiments were performed. In a two-alternative

forced choice procedure (2AFC) using the method of con-

stant stimuli the subjects had to discriminate the global speed

of motion of two patterns. In Experiment 1 the stimuli were

presented simultaneously in two circular apertures with di-

ameter of 6 deg positioned to the left and to the right of the

central fixation point. The closest edges of the apertures were

separated by a distance of 3 deg. All elements of the standard

moved at speed Vst. Two speeds of standard motion were

used: Vst1 = 6.2 and Vst2=9.2 deg/s. The dots in the test stim-

uli moved with speeds randomly selected from a uniform

distribution with a predefined range centered on their mean

speed Vt. This range determines the speed noise level. For

each standard speed we presented five speed noise levels: for

Vst1 they were: 0%, 14%, 21%, 29% and 36% and for Vst2 -

0%, 10%, 19%, 29% and 38%, where the noise levels are

expressed as percentage of Vst. The mean speed Vt of the

test stimuli could be faster or slower than Vst. Seven differ-

ent values of Vt were used for each standard.

The same stimuli were presented in Experiment 2. The fol-

lowing adaptation protocol was used: an initial adapting

stimulus lasting 32 s was presented before each block of tri-

als at the position of the standard; a top-up adapting stimu-

lus with duration of 2 s was presented 500 ms before each

pair of standard-test stimuli. The adapting stimuli were the

same as the standard stimuli.

11



B. Genova, Dept. of sensory neurobiology, Institute of Neurobiology,

BAS, 23 Acad. G. Bonchev St. Sofia Bulgaria 1113


Inbothexperiments theobserverhad to reportwhichof themo-

tions – the left or the right appeared as moving faster. The stan-

dardstimuluswasalwayspresented to the left sideof thescreen.

Each experiment was divided in two experimental sessions

that differ by the value of the standard speed. Half of the

subjects started with the slower, the other half – with the

higher speed. Experiment 1 (without adaptation) and Ex-

periment 2 (with adaptation) were performed at different

days. Each combination of the experimental variables –

magnitude of the standard and of the test speed and noise

level was presented 16 times in random order in each

experiment.

RESULTS

The proportion of responses “test speed faster than the stan-

dard” was use to evaluate the parameters of the psychometric

function: the point of subjective equality (PSE) and the dif-

ferential threshold. Each individual psychometric function

was fitted by a normal cumulative distribution function. To

compare the data for the different standards we transformed

the PSE and the differential thresholds in relative measures:

Weber fractions and relative constant errors. The Weber

fraction is determined as (differential threshold/ standard);

the relative constant error is (PSE-standard)/standard. Fig-

ures 1 and 2 show the effect of the experimental factors on

these characteristic of performance.

Amixed model ANOVAwith factorsage (asbetween-groups

factor) and standard speed, noise level and adaptation (as

within-groups factors) was applied separately to the Weber

fractions and to the relative constant error (CE). A multivariate

approach was applied to the mixed model ANOVA.

The results show that the main effect of age is not signifi-

cant (F(1,18)<1; p=0.05) both for Weber fractions and CE.

The adaptation to the global speed significantly changes the

performance: it reduces the Weber fraction (F(1,18)=5.6;

p<0.05) and increases the CE (F(1,18)=10.6; p<0.01). The

interaction of age and speed noise level is significant

(F(4,15)=6.3; p<0.01) revealing that CE decreases with

noise level for the older observers while for the younger

ones it increases (Fig. 2). As a result, at higher levels of

noise the older observers become more accurate than youn-

ger. The other main factors and their interactions are

insignificant at p=0.05.

The separate analyses of the data for the two age groups show

that for the older observers the sensitivity to speed differences

significantly increases after adaptation for both standard

speeds (F(1,9)=7.5; p<0.05 and F(1,9)=8.5; p<0.05). This ef-

fect is clearly visible in Fig.1 as a reduction in the Weber frac-

tions after adaptation. The younger group shows similar trend

(Fig. 1), however, for them this effect is insignificant.

Even in the unadapted condition the PSE is lower than the

standard for the two age groups. This is expressed by nega-

tive CE (Fig. 2). After the adaptation CE significantly in-

creases due to the significant shift in PSE of older observers

for the two speeds of the standard (for Vst1: F(1,9)=11,7;

p<0.01 and for Vst2: F(1,9)=5.8; p<0.05). For the younger

observers the CE changes significantly only for the faster

standard speed (F(1,9)=5.6; p<0.05). The older observers

are affected also by the noise level when the standard speed

is low (F(4,6)=4.6; p<0.05). No other main factors or inter-

actions are significant at p=0.05. It should be noticed that

interaction between speed noise level and adaptation is no

significant.

12

Effects of adaptation in visual perception of global speed

Figure 1. Decrease in Weber fractions after adaptation.

A. Data for Vst1=6.2 deg/s

Figure 1. Decrease in Weber fractions after adaptation.

B. Data for Vst2=9.2 deg/s

Figure 2. Lowering in PSE presented by CE normalized

to Vst. The values <0 indicate an increase in perceived

test speed after adaptation. A. Data for Vst1=6.2 deg/s

The observed changes in PSE and in the sensitivity to speed

differences after adaptation modify the position and steepness

of the psychometric curve. It shifts toward lower speeds, and

becomes steeper reflecting a reduction in absolute speed sensi-

tivity and improvement in relative speed sensitivity (Fig. 3).

Our findings for influence of adaptation to global speed per-

ception are similar to the known results obtained in local-mo-

tion perception. In the present study we adapted to a single

speed and compared the perceived speed of the adaptor to

stimuli with different range of speeds. In this way we used as a

measuring device the test stimulus and the performance was

determined not only by the effect of adaptation but also by the

ability of the observers to pool the local velocity in a global es-

timate. Two questions arise: how much the observed effects

are related to the perception of global speed and whether the

adaptation spreads to other parts of the visual field. In our pre-

vious study (3) we obtained no effect of the noise level on the

Weber fractions in global speed discrimination, smaller

Weber fractions for higher standard speed and an overestima-

tion of global speed for high levels of speed noise for the older

group. This implies that the sensitivity to differences in the

global speed of motion is independent of the range of speeds.

The older observers, however, seem to disregard the slowest

speeds which leads to a misperception of the global speed at

high noise levels. Extrapolating to the present study we would

expect an overestimation of the test speed for high noise lev-

els. Since the adaptation reduces the speed of the standard (CE

is negative at noise level =0%), the bias in the perceived speed

should increase, not decrease with the noise level.

DISCUSSION

Our results show that adaptation affects more the sensitivity to

differences in speed of the older observers – the Weber

fractions in the adapted and in the un-adapted condition differ

more than those of the younger observers. This finding could

not be explained by differences in the abilities of the two age

groups to integrate the speeds of the local motions in a global

estimate. It suggests that the effect of adaptation has spread to

un-adapted regions affecting the perceived speed of the test

stimulus. McGraw and Roach (6) have shown that adaptation

to motion propagates across space. When the adaptor is

presented close to the fixation point the adaptation effect

spreads centrifugally. These finding suggest that neural

populations from non-overlapping spatial regions interact.

The larger effect of the adaptation on the performance of the

older observers might be related to the lower inter-cortical

inhibition with age (e.g. 8, 9) that would allow the spread of

the adaptation over larger area.

If adaptation affects the local speeds in the test stimulus in

proportion to their difference with the adapting speed, no

differences in sensitivity or in bias would be expected due to

the symmetry of the speed range with respect to the mean

speed. The different effect of the noise level on the bias for the

two age groups reveals specific adaptation effects. Hietanen et

al (4) showed that the PSE is affected by the adapt/test speed

ratio. When the test speed is equal or slower than the adaptor,

the perceived speed is reduced; however, when the test speeds

are faster than the adaptor, the perceived speed may even

increase. Such effect would imply small negative bias in the

perceived speed in our experimental conditions. When the

range of speed increases, the variability of the global speed

percept might increase due to random deviations of the local

speeds from the uniform distribution, but there are no reasons

to expect that this would change the trend in the bias.

The different effects of the noise level on the bias for the two age

groups might be related to changes in the neural populations

coding speed. Aging shifts the preferred speeds of motion of the

MT neurons to lower speed (11). As adaptation affects more the

neurons that are not optimally tuned to the adapting speed (e.g.

(5)), its effect might be larger for the faster speeds in the test

stimulus for the older observers. This would reduce the perceived

global speedandwouldbemoreevidentathigh levelsofnoise.

CONCLUSIONS

In summary, the results of the experiments indicate that: The

adaptation to mean speed increases the sensitivity to differences in

global speed and reduces the accuracy in speed estimation; The

effects of adaptation are stronger for the older people; The accuracy

of speed estimation is affected differently by the noise level for the

two age groups; The effect of noise on the sensitivity to speed does

notdependonadaptation;Theeffectsofadaptationontheperception

of global speed are similar to the findings in the available literature

about the effects of adaptation on the perception of local velocity.

13

Genova B., N. Bocheva, M. Stefanova ...

Figure 3. After adaptation psychometric curve is

steeper and shifted on the left

Figure 2. Lowering in PSE presented by CE normalized

to Vst. The values <0 indicate an increase in perceived

test speed after adaptation. B. Data for Vst2=9.2 deg/s

ACKNOWLEDGEMENTS

This research is supported by Grant TK01/200-2009 of the

National Science Fund, Bulgaria.

REFERENCES

1. Bravo, M. J. , S. N. J . Watamaniuk. Evidence

for two speed signals: a coarse local signal for segre-

gation and a precise global signal for discrimination.

Vis. Res., 1995, 35, 1691–1697.

2. Cli f ford, C. W. G. , K. Langley .

Psychophysics of motion adaptation parallels insect

electrophysiology. Curr. Biol. 1996, 6, 1340–1342.

3. Genova, B. Z. , N. B. Bocheva, S. Stefanov.

Effects of aging on speed discrimination in the pres-

ence of noise. Perception, 2011, 40, ECVP Abstract

Suppl., p. 91

4. Hietanen, M. A. , N. A. Crowder , M.R.

Ibbotson. Differential changes in human perception

of speed due to motion adaptation. J. Vision, 2008, 8,

1–10.

5. Krekelberg, B. , R.J . van Wezel , T.D.

Albr ight . Adaptation in macaque MT reduces per-

ceived speed and improves speed discrimination. J.

Neurophysiol, 2006, 95, 255–270.

6. McGraw, P. V. , N. W. Roach. Centrifugal

propagation of motion adaptation effects across visual

space. J. Vision, 2008, 8 (11), 1-11.

7. Norman, J . F. , H. E. Ross, L.M. Hawkes, J .

R. Long. Aging and the perception of speed. Per-

ception, 2003, 32, 85–96.

8. Schmolesky, M. T. , Y. Wang, M. Pu, A.G.

Leventhal . Degradation of stimulus selectivity of

visual cortical cells in senescent rhesus monkeys. Nat.

Neurosci., 2000, 3, 384–390.

9. Tadin D. , R. Blake. Motion perception getting

better with age? Neuron, 2005, 45, 325–327

10. Thompson, P. Velocity aftereffects: the effects of

adaptation to moving stimuli on the perception of sub-

sequently seen moving stimuli. Vis. Res., 1981, 21,

337–345.

14

Effects of adaptation in visual perception of global speed

VISUAL EVENT-RELATED POTENTIALS BY AN IDENTIFICATION

OF GRATING ORIENTATION DIFFERENCE

Dushanova J., D. Mitov

Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

SUMMARY

Visual event-related potentials (VERP) to sinusoidal gratings employed in orientation discrimination

task were recorded. Stimuli were with spatial frequency of 2.9 cycles deg-1 and contrast of 5%, presented

in a Gaussian window with spatial constant of 0.483 deg. In each trial stimulus orientation varied ran-

domly between two values – 900 and 00, 900 and 750 as well as 900 and 850. The subject task was to press

one of the two keys by the left or the right forefinger according to the stimulus orientation (binary motor

task). It was found that reducing of orientation difference between the test stimuli decreased the ampli-

tude of N1 and N2 waves and increased the amplitude of P2 and in less extent – the amplitude of P3 com-

ponents. The latency of N1, N2 and P2 decreased with decrease of the orientation difference. The latency

of P3 wave prolonged at the smallest orientation difference. The effect of orientation difference on VERP

was negligible when the difference was decreased from 900 to 150, but it becomes substantial when the ori-

entation difference was decreased from 150 to 50.Thus the changes in ERP evaluated the selectivity of ori-

entation-specific channels and as a sign for a transition from detector to computational mode of

operation in orientation perception.

Key words: visual event-related potentials, sinusoidal gratings, orientation discrimination

INTRODUCTION

Many experimental data have given reasons to consider that

two main mechanisms are involved in the identification of

grating orientation – a detector mechanisms at large orienta-

tion differences and a computational mechanismat small ori-

entation differences between the stimuli (1,2,3,5).

In the present study the hypothesis was tested in experiments

based on detection of visual event-related potentials

(VERPs). The purpose of this study was to find a correlation

between the changes of VERP’s parameters, observed by

different orientation differences between the stimuli and the

selectivity of orientation-specific channels based mainly on

previous psycho-physiological investigations (2, 5,8,9).

It was ascertained appropriateness the changes of VERP

components as a sign for a transition from detector to com-

putational mode of operation in orientation perception.

MATHERIALS AND METODS

The EEG was recorded from 12 positions (10/20 system)

Fz, Cz, Pz, Oz, C3, C4, T3, T4, P3, P4, O1, O2 and EOG

for detection of blink artifacts. 20 subjects (11 females, 9

males, 31±7 years) had normal or corrected to normal vi-

sual acuity and no known ophthalmological or neurological

diseases. Handedness was assessed by a questionnaire

adapted from the Edinburgh Handedness Inventory.

Visual event-related potentials (VERP) to sinusoidal gratings

employed in orientation discrimination task were recorded.

Stimuli were with spatial frequency of 2.9 cycles/deg and

contrast of 5%, presented in a Gaussian window with spatial

constant of 0.483 deg for 100 ms in the centre of the visual

field. In each trial stimulus block orientation varied randomly

between two values – 900 and 00, 900 and 750 as well as 900

and 850, i. e. the orientation difference was 900, 150 and 50.

The subject task was to press one of the two keys by the left

or the right forefinger according to the stimulus orientation

(binary motor task, in all tasks by the right forefinger to

stimuli with orientation different from 900).

The VERPs were computed by averaging the artifact-free

trials for each stimulus/task combination and mean interval

across stimulus/task combination for each wave was N1

(80,140), P2 (130,200), N2 (190,298), P3 (290,550). The

statistical analyses of the parameters (amplitudes and laten-

cies of the waves) of VERPs were performed during the

above mentioned post-stimulus intervals and the statistical

difference between the groups (900/850 vs. 900/00, 900/750

vs. 900/00, 900/750 vs. 900/850) was assessed for each task

and interval by means of nonparametric procedure

(Kruskal-Wallis test) for pairs comparison of the scalp

leads between stimulus datasets.

15



K. Trifonova, Dept. of ophthalmology and general practiceStara

Zagora, Faculty of medicine, 47 Gen. Stoletov str, entr A, app 19,


RESULTS

When binary sensorimotor task was employed, the reduc-

ing of orientation difference between the test’s stimuli de-

creased the amplitudes of N1 and N2, as well as increased

the amplitude of P2 components and in less extent – the

amplitude of P3 components (Fig. 1).

The latency of N1, N2 and P2 decreased and the latency of

P3 wave prolonged at the smallest orientation difference

(Fig. 2).

DISCUSION

The interest to the spatio-temporal organization of the vi-

sual system and to the manner of coding different aspects of

the visual objects is not due to theoretical reasons only.

However, the results of this research could find some prac-

tical application. It is widely accepted in the literature that

the early N1, P2 components are related to the early infor-

mation processing, automatic or task induced, related to the

sensory analysis after the stimulus, while late N2 and P3

components reflect the late cognitive processes. The com-

ponents latency reflects the time for information processing

whereas the amplitude reflects the level of activation of the

brain structures (4). It is well known that perception is im-

paired when some neurological and other diseases exist (6,

7). The investigation of the characteristics of VERPs, regis-

tered under discrimination tasks conditions could be ap-

plied as an adequate and non-invasive method for studying

the sensory and motor information processing at central

brain level in healthy persons and patients with neurologi-

cal and other diseases (7). The research of the visual and

cognitive information processing for instance in diabetics is

of substantial importance since the retina cells are ex-

tremely susceptible to the change of the blood glucose level

and the disease often affects the periphery as well as central

nervous systems (6).

CONCLUSION

The effect of orientation difference on VERPs was negligi-

ble when the difference was decreased from 900 to 150, but

it becomes substantial when the orientation difference was

decreased from 150 to 50.

Thus the changes in VERPs evaluated the selectivity of

orientation-specific channels and as a sign for a transition

from detector to computational mode of operation in orien-

tation perception.

ACKNOWLEDGMENTS

The study was supported by NSF, Bulgaria, contract

0475/2008

REFERENCES

1. Blakemore C. and R. W. Campbel l , “On the

existence of neurones in the visual system selectively

sensitive to the orientation and size of retinal images,”

J. Physiol., 1969, 203, 237–260

2. Campbel l F. W., Robson J. G. , Application of

Fourier analysis to the visibility of gratings. Journal

of Physiology, 1968, 197, 551 - 556

3. Hubbel l D. H. , Wesel T. N. Receptive fields of

single neurons in the cat’s striate cortex. Journal of

Physiology, 1959, 148, 574 - 591.

16

Visual event-related potentials by an identification of ...

Figure 1. Statistical comparisons between theamplitudes of N1, P2, N2, P3 waves (rows) forpairs grating orientation differences duringsensory-motor task (columns).

Figure 2. Statistical comparisons between thelatencies of N1, P2, N2, P3 waves (rows) for pairsgrating orientation differences during

4. Êîê À. Event-related-potential (ÅRP) reflections of

mental resources: a review and synthesis. Biol.

Psychol., 1997, 45, 19 - 56.

5. Legge G. E. , “Sustained and transient mechanisms

in human vision. Temporal and spatial properties,”

Vision Res., 1978, 18, 69–81

6. Mc Crimon R. J . , Deary I .J . , Huntly l . J .

P. , MacLeod K.J. , Fr ier B.M. Visual informa-

tion processing during controlled hypoglycemia in

humans. Brain, 1996, 119 (4), 1277 – 1287.

7. Pol ich J, Herbst K L. , P300 as a clinical assay:

rationale, evaluation, and findings. Int J

Psychophysiol., 2000, 38 (1), 3 - 19.

8. Vassi lev A. , Simeonova B. , Zlatkova M.

Recognition of line’s orientation at the detection

threshold. In V.D. Glezer (Ed.). Pererabotka

informacii v zritelnoj sisteme, 1982, 35 – 40.

17

Dushanova J., D. Mitov

SEX-DEPENDENT EFFECT OF A NEW PEPTIDOMIMETIC ON

COGNITIVE FUNCTION OF ISOLATED RATS AFTER MATERNAL

DEPRIVATION

Tancheva L.1, E. Encheva

2, M. Novoselski

2, V. Petkov

1, R. Klisurov

2

1Institute of Neurobiology, BAS; 2Medical University, Sofia, Dept. of Physiology

ABSTRACT

INTRODUCTION: Maternal deprivation and social isolation lead to stress-related cognitive changes in rats.

The aim of the study was to test the effect of the peptidomimetic M6 on postnatal stress in female and male rat

offspring. MATERIAL AND METHODS: - Wistar pups were separated from their mothers on the 21st

day,

followed by 5-week isolation. Control rats were grouped in 2 cages: 6 male and 6 female. - Half the animals

(grouped and isolated) received M6, 150 mg/kg/d (i.p.) for 3 days. - Exploratory activity, and learning and

memory were tested with hole-board and step-through tests. - Data analysis was performed with SPSS,

ANOVA (mixed design). RESULTS: The isolated rat pups were more exploratory active; in the same time

they had better learning and memory than grouped animals. Cognitive functions were significantly better in

male than in female isolated rats. Exploratory activity was less in male than in female grouped rats. The M6

effect on cognitive functions was sex-dependent: improved memory in male grouped animals and also in iso-

lated female rats, but decreased memory in grouped female rats. CONCLUSION: Combined postnatal stress

exposure (maternal deprivation, followed by social isolation) affects cognitive functions differently, depend-

ing specifically on gender and stress exposure.

Key words: maternal deprivation, social isolation, peptidomimetic, cognitive functions

INTRODUCTION

The importance of early life stress for mental health is well

known, even though the underlying neurobiological mech-

anisms are not completely clear. Postnatal stress exposure

in rodents affects critical periods of brain development that

persistently alter structural, emotional and neuroendocrine

parameters in the adult offspring [1,2,5].

Maternal deprivation followed by social isolation is used as

an animal model for damaged cognitive function. On this

model we study the possibility for pharmacological modu-

lation and protection of cognition with the application of an

amino acid derivative.

In our earlier studies some newly synthesized aminoacids

(L-valine derivatives) demonstrated a significant

neuroprotective effect on adult rodents, especially the com-

pound M6 [4].

We suggest that this preventive effect may be even stronger

in young animals.

The purpose of the study was to examine the ability of M6

for pharmacological modulation and prevention of early

life postnatal stress in female and male rat offspring with

maternal deprivation.

MATERIALS AND METHOD

Experimental model and methods: Wistar male and female

rats were deprived from their mothers on the 21st day after

birth. Immediately after that, the animals were hosted in iso-

lated cages for 5 weeks (social isolation model according to

Valzelli (1978), and modified by Petkov [3]).

The control groups (after mother deprivation) were hosted in

commoncages-6rats incage,maleandfemaleseparatecages.

Half of the animals (grouped and isolated) received à newly

synthesized compound (L-valine derivative, M6) for 3 days

at the end of the isolation period, in a daily dose of 150

mg/kg intraperitoneally (i.p.).

The control groups of rats (male and female) received the

solvent Oleum Helianthi (in the same volume).

Comparative study on their cognitive functions was per-

formed on the 24th hour after the last treatment – namely:

1. Exploratory activity (Hole board test, on the 1st, 2nd and

3rd minute).

2. Learning and memory (on the 1st and on the 24th hour)

with the step-through test.

Statistical evaluation. Experimental data were analyzed

by statistical software SPSS 11.0 for Windows, ANOVA

(mixed design).

19



Eleonora N. Encheva, Department of Physiology, Medical Univer-

sity of Sofia 2, Zdrave Str., 1431 Sofia, Bulgaria

Tel: +359 885 089 175


RE SULTS AND DIS CUS SION:

The com bi na tion of 2 kinds of stress fac tors- ma ter nal de -

pri va tion (5 weeks) with so cial iso la tion - pro duced dif fer -

ent cog ni tive changes in male and female rats.

Our data show very in ter est ing re sults, like other re ports in

this field, which used dif fer ent mod els of early stress (swim

test and ex po sure to ad verse novel sit u a tion [1,2]).

Re gard ing the ef fect of so cial iso la tion, we found out that

so cially iso lated rats had:

- in creased ex plor atory ac tiv ity (Fig. 1)

- in creased pro cesses of learn ing and mem ory

(step-through test) in com par i son to the grouped an i mals

(on both the 1st and the 24th hour – Fig. 2 and 3)

The role of sex:

We found that the ef fect of the so cial iso la tion in young an i -

mals (56-57 days) on the cog ni tive func tion in rats is

sex-de pend ent. Ef fect of iso la tion on the learn ing and

long-term mem ory (on the 24th hour) is more sig nif i cant in

male than in fe male animals (Fig. 3).

Ex plor atory ac tiv ity is big ger in fe male than in male grouped

an i mals, but in iso lated the dif fer ence is in sig nif i cant (Fig. 4).

The ef fect of M6 treat ment:

Com pound M6 dem on strates pre ven tive ef fect on the cog -

ni tive func tion in rats - better in iso lated than in grouped an -

i mals. (Fig. 5)

We found also that in grouped an i mals the ef fect of M6 on

learn ing and mem ory is dif fer en tial, sex-de pend ent: in male

rats it is pre ven tive on the 1st hour, but in fe male rats, it is

the op po site- there is a mem ory de cline ob served dur ing the

step-through testing.(Fig.6)

Treat ment with M6 in grouped an i mals in creased the learn -

ing and short-term mem ory in male, but not in fe male (de -

creased it).The ten dency is quite the op po site in iso lated an -

20

Sex-dependent effect of a new peptidomimetic on cognitive function of ...

Fig ure 1

Fig ure 2

Fig ure 3

Fig ure 4

Fig ure 5

i mals- M6 de creased short-term mem ory in males and in -

creased it in fe males (Fig. 6)

M6 does not ex hibit any ef fect on long-term mem ory, ac -

cord ing to the step-through test (on the 24th hour af ter M6

treat ment) (Fig.7).

CON CLU SIONS

The com bined postnatal stress ex po sure (ma ter nal de pri va -

tion fol lowed by so cial iso la tion) af fects in dif fer ent way

the cog ni tive func tion of male and fe male ro dents and

prob a bly has a crit i cal in flu ence on brain de vel op ment and

neu ral plas tic ity.

The pro tec tive ef fect of M6 is also sex-de pend ent:

In male grouped an i mals the mem ory was im proved, but it

de creased in iso lated rats. The op po site was ob served in fe -

males- the pep tide mi metic M6 im proved mem ory in the

iso lated rats, but de creased it in grouped animals.

REF ER ENCES

1. Oomen CA, Soeters H, Audureau N,

Vermunt L, van Hassel t FN, Manders EM,

Joels M, Lucassen PJ, Krugers H. Se vere

early life stress ham pers spa tial learn ing and

neurogenesis, but im proves hippocampal syn ap tic

plas tic ity and emo tional learn ing un der high-stress

con di tions in adult hood. J Neurosci 2010; 30:

6635–6645.

2. Oomen CA, Soeters H, Audureau N,

Vermunt L, van Hassel t FN, Manders

EMM, Joels M, Krugers H, Lucassen PJ.

Early ma ter nal de pri va tion af fects dentate gyrus

struc ture and emo tional learn ing in adult fe males.

Psychopharmacology 2011; 214: 249–260.

3. Petkov, V.V. , S. Stancheva, Acta Physiol.

Pharmacol. Bulg., 1984, 10, 3-8

4. Stancheva S. L. Alova, L. Tancheva, V. V.

Petkov, E. Encheva, D. Tsekova, M.

Novoselski , Learn ing, Mem ory and Biogenic

Amine Lev els in Rat Hip po cam pus af ter Treat ment

with New L-Valine De riv a tives, J. Med.CBR I, vol 8,

Feb. 2010, 293-297, ISBN 978-954-9993-91-2

5. Yashmin J .G. Kar ten, Ana Olar iu, Heather

A. Cameron, Stress in early life in hib its

neurogenesis in adult hood, Trends in Neurosciences,

Vol ume 28, Is sue 4, April 2005, Pages 171-172,

(http://www.sciencedirect.com/sci ence/ar ti -

cle/pii/S0166223605000299)

21

Tancheva L., E. Encheva, M. Novoselski ...

Fig ure 7

Fig ure 6

EFFECTS OF D1 RECEPTOR BLOCKADE ON THE

INTENSITY-RESPONSE FUNCTION OF FROG DARK ADAPTED

ELECTRORETINOGRAM

Popova E.1, P. Kupenova

1, I. Ivanov

2

1Department of Physiology, Medical Faculty, Medical University - Sofia;2Specialized Hospital for Infectious and Parasitic Diseases “Prof. Ivan Kirov” - Sofia

ABSTRACT

The effects of dopamine D1 receptor blockade by SCH 23390 on the V – log I function of the ERG b- and

d-waves were investigated in dark adapted frog eyes. An enhancement of the b- and d-wave amplitude was ob-

tained during the blocker application. The effect was more pronounced on the rod- than cone-dominated re-

sponses. A clear ON-OFF asymmetry of the blocker’s action on the absolute and relative sensitivity of the

responses was demonstrated. The b-wave V – log I function had a steeper slope and narrower dynamic range.

The implicit time of the ERG waves was lengthened especially at lower stimulus intensities.

Key words: dark adaptation, dopamine, electroretinogram, frog, retina

INTRODUCTION

Dopamine is the predominant catecholamine in the verte-

brate retina, which is released by a unique set of

dopaminergic amacrine/interplexiform neurons (for review

16). Its participation in the overall retinal sensitivity control

can be readily evaluated by blocking the dopaminergic

transmission and following up the changes of the

electroretinogram (ERG). The most prominent ERG com-

ponents are the b-wave (in response to stimulus onset) and

the d-wave (in response to stimulus offset). These compo-

nents are usually used for assessment of the retinal ON and

OFF channel activity. Most of the authors reported that de-

pletion of retinal dopamine (with 6-OHDA treatment) or

application of the nonselective dopamine antagonist

haloperidol had no effect on the threshold dark adapted

ERG (6,7,10,17). But there is no agreement about the ef-

fects of the dopaminergic blockade on the supratheshold

dark adapted ERG, obtained with different stimulus inten-

sities, which evoked pure rod, mixed rod-cone or pure cone

mediated responses. Some authors failed to observe any ef-

fect on the b-wave amplitude (6,7,17), while other authors

reported its decrease (1,5,15). Still other authors obtained

an increase of the b-wave amplitude (2,10,13) and a left

shift of its V - log I curve, indicating increased relative sen-

sitivity of the response (13). The discrepancy in the results

cited might be due to species differences including involve-

ment of different kinds of dopamine receptors. It is known

that dopamine acts through five subtypes of dopamine re-

ceptors, which are grouped into two subfamilies: the

D1-like receptors (D1 and D5) and the D2-like receptors (D2,

D3 and D4). A few ERG studies were performed with appli-

cation of selective D1 receptor antagonist SCH 23390 and

the results obtained are contradictory. Some authors re-

ported diminution of the b-wave amplitude (9), while other

authors observed no effect on it (8). Changes of the d-wave

amplitude were not followed up in these studies.

In the present study we investigated the effects of SCH

23390 on the intensity-response function of the ERG b- and

d-waves, obtained in dark adapted frog retina.


The experiments were carried out on 43 eyecup prepara-

tions of frog (Rana ridibunda), continuously superfused

with Ringer solution and supplied with moistened O2. The

dopamine D1 receptors were blocked using 10 mM SCH

23390 (Sigma). Eyecups were stimulated by diffuse white

light stimuli with 5 s duration and interstimulus interval of

25 s, presented in the dark. The test stimulus intensity (It)

was changed in an ascending manner over a range of 11 log

units by means of neutral density filters. The maximal in-

tensity (denoted by 0) was 6 x108 quanta. s-1. ìm-2 at the

plane of retina. These light stimulation conditions allowed

us to obtain rod-dominated responses (using low It), mixed

rod-cone (using middle It) and cone-dominated responses

(using high It). The ERG was recorded by means of non

polarized Ag/AgCl electrodes at bandpass of 0.1 - 1000 Hz

and digitized at 1 kHz. The absolute sensitivity of the ERG

responses was assessed by their thresholds (5 mV criterion

amplitude) and the relative sensitivity – by stimulus inten-

sity (I�) required to produce half-maximum amplitude. The

23



E. Popova, Dept. of Physiology, Medical University,

1 “G. Sofiiski” st., 1431 Sofia


dynamic range of the b-wave was estimated as intensity

span of the responses with 5 - 95% of the maximal ampli-

tude. The dynamic range of the d-wave could not be deter-

mined because of the more complex character of its V - log

I function (for details see 12). The V - log I function of the

ERG waves was obtained twice in one and the same eye-

cup – first in a control period with Ringer solution perfu-

sion (first series) and then in a test period (second series)

during perfusion with SCH 23390 (test experiments) or

Ringer solution (control experiments). For statistical evalu-

ation of the data, Student’s t-test, One- and Two-Way

ANOVA with Bonferroni test (alpha = 0.05) were used.

RESULTS

In a preliminary group of experiments the effects of SCH

23390 were followed for a period of 26 min (using It = -6.0)

in order to evaluate the time course of the effects. SCH

23390 caused marked increase of the b- and d-wave ampli-

tude, which reached a plateau at the 12th minute from the

beginning of the application (Fig. 1 a). The SCH 23390 ef-

fects on the ERG waves were relatively stable until the end

of the perfusion period. The b- and d-wave amplitudes re-

covered to a great degree during reperfusion with Ringer

solution (Fig. 1 b).

In the control experiments the V - log I function of the b-

and d-waves showed no significant differences between the

first and second intensity series in one and the same eyecup

(Fig. 1 c). This allowed us to evaluate the effect of SCH

23390 on the V- log I function using the first series of the

test experiments as a control one.

In the test experiments SCH 23390 caused significant in-

crease of the b- and d-wave amplitude at all stimulus inten-

sities (Two-way ANOVA p<0.000001) except for the low-

est ones for the b-wave ( It = -11.5, -11, -10.5) and the mid-

dle ones for the d-wave (It = -8, -7.5, -7) (Fig. 1 d). The ab-

solute sensitivity of the b-wave was not significantly al-

tered, but that of the d-wave was increased (Table 1). The

stimulating effect of SCH 23390 on the b- and d-wave am-

plitude showed clear intensity dependence. It was greatest

in the lower part of the intensity range, where the responses

were mediated by rods and was less pronounced at higher

intensities, where the responses were mediated by cones

(One-Way ANOVA with Bonferroni test P<0.05; Fig. 2 a).

The V - log I curve of the b-wave had steeper slope and nar-

24

Effects of D1 receptor blockade on the intensity-response function of ...

Figure 1. (a) Time-course of the SCH 23390 effects on

the amplitude of the b- and d-wave, obtained with It =

-6.0. Results of both control experiments (open symbols;

n = 11) and test experiments (filled symbols; n = 10)

are represented. The amplitudes of the ERG waves are

normalized to the values obtained just prior to blocker

application. The time, when the perfusion was switched

to SCH 23390, is indicated by an arrow. The period,

when the V-log I function was tested in the main group

of experiments, is indicated by a rectangular above the

curves. Mean values ± SEM are shown. (b): Original

ERG records, obtained during the perfusion with

Ringer solution in the control period (upper row), SCH

23390 (middle row) and Ringer solution in the recovery

period (lower row). Calibration: time – 200 ms;

amplitude – 100 �V. (c) and (d): Effects of SCH 23390

on the V - log I function of the b- and d-wave. Results of

both control experiments (c; n = 10) and test

experiments (d; n = 12) are represented. The

amplitudes of the ERG waves are normalized to Vmax of

the responses obtained during the first series of the

experiments. Mean values � SEM are shown.

Figure 2. (a): Relative change of the ERG b- and d-wave

amplitude in the control (open symbols) and test

experiments (filled symbols). The amplitudes of the ERG

waves, obtained at each It during the second stimulus

intensity series were normalized to those, obtained

during the first series. Means � SEM are represented.

(b): Changes in the b/d amplitude ratio during the first

(R) and second (SCH) series in the test experiments. (c):

Normalized V - log I curves obtained during the first (R)

and the second (SCH) series of the test experiments. The

values in each series are normalized to the Vmax obtained

in the same series. This manner of representation

demonstrates the changes of the position of the V - log I

function along the intensity axis and its slope under the

influence of SCH 23390. (d): Original ERG records,

obtained with different stimulus intensities during the

control period (grey lines) and during the treatment with

SCH 23390 (black lines). The numbers on the left side

indicate stimulus intensity (lg It).

rowed dynamic range during the D1 receptor blockade (Ta-

ble 1). The curve was shifted to the left along the intensity

axis, indicating increased relative sensitivity of the ON re-

sponse (Fig. 2 c; Table 1). However, the relative sensitivity

of the d-wave remained unchanged. The b/d amplitude ra-

tio was significantly lowered in the rod-dominated part of

the intensity curve (Two-way ANOVA p<0.01), but it was

significantly increased (p<0.03) in the intensity range (It

=-8; It = -7.5; It = -7), where transition from rod to cone

dominated responses occurred. The b/d amplitude ratio re-

mained unchanged in the higher intensity range, where the

responses were mediated by cones (Fig. 2 b). The perfusion

with SCH 23390 did not change the latency of the ERG

waves, but it increased their implicit time especially at

lower stimulus intensities (Fig. 2 d, Table 1).

DISCUSSION

Our results clearly demonstrate that the blockade of dopa-

mine D1 receptors by SCH 23390 enhances the amplitude of

the suprathreshold ERG b- and d-waves, obtained over a

wide range of stimulus intensities in dark adapted frog retina.

This indicates that endogenous dopamine, acting through D1

receptors, has inhibitory action on the mechanisms, responsi-

ble for generation of these ERG waves regardless of the type

of the photoreceptor input. However, the relative strength of

the dopamine action seems to be greater on the rod- than

cone-mediated responses. It is generally assumed that the

ERG b- and d-waves depend mainly on the activity of ON

and OFF bipolar cells, respectively, with minor direct contri-

bution of proximal retinal activity. Our results are in agree-

ment with the results obtained in amphibian retina showing

that dopamine, acting through D1 receptors, increases the ra-

tio of amplitudes of the cone-driven to rod-driven compo-

nents of the bipolar cell responses (4). Inhibitory action of

dopamine on the rod ON bipolar cells, concomitant with a

decrease in the amplitude of the b-wave, has been demon-

strated also in fish retina (14).

Our results show a clear ON-OFF asymmetry of SCH

23390 effects on the absolute and relative sensitivity of the

ERG responses. The absolute sensitivity of the b-wave is

not changed, but that of the d-wave is significantly in-

creased. This result supports the suggestion that endoge-

nous dopamine, acting through D1 receptors, contributes to

the lower absolute sensitivity of the OFF as compared to

the ON response in dark adapted frog ERG (3). On the

other hand, the relative sensitivity of the b-wave is in-

creased, while that of the d-wave is not significantly

changed. This result is consistent with the greater enhance-

ment of the b-wave amplitude at lower stimulus intensities

during the dopaminergic blockade reported by other au-

thors (11, 13). The increased relative sensitivity of the

b-wave in our present study is accompanied by a steeper

slope and narrowed dynamic range of the response. This re-

sult suggests that endogenous dopamine, acting through D1

receptors, is involved in widening of the intensity range,

where the b-wave amplitude is a linear function of log It.

This might be the contribution of this neurotransmitter to

the retinal sensitivity control in frog retina.

It is well established that the rod- and the cone-dominated

suprathreshold electroretinograms differ markedly in re-

spect to their b/d amplitude ratio and implicit time of the re-

sponses. They have much higher values in the rod- than the

cone-dominated ERG. Our present results indicate that do-

pamine action through D1 receptors may contribute to this

phenomenon as regards to b/d amplitude ratio. We demon-

strate that the initial difference between the b/d ratio values

obtained in the rod- and cone-dominated ERG becomes

smaller under the influence of SCH 23390. The opposite is

true for the dopamine effect on the time course of the re-

sponses, where the initial difference between the implicit

times of the rod- and cone-dominated ERG waves is aug-

mented during the D1 receptor blockade.

CONCLUSION

The results of our study clearly show that D1 receptor

blockade has similar enhancing effect on the

suprathreshold b- and d-wave amplitude irrespective of the

type of the photoreceptor input, although the rod-mediated

responses are affected to a greater degree. Endogenous do-

pamine acting through D1 receptors changes in a specific

manner the intensity-response function of both the ERG b-

and d-waves. A clear ON-OFF asymmetry of the dopamine

25

Popova E., P. Kupenova, I. Ivanov

ERGwaveThreshold (lgIt)

Ringer SCH 23390

I � (lgIt)

Ringer SCH 23390

Dynamic range (log units)

Ringer SCH 23390

Implicit time (ms)

Ringer SCH 23390

b-wave

d-wave

-11.10 ± 0.08 -11.02 ± 0.05

-9.99 ± 0.27 -10.57 ± 0.21

p<0.0004

-7.66 � 0.16 -8.28 � 0.1

p<0.000005

-5.61 � 0.07 -5.72 � 0.04

6.86 � 0.28 6.07 � 0.34

p<0.04

It -9.5 777 � 40.16 883 � 35.57

p<0.025

It-4 296 � 13.36 283 � 21.08

It -9.5 850 � 34.08 1020 �58.94

p<0.005

It -4 213 � 16.69 235 � 19.92

p<0.002

Table 1. Effects of SCH 23390 on the threshold, I�, dynamic range and implicit time of the ERG b- and d-wave

action on the absolute and relative sensitivity of the ERG

responses is demonstrated.

ACKNOWLEDGMENTS

This work was supported by grant ¹ 6/2010 from the

Council for Medical Science, Medical University Sofia.

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Dopaminergic control of light-adaptive synaptic plas-

ticity and role in goldfish visual behavior. -Vision

Res., 36, 1996, 4045-57.

26

Effects of D1 receptor blockade on the intensity-response function of ...

DYNAMICS OF HEART RATE RECOVERY AFTER CYCLE

ERGOMETRIC PWC170 TEST IN SOCCER PLAYERS

Pavlova E., G. Uzunova, P. Somlev

Department of Physiology and Biochemistry, National Sports Academy “Vassil Levski”- Sofia

ABSTRACT

Relation of heart rate (HR) recovery dynamics with physical work capacity and maximal oxygen uptake

(VO2max) is important to understand more clearly adaptation to PWC170 test. The present aim is to study

correlations between PWC170, indirect measured VO2max and Dobrev’s recovery coefficients through cycle

ergometer PWC170 test and 5-minute recovery period. METHODS: Fifteen soccer players (age 21.5±1.43

years; weight 74.40±1.43 kg) performed two-load cycle ergometer PWC170 test and they have been examined

in 5-minute recovery period after the test. HR was monitored by Suunto t6c. PWC170, indirect VO2max (by

Karpman et al.), Dobdev’s coefficients, recovery HR sum, and BMI were calculated by equations. RESULTS:

It was found significant correlations between parameters (absolute and relative) of physical work capacity

and maximal oxygen uptake. PWC170 and VO2max depend significantly on BMI. There are not correlations

of HR recovery parameters (coefficients, sum of HR in 5-minute recovery) with working HR, VO2max and

PWC170. CONCLUSIONS: These findings support the suggestion that exercise cardiorespiratory parame-

ters and HR recovery dynamics after PWC170 test have independent diagnostics value.

Key words: HR recovery, PWC170, VO2 max, coefficient of recovery, soccer players

INTRODUCTION

Physical work capacity is the maximum amount of work a

person can perform. Increasing physical work capacity is a

basic element of any athletic training program. It is usually

related to a specific heart rate and is used as a measure of

aerobic fitness. Physical work capacity is primarily deter-

mined by the coordinated functioning of the neuro-muscu-

lar, cardiorespiratory, and energy producing systems. The

most common test of physical work capacity is called the

PWC170, which relates to the maximum amount of work

that can be done at a heart rate of 170 beats per minute.

Since originally designed, PWC170 tests (18,19) measur-

ing the power of physical activity required subjects to com-

plete five to seven stages of exertion lasting six minutes

each till nowadays various modified PWC170 tests have

been worked out and applied in functional laboratories

(1,3,4,14,) Their physiological basis is a linear relationship

between heart rate, oxygen uptake and physical activity for

the range between approximately 105-110 bpm and 170

bpm. Later studies by Karpman and others resulted in de-

velopment of the Sjostrand method that reduced testing

time considerably by utilizing only two sessions of load at

moderate power (6,7,8,11,16,17). PWC170 has been uti-

lized in predicting VO2max as well (3,5,9,11,13). In our

previous studies Margaria’s aerobic step-test has been

adapted for estimating physical work capacity and maximal

oxygen uptake (15). Studies about recovery heart rate after

PWC170 tests in athletes are scanty. The recovery heart

rate concept is mostly for people who are out of shape. In

our previous work we reported some data about dynamics

of HR recovery in athletes subjected to PWC170 tests (7). It

is important to clarify the interrelations between heart rate

(HR) recovery dynamics, physical work capacity and max-

imal oxygen uptake (VO2max). The present aim is to study

correlations between PWC170, indirect measured

VO2max and Dobrev’s recovery coefficients through cycle

ergometer PWC170 test and 5-minute recovery period.

METHODS

Fifteen soccer players (age 21.5±1.43 years; weight

74.40±1.43 kg) performed two-load cycle ergometer

PWC170 test (3) and they have been also examined in

5-minute recovery period after the test. The power output

for the load is set based on the subject’s weight and level of

fitness. Experimental design: The duration of the first load

is five minutes which is sufficient time for the heart to reach

a steady state of work. Then there is a three minute rest pe-

riod after completion of the first load. Then there is a sec-

ond five minute period of exertion, and 5-minute recovery

period after stop exercising. Two loads are with a constant

power output and a constant speed (rpm).The tests were

implemented on Monark 828E cycle ergometer. Heart rate

27



E. Pavlova. Dept. of Physiology and Biochemistry, National

Sports Academy”V.Levski”

Studentski Grad, Sofia 1710, Bulgaria


(HR) monitoring has been done by Suunto t6c. Studied pa-

rameters are absolute and relative physical work capacity

(PWC170, PWC170/kg), absolute and relative maximal

oxygen uptake (VO2max, VO2max/kg), pre-exercise HR,

submaximal HR, Dobdev’s coefficients, recovery HR sum

and BMI. Physical work capacity and indirect measured

VO2max are computed by following equations (3):

1. Absolute PWC170

PWC N N NHR

HR HR170 1 2 1

170 1

2 1� � �

�

�( )

PWC170 [kgm.min-1] - physical working capacity at a

heart rate of 170 beats per minute

N1 [kgm.min-1] - power of 1st step load; N2 [kgm.min-1] -

power of 2nd step load

HR1 [bpm] - steady state heart rate at 1st step load

HR2 [bpm] - steady state heart rate at 2nd step load

2. Relative PWC170/kg

PWC kgPWC

kg170

170/ �

PWC170/kg [kgm.min-1.kg-1] - PWC170 per kg body

mass

3. Absolute VO2max

VO2max = 2.2XPWC170 + 1070

4. Relative VO2max/kg

VO kgVO

kg2

2max/

max�

VO2max/kg [ml.min-1.kg-1] - VO2max per kg body mass

For HR recovery dynamics after PWC170 test are used

Dobdev’s coefficients, calculated by equations (2):

1. HRcoeff.1

HRcoeffHR HR

HR.

.( )�

� �

�100 6 10 60

2. HRcoeff.2

HRcoeffHR HR

HR

HR HRV

HR.

.( ) .( )�

� ��

� �

�100 6 10 60 100 6 10 10

I V��

HR10 – HR for first 10 s in 1st minute of recovery

HR60 –HR for last 10s in 1st minute of recovery

HRV10 – average HR for 10s of 5th minute

�HR – HR for 1st minute

�HRI-V – HR for five minutes of recovery

�HRI-V = HR1 + HR2 + HR3 + HR4 HR5Body mass index (BMI) is computed as follows:

BMIweight kg

height m�

( )

( )2

Statistics analysis has been done through Descriptive Sta-

tistics, Two-Related Samples Tests and Nonparametric

Correlations (Spearman’s rho).

RESULTS

Descriptive statistics (Mean±SD) of variables PWC170,

PWC170/kg, VO2max, VO2max/kg, pre-exercise HR,

submaximal HR, Dobdev’s coefficients, recovery HR sum

and BMI are shown in Table 1.

Submaximal HR reaches the work level before ceasing lin-

ear relationship between HR, physical activity and VO2

and namely 170 bpm. Pre-exercise mean values of HR

have been determined as normocardia. In the end of 5-min-

ute recovery period HR decreases and gets values below

the pre-test ones but the comparison between HR values

shows insignificant difference (p0.05).

The PWC170, PWC170/kg, VO2max, weight and BMI

significant correlations (NPar Spearman’s Rho) have been

considered (Fig.1).

The variable physical work capacity correlates positively

with relative physical work capacity, maximal oxygen up-

take, weight and BMI. PWC170 and VO2max depend sig-

nificantly on BMI. There are insignificant correlations of

28

Dynamics of heart rate recovery after cycle ergometric ...

Variable Mean ±SD

PWC170 [kgm. min-1] 1166 ± 224.97

PWC170 / kg [kgm. min-1. kg-1] 15.65 ± 2.17

VO2 max [ml. min-1] 3493 ± 549.39

VO2 max / kg [ml. min-1. kg-1] 47.04 ± 5.24

HR pre-exercise 61.6 ± 9.26

Submaximal HR 164.8 ± 5.95

K1 0.93 ± 0.11

K2 1.3 ± 0.13

HR Sum 551.4 ± 33.46

BMI 22.82 ± 2.33

Table 1. Descriptive statistics (Mean±SD) of variables

Figure 1. PWC170 correlations with PWC170/kg,

VO2max, weight and BMI

HR recovery parameters (coefficients, sum of HR in 5-min-

ute recovery) with working HR, VO2max and PWC170.

DISCUSSION

The obtained data from the investigation give the reason to

accept that the subjects demonstrate adequate cardiovascu-

lar response to the applied PWC170 test (see Tabl.1). The

workload HR really marks the beginning of the optimal

zone for the cardiorespiratory function in response to physi-

cal activity. According to published data the demands dur-

ing a soccer match may become so high that they lead to fa-

tigue, with impairments of the physical performance poten-

tial and the technical performance even at submaximal ex-

ercise intensities (12). It has been reported, the mean rate of

aerobic energy production for elite players is estimated to

be � 70% VO2max during match-play. Nevertheless, in-

vestigation of aerobic capacity of soccer players is essential

for those athletes to assess basic aspect of their specific

sports abilities. It is well-known the increase in heart rate

that accompanies exercise is due in part to a reduction in

vagal tone. Recovery of the heart rate immediately after ex-

ercise is a function of vagal reactivation. The results enable

us to analyze the relations between workload HR and HR

recovery after PWC170 test. Therefore, an objective quan-

titative assessment of physical work capacity can be an im-

portant tool in measuring the effectiveness of training.

Physical work capacity and maximal oxygen uptake of the

soccer players estimated from the PWC170 test confronted

our previous results and other authors’ research could de-

fine their level of physical and aerobic capacities.

Comparison of PWC170 mean values of examined soccer

players with the published results of physical work capacity

by Karpman (3) and our previous data (7,17) illustrated in

Figure 2 indicate lower level of PWC170 for our contin-

gent than the level of the cited data. One possible cause for

such physical capacity is that the investigated subjects are

students-athletes and they do not train so regularly like the

soccer players from elite teams. The measured indirect

VO2 max from PWC170 test shows lower aerobic capacity

in students toward the maximal oxygen uptake in soccer

players (3,10) using the same procedure (Fig. 3).

Relation of heart rate (HR) recovery dynamics with physi-

cal work capacity and maximal oxygen uptake (VO2max)

is important to understand more clearly adaptation to

PWC170 test. The relationship between PWC170 and re-

covery parameters (k1, k2) are similar to obtained correla-

tions between PWC170 and Dobrev’s coefficients in ath-

letes of different kind of sports in other research (7). Ac-

cording to the authors of this study the cases of high

PWC170 and greater value of coefficients are associated

with better cardiac adaptation to exercise.

CONCLUSION

This study, investigating physical work capacity, aerobic

capacity and HR recovery parameters through PWC170

test and reevaluation older data in light of current knowl-

edge, indicates that such approach assesses more complex

adaptive reactions. Therefore, this objective quantitative

assessment of physical work capacity can be an important

tool in measuring the effectiveness of training. Moreover,

these findings support the suggestion that exercise

cardiorespiratory parameters and HR recovery dynamics

after PWC170 test have independent diagnostics value.

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Pavlova E., G. Uzunova, P. Somlev

Figure 3 Comparison of VO2max values of examined

soccer players with published data

Figure 2 Comparison of PWC170 values of examined

soccer players with published data

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30

Dynamics of heart rate recovery after cycle ergometric ...

URIC ACID, THIOLS AND BURN-INDUCED OXIDATIVE INJURY

OF GASTRIC MUCOSA IN RATS

Bekyarova G., M. Hristova

Department of Physiology and Pathophysiology, Medical University -Varna, Bulgaria

ABSTRACT

Thermal skin injury causes oxidative stress and damage of organs distant from original burns wound. The

mechanisms of burn-induced oxidative damage of gastric mucosa remain unclear. This study was designed to

investigate the gastric mucosal injury after burns in rats using gastric mucosal concentration of

malondialdehyde (MDA), uric acid and thiol groups as biomarkers of the oxidative status. In order to accom-

plish 30 % third degree burn hot boiling water (90oC) as applied on the back of the animals during a period of

ten seconds under thiopental anesthesia. In the present study we found that the concentrations of MDA and

uric acid in gastric mucosa were significantly increased after burns. The decrease of mucosal thiols impairs

the redox balance and stimulates the oxidative injury of gastric mucosa induced by thermal trauma. In con-

clusion, the thermal trauma causes oxidative injury of gastric mucosa evidenced by significant increased con-

centrations of MDA and uric acid as well as a significant reduction of the levels of thiols and uric acid

occupying the front line of the antioxidant defense. Based on our findings we could assume that the introduc-

tion of antioxidants that maintain the concentration of thiol groups in gastric mucosa represents a promising

therapeutic approach for restoring the redox balance in gastric mucosa and protecting it from burns-induced

oxidative injury.

Key words: uric acid, thiol groups, malondialdehyde, gastric mucosa, burns

INTRODUCTION

Ischemia due to transitory and selective vasospasm in the

splanchnic area after major burns causes oxidative damage

in organs of gastro-intestinal system followed by

reperfusion injury (12,22). Increased evidence has been

presented connecting with hepatic oxidative damage after

thermal trauma (13,14). Data about oxidative damage of

gastric mucosa are little. The mechanisms of burn-induced

oxidative mucosal injury are not completely clarified yet.

The changes of the activity of pro-oxidantive enzymes such

as xanthine oxidase (XO) and myeloperoxidase and

antioxidantive enzyme such as superoxide dismutase in

burns were studied (13). The changes of the other end prod-

uct of XO such as uric acid, remain insufficiently investi-

gated yet, too. Certain authors mention uric acid as 'oxida-

tive stress marker' for tissue damage (19). According to

other authors, it has an antioxidant action in the plasma (7).

Being a main non-protein thiol component, glutathione

plays an important role of a cellular antioxidant and

protector of the mucosa from oxidative injury (9).

Experimental and clinical data demonstrate dual role of the

uric acid as a marker of the oxidative damage in tissues as

well as plasma antioxidant (7,19). Both uric acid and pro-

tein thiols accomplish to a great extent the antioxidant

power of the plasma. However, the results about the

changes of the plasma level of uric acid after burns are very

contradictory, indeed (10,21).

This study was designed to investigate the burn-induced gastric

mucosal injury using malondialdehyde (MDA), uric acid and

thiols in gastric mucosa as biomarkers of the oxidative status.


Experimental procedures

The experimental procedures was approved by the Home

Office for Care and Use of Laboratory Animals and per-

formed with a strong consideration for ethics of animal ex-

perimentation according to the International Guiding Prin-

ciples for Animal Research approved in Bulgaria.

Same-age male rats weighing between 220 and 250g fasted

for 12 h were allowed free access to water before injury

were used. Animals were housed in a 20oC room and of-

fered rat chow and water ad libitum. They were kept in the

individual wire-bottomed cages and fed standard rat chow.

After light ether inhalation, general anesthesia was per-

formed using thiopental 30 mg/kg intraperitoneally (i.p.). In

order to accomplish 30 % third degree burn hot boiling wa-

ter (90oC) as applied on the back of the animals during a pe-

riod of ten seconds. For those rats which were subjected to

31



G. Bekyarova, Ph.D, MD Department of Pathophysiology

Medical University of Varna, 55 Marin Drinov st.

Varna 9002, Bulgaria

E-mail: [email protected]

burn injury 4 ml physiological saline was applied i.p. for

immediate resuscitation following burn injury. No animals

died within the first 24 h post-burn period. Twenty nine

male rats were randomly assigned to two groups: 1.con-

trols-non burned rats (C) (n =10); 2.burned rats (B) (n =

19). All animal received buprenorphine 0,30 mg /kg. body

weight, i.p., twice daily for pain control post-burn. Animals

were reanesthetized with thiopental and scarified 24 h after

burns and stomachs were sampled.

Biochemical analysis

Gastric mucosa was gently separated from the underlying

tissue homogenized in 1:5 w/v 50 mM phosphate buffer (pH

7,4) containing 0,1mM EDTA, at 4000rpm for 10 min. The

homogenate was centrufugated at 800 x g rpm/15 min to dis-

card the sediment and supernatant was frozen until analysis.

All manipulations were performed at 4-8oC. Analysis was

performed immediately after thawing of the samples.

Membrane lipid peroxidation was assayed by MDA mea-

sured by its thiobarbituric acid (TBA) reactivity in gastric

mucosa hemogenates using the method of Porter et al (15).

Results were expressed as nmol MDA/g tissue were deter-

mined using the extinction coefficient of MDA-TBA com-

plex at 532 nm =1, 56 x 10-5 cm-1 M-1 solution.

Uric acid in gastric mucosal homogenate was determined by

the method of Bergmeyer (6), based on the ability of urate to

reduce phosphowolframic acid in alkaline solution to a blue

colored product. Standart solutions of uric acid were used to

calculate the extent of gastric mucosal homogenates.

Gastric mucosal thiol (SH) groups were determined by the

method of Hu (11), based on the absorbtion of the color

complex between SH groups and DTNB at 412 nm. Stan-

dard solutions of reduced glutathione were used to calcu-

late the concentration of SH groups.

Statistical analysis

Data were analyzed statistically by one-way analysis of

variance (ANOVA) and expressed as mean + SEM. A

value of p < 0,05 was considered statistically significant.

The statistical procedure was performed with

GraphPadInStat software.

RESULTS

Thermal skin injury causes significant elevation of gastric

mucosal MDA levels by 42 % (p<0,05) (Fig1.), mucosal uric

acid ones by 29% (p<0,05) (Fig.2) while mucosal SH level

was decreased by 26% (p<0,05) Fig.3 in burned rats compared

with those of the controls.

DISCUSSION

The results demonstrate that mucosal MDA level as a

marker of the oxidative damage related by most authors to

the intensive production of free radicals predominantly in

ischemia /reperfusion by activated leukocytes in the acute

phase post-burn (12). MDA accumulation is accompanied

by increasing of uric acid levels in gastric mucosa which al-

lows us to assume that the activation of the lipid

peroxidation is, probably, due either to the XO activation,

or to the accumulation of the substrate hypoxanthine, or

even to both of them. It is known that the uric acid is a natu-

ral product of purine metabolism that influences upon XO

activity. Some authors establish XO activation already dur-

ing the initial minutes after the thermal injury (12) as a re-

sult from ischemia /reperfusion particularly well-mani-

fested in the gastric mucosa after the thermal skin injury,

well as an increased hypoxanthine level (destruction of

32

Uric acid, thiols and burn-induced oxidative injury ...

Fig.1. Gastric mucosal MDA levels at 24 th hour

post-burn in rats. *P<0,05 vs controls; C- controls (non

burned rats); B - burned rats. Results are given as

mean + SEM.

Fig.2. Gastric mucosal uric acid at 24 th hour

post-burn in rats. *P<0,05 vs controls. C- controls (non


mean + SEM.

Fig.3. Gastric mucosal thiol groups at 24 th hour

post-burn in rats. *P<0,05 vs controls; C- controls (non


mean + SEM.

ATP down to AMP) that is a substrate for XO (20).

Hypoxantine conversion to xanthine and finally to uric acid

produces hydrogen peroxide and superoxide, deleterious

oxygen derived free radicals. Several studies suggested that

the burst of superoxide radicals and hydrogen peroxide

overwhelm the scavenging capacity of protective endoge-

nous enzymes (12,13). Superoxide radicals formed during

these reactions produce even more toxic radicals such as

hydroxyl radicals (OH.), and peroxynitrite (ONOO.) by in-

teracting with other radicals such as NO thus activating the

lipid peroxidation and inflammatory response.

Peroxynitrite production increases during the acute period

after burns (16). This suggests that activation of xanthine

oxidase (XO) pathway under conditions of ischemia/

reperfusion in gastric mucosa might be responsible for

superoxide radical production and more toxic radicals such

as OH. ànd ONOO. during the early phase after thermal

skin injury. Lipid peroxidation mediated by these reactive

oxygen- and nitrogen-based species is believed to be the

important factor for blood cells and tissue oxidative

damage after thermal trauma, including gastric mucosa.

Antioxidants block lipid peroxidation and protect gastric

mucosa from oxidative injury in burns (1,3,5,8,23).

As a basic non-protein thiol component, glutathione plays

an essential role of a cellular antioxidant and protector of

the mucosa from oxidative damages (9). It is known that

during tissue hypoxia and burns an exhaustion of the

intracellular glutathione sets in and this is, most probably,

due to its increased consumption during the oxidative stress

(2,17). Our data indicating a diminished level of mucosal

thiols and elevated mucosal MDA are in conformity with

reduced glutathione (GSH) depletion reported by other au-

thors finding that lipid peroxidation is activated after burns

(18). It may be supposed that the diminishion of thiol levels

impairs the redox balance and stimulates the oxidative

injury of gastric mucosa induced by thermal trauma.

In conclusion, the increased levels of MDA and urates

along with the decreased level of thiols could serve as a

marker of oxidative injury in gastric mucosa after thermal

injury. The thermal trauma causes a significant reduction of

the thiol levels occupying the front line of the antioxidant

defense. Based on our findings we could assume that the

introduction of antioxidants that maintain the concentration

of thiol groups in the gastric mucosa represents a promising

therapeutic approach for restoring the redox balance in gas-

tric mucosa and protecting it from oxidative injury induced

by thermal trauma.

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1. Bekyarova G, Tantcheva T, Yankova T. Ef-

fect of alpha-tocopherol on lipid peroxidation,

hemolysis and serum ceruloplasmin in the early post

burn period. Anal Lab1994; 4: 275-277.

2. Bekyarova G,Yankova T. Alpha-tocopherol and

glutathione deficiency and decreased deformability

after burn skin injury. Acta physiol pharmacol bulg,

23, 1998, 55-59.

3. Bekyarova G, Yankova T, Galunska B.Increased anti-

oxidant capacity, suppression of free radical damage

and erythrocyte aggregability after combined applica-

tion of alpha-tocopherol and FC-43 perfluorocarbon

emulsion in early postburn period in rats. Artif Cells,

Blood Subst Immobil Biotech 1996; 24:629-642.

4. Bekyarova G,Yankova T, KozarevI ,Yankov

D. Reduced erythrocyte deformability related to acti-

vated lipid peroxidation during the early postburn pe-

riod. Burns 1996; 22:291-294.

5. Bekyarova, G. ,Yankova T, Marinov M.

Lipofuscin product accumulation, insufficient antioxi-

dant defencein erythrocytes and plasma and enhanced

susceptibility to oxidative haemolysis after thermal

trauma. Acta Chir Plast 1997; 39: 61-64.

6. Bergmeyer HU. Methods of enzymatic analysis.

Breatfield Beach, FL:Verlag Chemie Int.,1981

7. Glantzounis GK, Tsimoyiannis EC, Kappas

AM, Galar is DA..Uric acid and oxidative stress.

Curr Pharm Des 2005;11:4145-4151.

8. Gurbuz V, Corak A, Yegen BC, Kurtel H, Alican I.

Oxidative îrgan damage in a rat model of thermal in-

jury: the effect of cyclosporin A. Burns 1997;

23:37-42.

9. Hayes JD, Mc Lellan LI. Glutathione and

glutathione dependent enzymes represent a

coordinately regulated deffense against oxidative

stress. Free Radic Res 1999;31: 273-280.

10. Haycock JW, Ralston DR, Morris B,

Freedlander E, MacNeil S. Oxidative damage

to protein and alterations to antioxidant levels in hu-

man cutaneous thermal injury. Burns1997;23:

533-540

11. Hu M. Measurment of protein thiol groups and

glutathione in plasma. Methods Enzymol 1994;

53:380-385

12. Latha B, Babu M. The involvement of free radi-

cals in burn injury: a review. Burns 2001; 27:

309-317

13. Naziroglu M., I . Kokcam, S. Yilmaz. Benefi-

cial effects of intraperitoneally administered al-

pha-tocopheryl acetate on the levels of lipid peroxide

and activity of glutathione peroxidase and superoxide

dismutase in skin, blood and liver of thermally injured

guinea pigs. Skin Pharmacol Appl Skin Physiol.

2003;16 : 36-45.

14. Pintaudi AM, Tesoriete LD, Arpa N,

D'Amelio L, D'Arpa D, Bongiorno A, et al.

Oxidative stress moderate to extensive burning in hu-

mans. Free Radic Res 2000; 33: 139-136.

15. Porter NA, Nixon J, Isaac J. Cyclic peroxidase

and thiobarbituric assay. Biochem Biophys Acta 1976;

441: 596-599.

16. Rawlingson A, Greenacre SA, Brain SD.

Generation of peroxynitrite in localised, moderate

temperature burns.Burns 2000; 26: 223-227.

17. Sakarsan A, Serer l i O, Vel ioglu-Ovunc A,

Erkan F, Erkanl G. Ginko Biloba extract im-

proves Oxidative organ damage in rat model of ther-

mal trauma. J Burn Care Rehabil 2005; 26: 515-524.

18. Sener G, Sehir l i O, Vel ioglu Ogune A,

Erean F, Erkainl i G, Gedik N, Yagen BC.

Propylthiouracil (PTU)-induced hypothyroidism alle-

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Bekyarova G., M. Hristova

viates burn-induced multiple organ injury. Burns

2006;32: 728-736.

19. Siems WG, van-Kuijk FJ, Maass R, Brenke

R. Uric acid and glutatione levels during short term

whole body exposure. Free Radic Biol Med 1994;16:

299-305.

20. Woll iscrof t JO, Prasad JK, Thomson P, Til l

GO, Fox IH. Metabolic alterations in burn patients

and detection of adenosine triphosphate degradation

products and lipid peroxies. Burns 1990; 16: 92-96.

21. Yamazaki T, Ueda, T. Secondary hyperuricemia

in burn and trauma. Nippon Rinsho 2003; 61:

313-317.

22. Yoshida M, Wakabayashi G, Ishikawa H,

Kitahora T, Otani Y, Shimazu M, Miura S,

Ishi i H, Kitaj ima M. Rebamipide attenuates gas-

tric microcirculatory disturbances in the early period

after thermal injury in rats. Dig Dis Sci 1998;

43:148S-153S.

23. Yoshikawa Ò, Naito Y, Ueda S , Oyamada

H, Takemura T, Yashida N, Sugino S,

Kondo M. Role of oxygen derived free radicals in

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Clin Gastroenterol 1990;12:65-71.

34

Uric acid, thiols and burn-induced oxidative injury ...

CYTOPROTECTIVE EFFECT OF MELATONIN AND ANTIOXIDANT

DEFENSE AFTER THERMAL SKIN INJURY

Bekyarova G.1, M. Hristova

1, M. Tsaneva

2

1 Department of Physiology and Pathophysiology, Medical University -Varna, Bulgaria;2Department of General and Clinical Pathology, Medical University-Varna, Bulgaria

ABSTRACT

Melatonin protects against hepatic injury and this effect has mainly attributed to the antioxidant properties of

the indoleamine. The aim of this study is to investigate the expression of antioxidant enzyme CuZn superoxide

dismutase (CuZnSOD), total thiol levels in liver and their relationship with melatonin-mediated hepatic pro-

tection after burns. Under anesthesia, 30% total body surface area (TBSA), third degree burn was applied on

the back of animals. Melatonin (N-acetyl-5-methoxytriptamine, Merck, Germany 10 mg/kg(-1)) was i.p. ad-

ministered immediately and 12 hours after thermal skin injury. The hepatic CuZnSOD expression was exam-

ined by using light immunohistochemistry. The hepatic malondialdehyde (MDA) and total thiols, as oxidative

markers, were investigated too. Hepatic MDA levels were increased while total thiol in liver did not changed

early post burns. An increased expression of hepatic CuZnSOD in burned group was found. Melatonin re-

stores the expression of increased CuZnSOD activity to the control levels, resulting in normal protection

against burn-induced oxidative damage. In conclusion, thermal skin injury causes lipid peroxidation activa-

tion and compensatory increase in CuZnSOD expression in liver. We suggest that melatonin as a free radical

scavenger improves oxidant /antioxidant balance and restricts burn-mediated injury in the liver.

Key words: superoxide dismutase, light immunohistochemistry, malondialdehyde, liver, burns, melatonin

INTRODUCTION

Thermal skin trauma leads to free radical overproduction

following ischemia reperfusion resulting in elevated risk of

damage to organs distant from the original burn wound, in-

cluding liver (1). Although the mechanisms of burn-in-

duced injury remain unclear increasing evidences indicate

depletion of glutathione peroxidase (GSH-Px), glutathione

and other antioxidants under condition of activated free

radical processes as factor for hepatic injury (15,16,18).

Data about hepatic superoxide dismutase (CuZnSOD) ac-

tivity early post burn are converse. Some authors reported

that hepatic CuZnSOD activity is increased while others

find a decreased activity of this enzymes early post burn (1,

19). Administration of antioxidants (18) and exogenous

SOD (10, 21) restricts burn-induced oxidative damage in

liver and other organs.

Melatonin, a basic secretory product of the pineal gland, is

a powerful scavenger and antioxidant. It scavenges reactive

oxygen species (ROS) and reactive nitrogen species (RNS)

(12). Melatonin has been shown to stimulate CuZnSOD

and GSH-Px expression (14). Melatonin has been found to

attenuate burn-induced oxidative damage evidenced by

lipid peroxidation and antioxidant enzymes such as

glutathione peroxidase (GSH-Px), glutathione in liver and

intestine (3). It also exerts a therapeutic effect on liver dam-

age induced by ischemia/reperfusion (9). There are not data

about melatonin’s effect on hepatic CuZnSOD expression

early post burn.

The aim of this study is to investigate the expression of

hepatic CuZnSOD using light histochemistry, total thiols in

liver and their relationship with melatonin-mediated

hepatic protection after burns.


Experimental burns and melatonin treatment

The experimental procedures were approved by the Home

Office for Care and Use of Laboratory Animals and per-

formed with a strong consideration for ethics of animal ex-

perimentation according to the International Guiding Prin-

ciples for Animal Research approved in Bulgaria.

Same-age male rats weighing between 220 and 250 g were

fasted for 12 hours, but were allowed free access to water

before injury. Animals were housed in a 20oC room and of-

fered rat chow and water ad libitum. They were kept on a

12-h dark/12-h light cycle, with lights turned off at 8:00

p.m. and turned on at 8:00 a.m. to achieve a satisfactory

photoperiod. After light ether inhalation, general anesthe-

sia was performed using thiopental 30 mg/kg

35



G.Bekyarova, Dept. of Pathophysiology

Medical University of Varna, 55 Marin Drinov st

Varna 9002, Bulgaria


intraperitoneally (i.p.).In order to accomplish 30% third-de-

gree burns, hot boiling water (90oC) was applied to the

back of the animals for a period of 10 s. For those rats

which were subjected to burn injury, 4 mL physiological

saline was applied i.p. for immediate resuscitation follow-

ing burn injury. No animals died within the first 24-hour

post burn period. Twenty-four male rats were randomly as-

signed to three groups of 8 animals each: 1. non-burned,

non- treated (N-B) (control), 2.burned, non- treated group

(B), 3.burned group, treated with melatonin (B+M).

Melatonin (Merck, Germany) at a dose of 10 mL/kg solved

in vehicle (2% ethyl alcohol diluted in physiological saline

in doses up to 5 mL/kg) was given i.p. to the treated animals

in the morning between 8:00 a.m. and 9:00 a.m. immedi-

ately after burns. Solvent vehicle was administered i.p. in

the burned and control rats (sham groups) immediately af-

ter burns in the morning. All animals received

buprenorphine in a dose of 0.3 mg/kg i.p. twice daily for

pain control post burn. The animals were re anesthetized

with thiopental and sacrificed 24 hours after burns, and

livers were sampled.

Each liver was gently separated from the underlying tissue

homogenized in 1:5 w/v 50mM phosphate buffer (pH =

7.4) containing 0.1 mM ethylene-diaminetetraacetic acid at

4,000 rpm for 10 min. The homogenate was centrifuged at

800 x g every 15 min to discard the sediment, and

supernatant was frozen until analysis. All manipulations

were performed at 4-8oC. Analysis was performed imme-

diately after thawing of the samples.

Biochemical analysis

Membrane lipid peroxidation was assayed by MDA mea-

sured by its thiobarbituric acid (TBA) reactivity in hepatic

homogenates using the method of Porter et al.(13). Results

were expressed as nmol MDA/g tissue and were deter-

mined using the extinction coefficient of MDA–TBA com-

plex at 532 nm = 1.56 x 10–5 cm–1 M–1 solution.

Hepatic total thiol level were determined by the method of

Hu (7), based on the absorbtion of the color complex be-

tween SH groups and DTNB at 412 nm. Standard solutions

of reduced glutathione were used to calculate the concen-

tration of SH groups.

Immunohistochemistry

Rat liver specimens were fixed in 10% neutral buffered for-

malin and embedded in paraffin. The deparaffinized and

rehydrated sections (5 ìm thick) were treated in 1% hydro-

gen peroxide for peroxidase activity inhibition for 5 min.

Then they were rinsed in 0.1 M phosphate buffered saline

(PBS) pH 7.4 and treated in normal goat serum for 20 min.

Subsequently the sections were incubated with polyclonal

primary antibody for 24 hrs at room temperature. The used

antibody was rabbit anti-SOD (DAKO, USA). After rins-

ing with PBS the sections were incubated for 20 min in goat

anti-rabbit immunoglobulins at room temperature. Then

they were rinsed in PBS again and treated with rabbit

peroxidase-anti-peroxidase complex for 20 min at room

temperature, after which, rinsed in PBS. Finally, peroxidase

activity was developed by the diaminobenzidine-tetrachlo-

ride H2O2- method.

Negative controls. Controls were incubated with non im-

mune sera instead of primary antibody.

Statistics

The liver enzyme data were log transformed to satisfy the

assumptions required to perform parametric tests and are

therefore presented as geometric mean and 95% confi-

dence intervals of the mean. Orthogonal contrasts in

ANOVA were then used to analyze statistically the differ-

ence between any two specified groups

RESULTS

The levels of hepatic MDA were found to be higher by

48% (p<0,05) in the burned group than that of the control

group (fig. 1). Treatment with melatonin signi?cantly in-

36

Cytoprotective effect of melatonin and antioxidant defense ...

Fig. 2 – Effect of melatonin on total thiol levels in liver

after burns. Results are given as mean + SEM. *p <

0.05 vs. control rats; p < 0.05 vs. burned, non-treated

rats. C, Controls; B, burned rats; B + M, burned rats,

treated with melatonin.

Fig. 1 Effect of melatonin on liver MDA levels after

burns. Results are given as mean + SEM. *p < 0.05 vs.

control rats; p < 0.05 vs. burned, non-treated rats. C,

Controls; B, burned rats; B + M, burned rats, treated

with melatonin.

hibited the elevation in hepatic MDA level, maintaining the

level close to the control values. The total thiol levels did

not changed in liver after burns. In the melatonin treated

rats, the thiol level was similar that of the controls (fig. 2)

CuZnSOD expression was found in sinusoidal cells and

sometimes in hepatocytes. The staining intensity of the

SOD-positive cells was weak or moderate in the control

group (Fig. 3A). The expression in hepatocytes and sinu-

soidal cells of SOD was increased in the burned group (Fig.

3B). It was moderate to strong in the individually cells and

their mean content was significantly higher than this of the

control rats. The content of CuZnSOD varied from nega-

tive to weak or moderate in the burned group treated with

melatonin (Fig. 3C). The mean cell content was near to the

control one. (Fig. 3C).

DISCUSSION

Our data demonstrated that levels MDA (as (marker of oxi-

dative stress) increased, whereas the expression of

CuZnSOD increased in liver after burns, possibly to reduce

oxidative stress. The biological effect of free radicals is

controlled by wide range of antioxidant and antioxidant en-

zymes. CuZnSOD reacts with superoxide radicals and is on

the first line of antioxidant defense. We speculate that the

increased expression of CuZnSOD in liver may be acute

compensatory reaction and defense against overproduction

of superoxide radicals. Systemic oxidative stress was de-

fined as persistent imbalance between the formation of

highly reactive molecular species and organism’s antioxi-

dant defenses (11). The activation of oxidative stress path-

way in liver is more likely due to increased production of

free radical by burned skin, activated leukocytes activated

and xanthine/xanthine oxidase system (2).

Accumulating associative data suggest these reactive species

may act directly through oxidative damage on

macromolecules or indirectly activating single transduction

pathway sensible to stress mechanisms namely: nuclear fac-

tor kappa B (NF-kB), protein kinase B or Akt (PKB/Akt) (6).

The activation of these stress sensitive signal pathway is be-

lieved to be responsible for activation of antioxidant en-

zymes including CuZnSOD as adaptation response.

Our data about the increased hepatic CuZnSOD expression in

liver are in accordance with previous studies for increased ac-

tivity of this enzyme as compensatory reaction early post burn

when oxidative stress is increased (16,19). Agay et al (2005)

suggested that increased hepatic CuZnSOD activity is associ-

ated with mobilization of Cu and Zn in hepatic cells early post

burn. Among the major enzymes containing Cu and Zn, SOD

play a key role to counteract the oxidative stress-induced by

thermal injury. The increased CuZnSOD activity is known to

limit the diffusion of oxygen free radicals released by injured

tissues and therefore to avoid the activation of oxidative stress

(11). The expression of hepatic SOD increased while the total

thiol levels remained unchanged on the 24th hours. Previous

study showed an increased protein (SH) oxidation and oxida-

tive stress in the animals with Zn depletion diet (4). Others au-

thors did not find also any changes in total thiols in liver on the

first day (16). There are increased thiol oxidation and oxida-

tive damage of liver in the next days. The increased

CuZnSOD activity and reduced GSH-Px activity in liver to-

gether with revealed unbalanced hepatic antioxidant defenses

capacity contributing to liver oxidative injury (1).

We demonstrated that melatonin restores the expression of

increased CuZnSOD activity to the control levels, resulting

in normal protection against burn-induced oxidative dam-

age. Thus, it is likely that the decreased of hepatic MDA

levels in melatonin-treated rats results from an antioxidant

effect and inhibitory effect of this peptide on oxygen free

radical formation. Melatonin eliminates oxygen free radi-

cals and attenuates lipid peroxidation measured by serum

aspartate amino transaminase (AST) and alanine amino

transaminase (ALT) and MDA, which levels are decreased

and, in this way, restricts burn-induced oxidative hepatic

injury (3). In additional melatonin enters cell membranes, it

mainly localizes in the superficial position in lipid bi layer

near the polar heads of membrane while in this position it is

obviously capable of functioning as a free radical scaven-

ger and may also provide an indirect means by which the

membranes resist oxidative damage. Other possible expla-

nation is that melatonin stimulates production of

glutathione (20). Unlike other antioxidants melatonin don’t

consume cellular glutathione in redox cycling after scav-

enging free radicals (17). Time course studies are needed to

37

Bekyarova G., M. Hristova, M. Tsaneva

Fig. 3 (A) SOD expression in liver of non-burned (control) group (peroxidase-antiperoxidase complex; original

magnification x 100). (B) SOD expression in liver and sinusoidal cells in the burned group (peroxidase-antiperoxidase

complex; original magnification x 100). (C) SOD immunoreactivity in the liver of melatonin-treated burned group

determine the effect of melatonin on antioxidant activities

in this experimental model. The antioxidant effect of

melatonin was shown in different organs such as liver, in-

testine, lung after burns (5). In the previous studies and the

present study, burn-induced tissue damage was shown to

be reduced with melatonin administration (2,3).

In conclusion, thermal skin injury causes activation of

hepatic lipid peroxidation and compensatory increase in

CuZnSOD expression in liver. We suggest that melatonin

as free radical scavenger improves oxidant /antioxidant bal-

ance and restricts burn-mediated injury in liver.

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4. Claeyssen R, Andriol lo-Sanchez M, Arnaud

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burned rats fed a low zinc diet. Biol Trace Elem

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5. De Fil ippis , D. , Iuvone, T. , Esposi to , G. et

al . Melatonin reverses lipopolysaccharide-induced

gastro-intestinal motility disturbances through the in-

hibition of oxidative stress. J Pineal Res 2008, 44(1):

45-51.

6. Horton JW. Free radicals and lipid peroxidation

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7. Hu M. Measurment of protein thiol groups and

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8. Hula NM, Chumak AA, Berdyshev AH,

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9. Kesik V, Guven A, Vurucu S, Tunc T,

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13. Porter , N.A. , Nixon, J .R. Isaac, R. Cyclic

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38

Cytoprotective effect of melatonin and antioxidant defense ...

UNDERSTANDING PATHOGENESIS OF RISKY BEHAVIOR:

A HOLISTIC APPROACH

Sarov G.

Department of Pathophysiology, Trakia University, Medical Faculty, Stara Zagora

ABSTRACT

Epidemiological investigations of risk factors associates some particular form of risk behavior with the high

probability of disease appearance. However, the pathogenic nature of such behaviors remains to be under-

stood. Using a holistic approach I offer a theoretical model that defines risk factors as pathogenic

constelations that initially change body only functionally. However long-lasting disfunctions cause

microstructural damages that summarizes over the time and then disease becomes detectable by medical ex-

aminations. From holistic point of view risky constelations change body identity from healthy into pathogenic

one and this deformed body identity is a real cause for chronic diseases.

INTRODUCTION

Despite the huge number of empirical data on the patho-

genic role of risk factors, the mechanism of action remains

unclear. The origin of the term risk factor is statistical in na-

ture and its pathogenic logic is still not well understood.

This is so may be because insufficient development of ho-

listic understanding about living and disease. Holism is in-

sufficiently defined althought increasing understanding of

the need for a synthetic approach in many areas of medicine

(1-7).Here we offer a holistic explanation of the causation

of diseases and risk factors in particular.

METHOD

One can confirme the truth in three ways: by confirming the

description through observation, by successful prediction

of the future, proven by the practice, and by logical consis-

tency of the theory. There is a huge number of observations

and experiments, confirming the role of risk factors, but still

no consistent theory. Here we use logical reasoning as a re-

search method. The main proposition in our logical re-

search is that every whole has three dimensions: substance,

function and coordination. Starting with this basic holistic

proposition, we will look for a new meaning to physiology,

pathophysiology and causality in diseases.

Physiological extension of the main

propositions

In the field of physiology the three dimensions of the hole

are respectively structures, functions and bioidentity.

Structures. The substance exists in the forms of substrate,

energy (ATP) and structures. Substrate is converted into

ATP and structures, and structures use ATP to function.

Normally consumption of ATP can not exceed the rate of

its synthesis and ATP deficiency inhibits functioning to

prevent cells desintegration. Damage to the structure also

makes functioning impossible.

Function: Life as a function has three dimensions: con-

sumption, production and regulation.

Consumption provides substrates, thus determines both

ATP production and synthesis of the structures. Insufficient

consumption prevents restoration of cellular structures and

down-regulates cellular functioning.

Production. Every cellular population impact some

homeostatic variable (HV). The maximum amount of HV,

produced by the cell population per unit time is its func-

tional capacity. It depends on the number of the cells in the

population and their capacity. The real HV quantity pro-

duced by the cell population at any moment is its functional

volume. The difference between the functional capacity and

the functional volume is functional reserve - additional

quantity of HV, produced by cellular population in

responce of extra stimulation.

Regulation defines the intensity of the cell function and

could be feedback, anticipatory and residual.

Feedback regulation is spontaneous and self-regulated:

HV level activated the sensitive structures that operate

function of the HV producing cells. HV consumption down

regulates HV level and HV production up-regulates it.

Anticipatory regulation determines the HV level above the

physiological values facing increasing needs in the future.

Residual regulation starts with a release of waste products (re-

sidual homeostatic variables - RHVs) and triggers feedback

control by activating cellular populations that decrease RHVs

to minimal levels, otherwise RHVs damage cell structures.

Bioidentity: Bioidentity is the organization of the body

functions in specific situations. The body periodically

changes its bioidentity, but always retains functionally and

structurally integrated. Besides the condition of structures

and functions, bioidentity is determined by the situation to

which the individual must adapt. Relationship between or-

ganism and environment is defined in two ways - through

internal and external locus of control.

39


The logic of external locus of control is: situation> evalua-

tion> preparation> action> homeostasis. If the individual

evaluates the situation as important, anticipatory regulation

and decision making are activated. Only after decision taking

individual can start actions, thus duration of decision making

determines the duration of the anticipatory control. If deci-

sion making prolongs, anticipatory control increase progres-

sively HVs and activates simultaneous feed-back regulation.

The logic of the internal locus of control is: homeostasis>

evaluation > preparation> action> homeostasis. It starts

with homeostatic problem that can need behaviour. The

need trigger anticipatory control and decision making. The

anticipatory control increases HVs in proportion with the

duration and intensity of decision making. After taking de-

cision, the actions decrease elevated HVs and increase

RHVs. These homeostatic changes activate feedback

regulations that restores homeostasis.

Holistic explanation of pathophysiology

The pathophysiologic transformation can start with disor-

ders in structures, functions and bioidentity. For that reason

three main pathogenic mechanisms exist: distruction,

disfunction, and disregulation.

Distruction: Every structure corresponds to a specifuc

function, so the magnitude of destruction determined the

severity of dysfunction. This is the law of structural func-

tionalism. Small structural damages can not produce

alarming symptoms, but every structural damage reduces

the functional capacity, and thus the functional reserve. Ir-

reversible destructions accumulate over time and functional

failure progresses irreversibly.

Dysfunction: When HVs deficiency increases, the likeli-

hood of structural damage (dystrophy and necrosis) in-

creases too. According to the law of functional

structuralism function can actively modulate structures and

severe dysfunction cause dystrophy and necrosis

(distruction).

Disregulation (pathogenic identity). Disregulation is a

process that creates pathogenic identity (PI) by two main

mechanisms.

1. Distructions and dysfunctions decrease initially HVs

production and all respective HVs consumers suffer in pro-

portion to their dependence on the HVs. This in turn leads

to secondary reduction in the production of HVs. This is the

law of dysfunctional expansion. Every pathological process

that triggered this low differently, which manifests as dif-

ferent diseases. The diversity and intensity of dysfunctional

expansion is proportional to the severity of the disease.

2. When individual is unable to solve an important adaptive

problem, anticipatory control is activated strongly and

protractedly. The more protracted and stronger is the anticipa-

tory regulation, the bigger is the corresponding feed-back coun-

ter regulation and bigger is the probability for dystrophy and/or

functional exhaustion. This is the law of dysadaptation.

Holistic explanation of causality

Pathogenicity is ability of the pathogenic factor (Fi) to in-

duce the three D (distructions (Ds), disfunctions (Df), and

disregulations (Dr)) in the body. Pathogenic power (PP) is

the intensity of the pathogenicity measured as:

PPFi=DsFi+DfFi+DrFi. Because of the dysfunctional expan-

sion, secondary changeo of the three D is superimposed to

the Fi action. So the pathogenic identity acquires cumula-

tive pathogenic power (CPP), which is calculated as the

sum of changes in the three D: CPPi=�Dsi +�Dfi+�Dri.

CPP varies over the time. When CCP>0, the identity is

pathogenic. Despite the absence of symptoms, the doctor

must imply the existence of an active pathological process

and to seek actively its pathogenesis. When CCP decreases

over the time (CCPt1>CCPt2) the body is in a process of res-

toration. When CCPt1<CCPt2, the dysfunctional expansion

increases the health risk and the patient needs intensive

care.

Structures, functions and identity participate in the

pathogenesis as constitutional factors. The constitutional

complex of genetic and phenotypic factors (CsC) deter-

mines the level of resistance and reactivity and promotes

health. The three ‘’D’’ depends on the contradiction be-

tween defence power (DP) of the constitutional complex

and PP. To be effective Fi should have destructive power

that is able to exceed physiological resistance and to gener-

ate Ds and/or Fi should have physiological activity that is

able to produce Df and/or Fi should put individual into Dr.

All the factors that affect the PP of Fi form the complex of

conditions (CdC). The disease prevention does not neces-

sarily require removal of the pathogen (Fi). It is enough to

reduce the magnitude of PP below the value of DP.

When the first sign of the disease is Dr> 0, the disease is in

preclinical stage and may involve asymptomatic

dysfunctions and structural damages.

When the first sign of the disease is Df> 0, the individual

suffers from a distinct symptoms (clinical stage).

When the first sign of the disease is Ds> 0, the magnitude of

symptoms and adaptive disorders is proportional to the lim-

itation of functional capacity and reserve of the damaged

cell population.

Holistic logic of pathogenesis

The magnitude and composition of PP, Ds, Df, Dr, and

CPP are central for the understanding pathogenesis. Model

proposed here is universal and allows explaining different

types of diseases.

The causal diseases starts with Ds>0. Because of the struc-

tural functionalism after some time (latent period) Df>0,

and PP=Ds+Df (clinical stage). In its turn Df activates dys-

functional expansion and secondary Df-s are activated. Ev-

ery additional Df starst its own chain of pathogenic or coun-

ter-pathogenic logics by changing regulations and structures

(functional structuralism and) and CCP

(CPPi=�Dsi+�Dfi+�Dri) increases (CCPt1<CCPt2) or de-

creases (CCPt1>CCPt2) depending on the contradictions be-

tween pathogenic and healing forces. Complex of conditions

and constitutional complex give specificity and individuality

of the pathogenetic processes. CdC determines the strength

of PP. CsC determines the strength of DP. CCP arises as a re-

sult from the contradiction between the PP and DP.

40

Understanding pathogenesis of risky behavior: a holistic approach

Risk-dependent diseases start with disregulation mainly be-

cause of inability of the individual to take right decisions.

The healthy decision is sometimes really difficult. It requires

knowledge, but also the ability to control. When individual

know the risk, but practice it, the problem is lack of self-con-

trol or ability to control circumstances. This part of the pa-

thology is most closely associated with humanity, since the

right and ability for making choices determine whether the

organism will be in state of regulation or disregulation. If the

choice is wrong disregulation occurs and this activates the

pathogenesis of risk-dependent disease.

The prolonged disregulation leads to dysfunctions

(Dr>Df). The prolonged dysfunction activates mechanisms

of functional structuralism and creates microstructural

damages, that increase with time (Df>Ds). When Ds-s re-

duce enough the functional reserve of the damaged struc-

ture, another set of dysfunctions appears following the logic

of dysfunctional expansion and disease enter into its clini-

cal stage (Ds>�Df’’). Then disease follows both

risk-and-causal logic. The logic of the risk stimulates the

permanent increase of the magnitude of the initial Ds-s by

the association Dr>Df>Ds. The causal logic activates the

mechanisms of dysfunctional expansion (Ds>�Df’’) and

secondary distructioins. The CCP increases progressively

(CCPt1<CCPt2) up to end-stage.

Dysfunctional diseases are three groups according to their

pathogenesis:

Genetic diseases arise due to synthesis of abnormal struc-

tures. During the intrauterine period Ds occurs, which then

manifests as Ds and Dr. Individuality and specificity of the

pathological process is determined by the ability to achieve

adaptation of the maladaptive function by an appropriate

modification of the environment.

Family diseases are caused by habits that create Dr or be-

cause of living in an unhealthy environment. Theses dis-

eases have social and economical background and cultural

specificity. Their individuality depends on the strength of

CsC that determines DP against Dr.

Constitutional diseases arise due to altered reactivity (Df),

which motivates the individual to react abnormally to nor-

mal circumstances and thus creates disregulation (Dr).

They have a psychological basis and relate mainly to the in-

dividual experience. Genetic and social factors contribute

to formation of this experience but not determine it.

CONCLUSION

The holistic model of diseases is universal, but each disease

has specific dimensions. The biggest advantage of the model

is its ability to explain risk-dependent diseases that are unex-

plained by the ‘’causal’’ logic. It also divides diseases into

three groups according to main mechanism: destructive

(causal), dysfunctional (hereditary and constitutional) and

risk-dependent (with disregulation as main mechanism).

REFFERENCES

1. Friboulet A. , D. Thomas. Systems biology-an

interdisciplinary approach. Biosens Bioelectron.

2005; 20(12): 2404-7.

2. Joyner M.J. Giant sucking sound: can physiology

fill the intellectual void left by the reductionists? J

Appl Physiol. 2011; 111(2): 335-42.

3. Joyner M.J. , B.K. Pedersen. Ten questions

about systems biology. J Physiol. 2011; 589(Pt 5):

1017-30.

4. Mesarovic M.D. , S.N. Sreenath, J .D.

Keene. Search for organising principles: understand-

ing in systems biology. Syst Biol (Stevenage).

2004;1(1): 19-27.

5. Neugebauer E.A. , C. Wil ly , S. Sauerland.

Complexity and non-linearity in shock research:

reductionism or synthesis? Shock. 2001; 16(4): 252-8.

6. Pezard L. , J .L. Nandrino. Dynamic paradigm in

psychopathology: “chaos theory”, from physics to

psychiatry. Encephale. 2001; 27(3): 260-8.

7. Pi t t B.R. , J .W. Chris tman, S.J . Gunst , M.A.

Matthay, T. Stevens, L.B. Ware. Physiology,

reductionism, and translational medicine: the right

mix. Am J Physiol Lung Cell Mol Physiol. 2011;

301(4): L389-90.

41

Sarov G.

COMPARISON OF FUNCTIONAL AND STRUCTURAL

CLASSIFICATION IN PATHOPHYSIOLOGICAL THEORY

AND TEACHING

Sarov G.

Department of Pathophysiology, Trakia University, Medical Faculty, Stara Zagora

Nowadays teaching pathophysilogy is based on structural functionalism, although disease is a functional mat-

ter. This inconsistency makes understanding of the subject matter difficult for students. Functional concepts

demand classification of diseases by the similarities in the body disfunctions while the structural aproach mo-

tivates classification by the similarities in the initially damaged body structures. Both classifications have

their reasons but expecially for the pathophysiology and internal medicine functional approach seems to be

better. Since 2003 I have applied a combination of functional and structural approaches into my teaching.

The functional approach resulted in better and faster understanding of the pathophysiological matter and

students improved their performance on examinations.

Key words: holistic, structural, functional, pathophysiology

INTRODUCTION

Every science becomes complete by organizing its content

into consistent system of notions. Otherwise its fundament

concepts are incomplete or even wrong. Since Aristotle one

believe that the scientific systematization should be con-

structed by genus (general) and species (unique variations

of the genus). According to this concept the knowledge

about the body could be systematically organized by

structural or functional point of view.

The first one makes the structure genus and its functions –

species, as it believes that structure determines the function.

This is now the dominant approach in physiology (7,13),

pathophysiology (1,2,4,5,8,10,11), and clinical medicine

(6,9). In the available literature, we saw only one attempt

(14) to a holistic organization of content, which is still dom-

inated by structural approach in classification.

Functional viewpoint is that the function organizes struc-

tures and organises the knowledge just in opposite way (3).

As the science could not be hole without consistent system

of notions, the obvious question is which one concept is

more consistent and reliable.

METHOD

Every classification is questionable if is internally incon-

sistent. The inconsistency becomes obvious by finding log-

ical contradictions. When use this rule we analyse some in-

consistency in the structural classification of diseases and

offer an alternative viewpoint (3), that is more consistent in

understanding and teaching pathophysiology and internal

medicine. As consistency is natural for thinking, more con-

sistent science means more effective teaching. We carry out

a pilot investigation on succesfull taking examinations by

medical students trained in conventional and functional

thinking in the pathophysiology. First experimental group

(n=6) was trained both conventional and functional way of

thinking on the basic pathologic processes. The second one

(n=13) – was trained in the same way in diseases’ mecha-

nisms, and the control group (n=35) was trained only con-

ventionally. The three groups were tested by independent

examiner. Statistics were comparison of percent distribu-

tion of successfully tested students in three consequent ses-

sions and Fisher exact test for insignificance.

RESULTS

About consistency of structuralistic classification. Table 1

illustrates the comparison of six diseases classified both in

structural and functional manner. As one can see first three

rows are functionally incompatible althought all they be-

long to the structural genus ‘’blood diseases’’. The basic

function of the thrombocytes is associated with the

impermeability of the blood vessels. Leucocytes participate

in immunity and leucosis functionally belongs to tissue

homeostatic disorders. Erythrocytes functionally belong to

the system of oxidation. This means that the structural clas-

sification is inconsistent with functional logic in these par-

ticular points. And vise versa, the last three rows belong to

one functional genus and different structural species . They

all have as common syndrom (functional disorder) the in-

sufficiency of oxygenation.

The same contradictions one can find in endocrine disorders.

Some of the hormones belong functionally to homeostasis

(aldosteron, insulin, glucagon,ect.), other could be determined

as stress hormones (epinephrine, corticosterone, ect), third

play important role in the physiological effect of the social in-

teractions (sex hormones, oxitocine, ect) and could be classi-

fied as social hormones. In the structural classification all they

43


belong to one functionally (and logically) inconsistant group

of endocrine diseases.

Empirical observations. All the first three diseases from

Table 1 could be learned by using only memory as they

have quite different mechanisms and are logically inconsis-

tent. Last three diseases could be learned easy by using

thinking and understanding, which means more logic and

less memory. Since the pathophysiology is a logical sci-

ence, we assumed that the second way will be more suc-

cessful in taking the exams.

Table 2 shows result from the anual tests of pathophysiology .

Additional functional teaching on basic pathologic processes

(group I) provides worst results and students need most time to

takeexaminations (althoughexpressive resultsdiffernot signifi-

cantly, probably because small number of n in this group). Ad-

ditional functional teaching on the mechanisms of diseases

(group II) provides twice better results during first session com-

pared with the control group (P<0.05). Even after the second

examination the control group can not reache the results of the

second experimental group (P<0.05). When we asked addition-

ally, the students from the first and second groups share that

functionalapproach ismoreunderstandable thanconventional.

CONCLUSIONS

Logical contradictions between both classification come

from the different viewpoint. In fact structural and func-

tional approaches are not in conflict. The object of some

medical sciences is description of the structures and ana-

tomical spaces, that is why structural classification is best

for them (for example, anatomy, surgery, pathoanatomy).

On the contrary, the object of pathophysiology and internal

medicine are the functions and functional manner of classi-

fication is best for them. As a bridge between the both clas-

sifications physiology seems to need both viewpoints: firs

one - to describe the structures functioning, second one - to

explain how the life is possible. Medical student and prac-

titioner must use appropriately both viewpoints to be profi-

cient in the field.

The genus in the functional approach are functions, and spe-

cies are structures. Starting with common functions and dis-

orders the functional classification divides into structure spe-

cific species. By using this classification the physician logi-

cally follows the symptoms of the patient to find functional

genus and then - to specify disease by seeking structural dif-

ferences. Thus diagnostic process is logically connected to

the patient’s symptoms. As symptoms are in fact signs of

functional disorders, they refer directly to the functional clas-

sification and direct the thinking process quickly and natu-

rally to the possible combination of structural damage.

The limitations of the pilot study do not allow final conclu-

sions, but results show that the functional method may be

able to increase the effectiveness of teaching

pathophysiology. The best performance of the second

group is possibly due to improved understanding of the dis-

ease logic. Without this advantage the students from the

control group must use more extensively memory, which

increases the time of preparation. The worst results of the

first group of students may be due to the fact that they know

that there is a functional approach but fail to implement it

without the help of a teacher in understanding the mecha-

nism of disease. Thus they probably unsuccessfully try to

find functional explanations and lose most time for

preparation.

REFERENCES

1. Èëó÷åâ Ä.Õ. , À.Ã. Ñòîéíåâ (ðåä) Îñíîâè íà

ïàòîôèçèîëîãèÿòà. ÌÈ Ðàéêîâ, Ñîôèÿ 2010

2. Ëîëîâ Ð. , Ä. Ìèòêîâ, Ïàòîëîãè÷íà

ôèçèîëîãèÿ. ÌÈ Àðñîâ, Ñîôèÿ, 1998.

3. Ñàðîâ Ã.Ì. Êðàòêà Èëþñòðèðàíà

Ïàòîôèçèîëîãèÿ íà âúòðåøíèòå áîëåñòè, ÌÈ

ÑÀÐ, Ñòàðà Çàãîðà, 2003.

4. Öåêîâ Ö.Ò. Ïàòîëîãè÷íà Ôèçèîëîãèÿ.

Êíèãîèçäàòåëñòâî Çîãðàô, Âàðíà 2011.

5. Cotran R.S. , V. Kumar, T. Coll ins . Robins

Pathologic basis of diseases. WB Saunders, 1974.

6. Fauci A.S. , E. Braunwald, D.L. Kasper ,

S.L. Hauser , D.L. Longo, J .L. Jameson, J .

44

Comparison of functional and structural classification in ...

diseasesructural

classification

functional

classification

thrombocytopenia blood diseases vessels disorders

lymphoma blood diseasestissue homeostasis

disorders

anemia blood diseases oxygen disorders

chronic obstructive

pulmonary diseasepulmonary diseases oxygen disorders

cardiac insuficiency cardiac diseases oxygen disorders

Table 1. Illustrative comparison of structural and

functional manner of classification

Groups

sessions

² group

(n=6) n (%)

²² group

(n=13) n

(%)

control

(n=35) n

(%)

All (n=54)

n (%)

First 1 (16,66%) 5 (38,46%) 6 (17,14%) 12 (22,22%)

Second 1 (16,66%) 4 (30,77%) 14 (40%) 19 (35,18%)

First and

second2 (33,33%) 9 (69,23%) 20 (57,14%) 31 (57,40%)

Last 4 (66,66%) 4 (30,77%) 15 (42,85%) 23 (42,59%)

Table 2. Sucessfull exams of the three groups of

students (see text for more explanations).

Loscalzo Harrison’s principles of internal medi-

cine, 17th edition. The MacGraw Hill Co. Inc. , 2008.

7. Guyton A.C. , J .E. Hall Textbook of medical phys-

iology. WB Saunders, 2000.

8. Huether S.E. Understanding Pathophysiology.

Mosby Inc., 2008.

9. Kumar P. , M. Clark. Clinical medicine. WB

Saunders, 2002.

10. Lazenby R.B. , Handbook of Pathophysiology.

Wolters Kluwer Health, Lippincott Wil-

liams&Wilkins, 2011.

11. McCann J.A.S. Professional guide to

pathophysiology 3th edition. Wolters Kluwer Health,

Lippincott Williams&Wilkins, 2011.

12. McPhee S.J . , G.D. Hammer. Pathophysiology

of Disease: An introduction to Clinical Medicine. The

MacGraw Hill Co. Inc., 2006.

13. Pocock G., C.D. Richards, Human Physiology.

The basis of Medicine. Oxford University Press,

2001.

14. Tortora GF, S.R. Grabovski . Principles of

anatomy and physiology. John Wiley&Sons Inc,

2000.

45

Sarov G.

ÅFFECT OF CHRONIC TREATMENT WITH MELATONIN ON

DIURNAL VARIATIONS OF SPONATENOUS MOTOR SEIZURES

AND BEHAVIOR OF WISTAR AND SPONTANEOUSLY

HYPERTENSIVE RATS IN KAINATE MODEL

OF TEMPORAL LOBE EPILEPSY

Tchekalarova J.1, D. Pechlivanova

1, Zl. Petkova

1, Al. Stoynev

2

1Institute of Neurobiology, Bulg. Acad. Sci.;2Department of Pathophysiology, MU - Sofia

ABSTRACT

The aim of the present study was to analyze the effect of long-term melatonin treatment during

epileptogenesis on diurnal rhythms of spontaneous recurrent seizures (SRS) during the chronic phase in

Wistar (WIS) and spontaneously hypertensive rats (SHRs) in kainate (KA) model of temporal lobe epilepsy

(TLE). Melatonin treatment (10 mg/kg diluted in drinking water, 8 wk) increased the latency for appearance

of SRS in WIS rats. Whereas epileptic WIS and SHRs treated with saline exhibited circadian fluctuations in

the frequency of the SRS with a prevalence of seizures during the light phase, melatonin-treated rats did not

show circadian rhythms of seizures during the 3th

and 4th

month after status epilepticus (SE). Taken together,

our data suggest that chronic treatment with melatonin after SE may have potential to aid in the prevention

and development of epilepsy particularly accompanied with high arterial blood pressure.

Key words: Wistar rats, spontaneous hypertensive rats (SHRs), kainate model of temporal lobe epilepsy;

melatonin; diurnal rhythms

INTRODUCTION

The hormone melatonin, which is the main secretory prod-

uct of the pineal gland, plays a modulatory role in numer-

ous physiological functions. Accumulating clinical and ex-

perimental data raise the point of potential therapeutic role

of melatonin in epilepsy. Clinical evidence revealed that

melatonin posses low toxicity and may be used in seizure

control in conjunction with anti-seizure medications (10).

On the other side, experimental data revealed that

melatonin act as an anticonvulsant against chemically-in-

duced seizures (7,15), maximal electroshock (3), and elec-

trically kindled stimulation of amygdala (8).

Spontaneously hypertensive rats (SHRs) are widely ac-

cepted as an experimental model of essential hypertension

and has extensively been explored in chronobiological as-

pect (5,11). In these rats cerebrovascular changes, brain at-

rophy, loss of nerve cells in cerebrocortical areas, and glial

reaction were documented (reviewed in:1). In vivo imaging

studies suggest that hypertension-induced brain aberrations

detected in SHRs are similar to those found in patients with

essential hypertension. Clinical data showed disturbances

of melatonin biosynthesis in hypertensive patients while

experimental studies reported antihypertensive effect of

melatonin in SHRs (9). Taken together, these data are con-

sonant with the idea that melatonin may hold potential for

preventing neuronal damage after brain insults such as

brain trauma, status epilepticus (SE) and

hypertension-evoked brain damages.

Recently, we have demonstrated a circadian rhythm of ap-

pearance of SRS in epileptic state and strain-dependent di-

urnal variations in behavioral changes of normotensive

WIS and SHRs in KA model of TLE (13,14). Considering

the important function of melatonin in the circadian timing

system and numerous evidence supporting melatonin role

in epileptic phenomena, in the present study we aimed to

analyze whether the chronic melatonin treatment during

epileptogenesis would be able to attenuate the frequency of

spontaneous recurrent seizures (SRS) during the chronic

epileptic state in kainate (KA) model of TLE.

METHODS

1. Subjects

Male normotensive Wistar (WIS) rats and SHRs were indi-

vidually housed under standardized conditions (12/12-h

light/dark cycle and 20±1 oC, 50-60% humidity). Food and

water were available ad libitum. The experimental design

was approved by governmental authorities fully in accor-

dance with the European Communities Council Directives

of 24 November 1986 (86/609/EEC).

47


2. Induction of status epilepticus with

kainic acid

Details of methodology were recently published (13,14). In

brief, SE was induced by a repetitive kainic acid (KA,

Sigma-Aldrich, Bulgaria) injection (5 mg/kg/h,

intraperitoneally). The first melatonin or saline

intraperitoneal (i.p.) injection was started 3 h after the onset

of SE. After recovery melatonin (Sigma-Aldrich, Bulgaria)

was administered chronically for a period of two months

(10 mg/kg in drinking water). Matched controls were

treated with an equivalent volume and number of injections

of sterile saline.

3.Video monitoring of spontaneous

reccurent seizures

The circadian distribution and the frequency of spontane-

ous recurrent seizures were ascertained by continuous (24

h/3 months) video-monitoring with a light-sensitive black

and white camera (S- 2016, AVTECH, Taivan, No

AVC307R).

Statistical analysis

ANOVA analysis of variance was used followed by a

post-hoc Bonferroni t-test to examine individual group dif-

ferences. P<0.05 was accepted as an index of statistically

significant differences.

RESULTS

Chronic melatonin treatment increased the latency for onset

of the first SRS in WIS rats (*p<0.05) (Fig. 1).

The distribution of SRS during the 24-h light-dark cycle at

the 3rd month after KA-induced SE is demonstrated in

Fig.2. Similarly to our previous studies (13,14) we have

found circadian distribution in the appearance of SRS with

a prevalence of seizures during the light phase

(08:00-20:00) (82%). At the 3rd month after SE both WIS

and SHRs exhibited higher seizure frequency during the

light phase (82 %).

However, melatonin treatment abolished circadian rhythms

of SRS in WIS and SHRs (Fig. 2 and Fig.3).

Although more SRS appeared during the light phase com-

pared to the dark one, significant difference was found for

the 3rd month (WIS: op<0.047; SHRs: op<0.013) and the

4th month (SHRs op<0.001), respectively. The total number

of SRS was statistically different between WIS and SHRs

at the 3rd month (11:00 p.m.), the 4th month (08:00;

10:00-16:00 p.m.) and the 5th month (12:00; 14:00 p.m.),

respectively, op<0.05 (Fig. 3). In 63 % of WIS rats and 100

% of SHRs, respectively, seizures appeared in clusters (i.e.

>3 seizures per day) (38). In 100 % of WIS rats and 63 % of

48

Sex differences in functional organization of ...

Fig. 1. The effect of melatonin on the latency for onset

of the first spontaneous recurrent seizure after kainite

(KA)-induced status epilepticus (SE) in Wistar and

spontaneous hypertensive rats (SHRs). Data represent

the mean ± SEM (n=10). *p<0.05 vs Wistar KA group.

Fig. 3. Total seizure frequency (mean ± SEM) during

the IIIrd, IVth and Vth month of KA-induced SE in Wistar

and SHRs. *p<0.05 light vs dark phase; op<0.05 vs

WIS with the same treatment.

Fig. 2. Dynamics of spontaneous reccurent seizures

(SRS) during the chronic phase of epilepsy counted for

12 weeks (starting three months after KA-induced SE in

Wistar and SHRs. Data shows the mean ± SEM of

weekly seizure frequency during the light (WISd, SHRd)

and dark (WISn, SHRn) phase of the twenty-four-hour

period of observation. *p<0.05 light vs dark phase;op<0.05 vs WIS with the same treatment.

SHRs the appearance of clusters was followed by a sei-

zure-free period of 2-5 days.

DISCUSSION AND CONCLUSION

Our results support previous reports demonstrating that

acute administration of melatonin in rats before and during

SE induced by either KA or pilocarpine displays

neuroprotective effects by reducing neuronal death,

supragranular mossy fiber sprouting, lipid peroxidation,

and microglial activation. In this line, Guisti et al. (1996) re-

ported that the cumulative dose of 10 mg/kg melatonin pre-

vented KA-induced neuronal death as well as behavioral

and biochemical disturbances in WIS rats. The hormone is

also able to reduce the spiking activity and seizure fre-

quency in patients with intractable epilepsy (2). In patients

with TLE high levels of salivary melatonin were observed

during the postictal period (4). The hormone is also able to

reduce the spiking activity and seizure frequency in patients

with intractable epilepsy (2). In patients with TLE high lev-

els of salivary melatonin were observed during the postictal

period (4). We have found that melatonin modified the de-

velopment, the frequency and the severity of SRS in a

strain-dependent manner. It could be speculated that

melatonin exerts its neuroprotective effect in KA model

through different mechanism in WIS and SHRs. Previous

clinical and experimental findings, including ours, suggest

a circadian rhythm of appearance of SRS in epileptic state

(12-14). However, we have fount that the hormone was

able to diminish the circadian rhythm of SHRs in both WIS

and SHRs. This effect might be a part of the

neuroprotective mechanism of melatonin action in KA

model of TLE.

In conclusion, the major findings of this study are that

melatonin exerts a potent neuroprotective effect on seizure

susceptibility in WIS and SHRs during the chronic epilep-

tic phase. While the hormone treatment increased the la-

tency for onset of the first SRS in KA-treated WIS rats it at-

tenuated the seizure frequency of SRS in SHRs.

REFERENCES

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Arterial hypertension and brain damage--evidence

from animal models (review). Clin. Exp. Hypertens.,

25, 2003, No 6, 359-380.

2. Anton-Tay, F. Melatonin: effects on brain func-

tion. Adv. Biochem. Psychopharmacol., 11, 1974, No

4, 315- 324.

3. Bazi l C.W., D. Short , D. Crispin, W.

Zheng. Patients with intractable epilepsy have low

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4. Borowicz, K.K. , R. Kaminski , M. Gasior ,

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Schiavo, M. Floream, H. Manev.

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891-896.

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attenuates age-associated hypertension and weight

gain in spontaneously hypertensive rats. Ann. N Y

Acad Sci., 1199, 2010, 114-20.

10. Rufo-Campos, M. Melatonin and epi lepsy .

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11. Stoynev, A.G. , O.C. Ikonomov, N.K.

Minkova, S.Z. Zacharieva, V.G.

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13. Tchekalarova J. , D. Pechl ivanova, D. I tzev,

N. Lazarov, P. Markova, A. Stoynev. Diur-

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hypertensive rats in kainate model of epilepsy. Epilep.

Behav. 17, 2010, No 1, 23-32.

14. Tchekalarova J. , D. Pechl ivanova, T.

Atanasova, P. Markova, V. Lozanov, Al .

Stoynev. Diurnal variations of depressive-like be-

havior of Wistar and spontaneously hypertensive rats

in kainate model of temporal lobe epilepsy. Epilep.

Behav. 20, 2011, 2, 277-285.

15. Yamamoto, H.A. , H.W. Tang. Melatonin at-

tenuates l-cysteine-induced seizures and peroxidation

lipid in the brain of mice. J. Pineal Res., 21, 1996, No

6, 108- 113.

49

Tchekalarova J., D. Pechlivanova, Zl. Petkova ...

DESIGN, SYNTHESIS AND ANALYSIS OF NOVEL

ENDOMORPHIN-2 AND MORPHICEPTIN ANALOGUES

Georgiev K.1, E. Miladinova

2, T. Pajpanova

2

1Medical University of Varna, 2Institute of Molecular Biology "Roumen Tsanev",

Bulgarian Academy of Sciences

Endomorphins (endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2, endomorphin-2, Tyr-Pro-Phe-Phe-NH2) are po-

tent and selective �-opioid receptor agonists. Morphiceptin (Tyr-Pro-Phe-Pro-NH2), a tetrapeptide present in

the enzymatic digest of bovine �-casein, is a selective ligand of the �-opioid receptor. New endomorphin-2 and

morphiceptin analogues incorporating halogenated phenylalanines in place of the native phenylalanine are

reported. In order to improve the membrane permeability of the parent molecule, deoxycholic acid was at-

tached to the N-terminus of the peptide chain.

Key words: endomorphins, morphiceptin, opioid receptors, halogenated phenylalanines, peptide mimetics

INTRODUCTION

The three opioid receptors (�, and �) belong to the G-pro-

tein coupled receptors (GPCRs), which are characterized

structurally with a core of seven helical transmembrane do-

mains connected by three extracellular and intracellular

loops (1) These receptors are extremely important clinical

target in the treatment of pain and additionally have pro-

found effects on the neuroendocrine system immune re-

sponse (2). The � opioid receptor (MOR) is proven to be the

major target of analgesics, (2) because of the particular phys-

iological characteristics of this receptor, development of the

synthetic MOR-specific ligand is of great importance.

µ-Opioid agonists, such as morphine and heroin, produce

potent antinociception through µ-opioid receptors. However,

they also produce serious side-effects such as respiratory de-

pression and physical and psychological dependence.

The endogenous peptide ligands for �-opioid receptor,

endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1) and

endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2) were dis-

covered in 1997 by Zadina et al. (3). Both endomorphins

(EMs) exhibited the highest affinity for �-opioid receptor

and extraordinarily high selectivity relative to - and

�-opioid receptor systems of all known opioid substances

(3). These tetrapeptides are involved in the regulation of

several behavioral, emotional (4), cardiovascular (5), gas-

trointestinal (6,7) and respiratory functions (8). Further-

more, both EMs display a strong antinociceptive effect on

acute pain (9), similar to that of morphine. They are more

effective than the majority of the opioid peptides in reliev-

ing neuropathic pain even at very low dose, opening up the

possibility of pharmaceutical uses of these peptides (10).

They are also important model peptides to determine the

structure-activity relationship based on their typical charac-

teristics of backbones and aromatic side chains (11,12).

Morphiceptin (Tyr-Pro-Phe-Pro-NH2), which was origi-

nally synthesized as an analog possessing the N-terminal

tetrapeptide fragment of �-casomorphin (13) and was later

isolated from the enzymatic digest of bovine �-casein (14)

is a selective ligand of the �-opioid receptor. Its structure

differs from endomorphin-2 only by the amino acids in the

fourth position (Pro and Phe, respectively).

Morphiceptin, among other opioid peptides, has been

shown to elicit strong supraspinal antinociception when

given intracerebroventicularly (i.c.v.), which means di-

rectly to its site of action, which is the central nervous sys-

tem (CNS). This effect is not observed after peripheral ad-

ministration due to relatively rapid degradation of

morphiceptin, resulting in a short duration of action, and

limited delivery to the CNS.

A close structural similarity of endomorphin-2 and another

atypical opioid peptide, morphiceptin, which both have a

Phe residue in the third position, encouraged us to study

antinociceptive activity of these two peptides and their ana-

logues.

In order to improve the affinity and chemical stability of these

opioid peptides, we have designed, synthesized, and analyzed

9 novel analogues incorporating halogenated phenylalanines

in position 3 or 4 as surrogates of the native phenylalanine.


Peptide synthesis

Peptides EM-2 and analogue 1 were synthesized by an ''in

solution, step-by-step'' method, starting with proline or

phenylalanine amides, respectively. To C-terminal frag-

ments respective Boc-amino acids were coupled using

dicyclohexylcarbodiimide and N-hydroxybenzotriazole.

Deprotection of the Boc-group was provided by 2 N HCl in

dioxane.

Peptides 2-9 in this study were synthesized by manual

solid-phase procedures using techniques for Fmoc-pro-

tected amino acids on Wang or MBHA Rink-Amide pep-

51

Georgiev K., E. Miladinova, T. Pajpanova

tide resins respectively. 20% Piperidine in DMF was used

for deprotection of Fmoc-groups and DIC and HBTU were

employed as a coupling agent. Simultaneous deprotection

and cleavage from the resin was accomplished by treatment

with TFA/TIS/water (95:2.5:2.5) for 3 h at room tempera-

ture. Crude peptides were purified by preparative TLC and

their purity was checked by analytical HPLC.

Peptide analysis

HPLC analyses were performed on Agilent Technologies

HP 1100 and Dionex, UltimateR 3000 LC instruments, us-

ing a Beckman UltrasphereTM RPC18 (250 � 4.6 mm, 5

�m) column with a mobile phase: mixture of 0.01 M

K2HPO4 (pH = 2.5) and acetonitrile (volume ratio = 80 :

20) over 30 min at a flow rate of 0.5 ml min-1, and

ZICR-HILIC (100 � 4.6 mm, 5 �m) column with a mobile

phase: mixture of 0.05 M CH3COONH4 (pH = 6.8) and

acetonitrile (volume ratio = 50 : 50), and mobile phase:

mixture of 0.005 M CH3COONH4 (pH = 6.8) and

acetonitrile (volume ratio = 20 : 80), respectively at a flow

rate 0.5 ml min-1.

Final purity of all peptides was >97%. Calculated values

for protonated molecular ions were in agreement with those

determined by FAB mass spectrometry.


Endomorphin-2 and morphiceptin are the same class of

opioid peptides which have a unique N-terminal

Tyr-Pro-Phe sequence different from the N-terminal

tetrapeptide sequence Tyr-Gly-Gly-Phe, characteristic for

typical opioid peptides, like enkephalins, endorphins, and

dynorphins. The aromatic amino acids (Tyr1, Phe3, or Phe4)

of EM-2 and morphiceptin have been shown to be impor-

tant structural elements that interact with the opioid recep-

tors besides the proline residue at the second position con-

ferring high selectivity on the �-opioid receptor.

In search of novel endomorphin-2 and morphiceptin analogues

with improved pharmacological profile we have synthesized a

series of peptide mimetics modified in position 1,2,3.

The first modification included EM-2 and morphiceptin

analogues (Fig. 1 and Fig. 2), where Phe was replaced with

Phe(p-CI) (2,4 and 8) or Phe(p-F) (3,5,7 and 9). The pep-

tides were synthesized on a Rink-amide resin using

Fmoc-strategy with DIC/HOBt activation.

The second surrogate that we choose, was deoxycholic acid

in order to improve membrane permeability of the parent

molecule (4 and 5). The deoxycholic acid was attached to

the N-terminus of the peptide chain on a Rink-amide resin

using Fmoc-strategy with DIC/HOBt activation.

A majority of bioactive peptides reveal their biological

functions by C-terminal amidation. As the deamination of

such peptides leads to considerable loss of activity, the am-

ide group may be strongly correlated with the bioactivity.

C-terminal amidation would significantly associate with

the bioactive conformation of a bioactive peptide and its in-

teraction with a receptor.

As a possible clue to the biological and structural implica-

tions of C-terminal amidation, we have investigated the

modifications of C-terminal amide group of EM-2 by sub-

stitution of the C-terminal amide group with biogenic

amines. The analogues 6 and 7 modified at C-terminus with

biogenic amines were obtained on a Wang resin using

Fmoc-strategy with HBTU/HOBt activation. The crude

peptides were purified by preparative TLC and their purity

was checked by analytical HPLC. The precise molecular

mass was confirmed by ESI-MS.

52

Design, synthesis and analysis of ...

Fig. 2. Design of Morphiceptin mimetics.

Fig. 1. Design of Endomorphin-2 mimetics.

CONCLUSIONS

In summary, we succeeded to incorporate the halogenated

amino acids into EM-2 and morphiceptine peptide moiety.

We are able to obtain the peptide mimetics with satisfactory

purities (typically > 97%), as assessed by analytical HPLC).

The investigation of the biological activities of the new an-

alogues is in progress also. They are object of pharmaco-

logical investigation, and this data will de presented latter.

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lecular cloning of a rat kappa opioid receptor reveals

sequence similarities to the mu and delta opioid re-

ceptors. - Biochem. J., 295,1993, 625-628.

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8. Yu, Y. , Y. Cui , X. Wang, Y.Z. Fan, J .

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Differential antinociceptive effects of endomorphin-1

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10. Przew �ocki , R. , D. Labuz , J . Mika,B.

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11. Okada, Y. , Y. Fuj i ta , T. Motoyama, Y.

Tsuda, T. Yokoi , T. Li , e t a l . Structural stud-

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logues: enhanced activity and cis orientation of the

Dmt-Pro amide bond. - Bioorg. Med. Chem., 11,

2003, 1983-1994.

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T� th , M. M�csai , G. Szab� , e t a l . Influence of

degradation on binding properties and biological ac-

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Commun., 284, 2001, 771-776.

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Cuatrecasas , J .K. Chang. Morphiceptin: a po-

tent and specific agonist for morphine (mu) receptors.

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14. Chang , K.J., Y.F. Su, D.A. Brent, J.K. Chang. Isola-

tion of a specific mu-opiate receptor peptide,

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53

Georgiev K., E. Miladinova, T. Pajpanova

PHYSIOLOGICAL AND NON-INVASIVE BIOCHEMICAL INDEXES

IN A MODEL OF EMOTIONAL STRESS IN SHOOTERS

Petrov L.1, P. Atanasov

1, N. Zaekov

1, A. Alexandrova

2,

Z. Zsheliaskova-Koynova1, I. Achkakanov

1

1National Sports Academy "Vassil Levski", Sofia, 2Institute of Neurobiology,


ABSTRACT

The use of noninvasive methods for assessment of stress under real and experimental conditions in people has

obvious advantages. Invasive methods cause stress and make difficult the interpretation of results. In sports

shooting psycho-emotional stress is severe and is associated with both the shooting performance and the reac-

tion to the reported results. The aim of this study was to evaluate the informative value of some stress bio-

chemical indexes, determined in saliva, and to compare them with suitable noninvasive physiological

parameters such as heart rate (HR) and heart rate variability. Twelve sports shooters (age from 14 to 19

years), divided into two groups: 5 in discipline air rifle and 7 in air pistol were tested. Two tournaments in

both disciplines were held. Each shooter performed 20 shots within 30 minutes. Samples of saliva were taken

by salivettes and the concentration of Na+, K

+, protein and salivary alpha-amylase activity (sAA) were mea-

sured. HR and physical activity were recorded, using GSM device in communication with a server. After the

tournament, in saliva the concentration of K+

and protein were increased significantly in all shooters. The

sAA activity demonstrated the same dynamics with two exceptions that are discussed. The concentration of

Na+

did not change. HR showed a typical increase in the middle and a gradual decrease to the end of the tour-

nament. rMSSD index showed a mirror mode dynamics. So we suggest that rMSSD represents the most stable

cardio vascular index of the emotional stress level in the model used.

Key words: heart rate, heart rate variability, saliva, salivary alpha-amylase activity, salivary potassium

INTRODUCTION

The use of noninvasive methods for assessment of stress

under real and experimental conditions in people has obvi-

ous advantages. Invasive methods cause stress and make

difficult the interpretation of results. In recent years more

often to evaluate the stress, changes of certain parameters,

measured in saliva are used. A parameter that has been sug-

gested to reflect the stress-related changes in the body is the

salivary enzyme alpha-amylase (sAA) (1,2,6,9). The sAA

release is known to be elicited by activation of the auto-

nomic nervous system (ANS) which controls the salivary

glands (7). Other relevant parameters are the concentration

of protein and some electrolytes in saliva. Their typical

changes in stress correlate with blood adrenaline and

cortisol levels. In particular, many researchers have re-

ported salivary K+ and protein increase in stress. While in

same conditions, the salivary Na+ did not change or even

has shown a reduction (5,8,10).

In sports shooting the psycho-emotional stress is severe and

is associated with both the shooting performance and the

reaction to the reported result - especially when the series of

successive shots are successful or unsuccessful. In turn, the

physiological changes in stress affect the shooting

performance, having in mind that the good players pull the

trigger in exact phase of breath holding and even at exact

time point of the cardiac cycle (3,4).

The aim of this study was to evaluate the informative value

of some biochemical indexes determined in saliva,

assessing the level of stress in shooters and to compare

them with suitable noninvasive physiological parameters

such as heart rate (HR) and heart rate variability (HRV).

MATERIALS AND METHODS

Twelve sports shooters, divided into two groups: 5 (2

women and 3 men) in the discipline air rifle and 7 (1

woman and 6 men) in the discipline air pistol were tested.

The age of the shooters varied between 14 and 19 years

(average age of 16 years). Two tournaments including the

both disciplines were held. Each shooter performed 20

shots from a distance of 10 m within 30 minutes. For better

motivation a small prize - 60 BGN for the first place and 40

BGN for the second place were provided. One day before

competition, the players were instructed not to use alcohol,

cigarettes and coffee, and to abstain from plenty eating just

before the tournament. Before and after the tournament,

samples of saliva were taken by salivettes. The samples

were centrifuged at 8000g and were stored at -20°C. On the

55


next day the concentration of Na+, K+, protein and activity

of alpha-amylase in the saliva were measured on biochemi-

cal analyzer Human80. Reagents were purchased from Hu-

man Ltd (France). The HR was recorded (as R-R intervals

with 1 ms accuracy) during the entire period of the investi-

gation, using GSM device in communication with a re-

search server. The device, produced by Security Solutions

Institute Ltd. (Bulgaria), registered the physical activity ev-

ery second by built-in accelerometer, too. The instanta-

neous HR and the index of HRV - the square root of the

mean squared difference of successive R-R intervals

(rMSSD), were calculated automatically.

Significance of the differences was tested by Student test

for paired data.

RESULTS

The changes in the concentration of saliva potassium of any

one of the tested persons before and after tournaments is

presented on Figure 1. On Figure 2 the average values of

saliva K+ concentration of both disciplines separately and

jointly for all athletes are presented.

The average concentrations of Na+ in the saliva, collected

before and after both tournaments are shown in Table 1. In

terms of this index, statistically significant differences be-

fore and after the tournaments are not registered.

The changes in protein content in saliva of any one of the

tested persons before and after tournaments are presented

on Figure 3. On Figure 4 the average values of saliva

protein content of both disciplines separately and jointly for

all athletes are shown.

The individual sAA activity before and after the air rifle

and air pistol tournaments are presented on Figure 5 and on

Figure 6 the average sAA activity before and after both

tournaments separately and jointly for all shooters are

summarized.

56

Physiological and non-invasive biochemical indexes in a model of ...

Figure 1. Individual saliva K+ concentration before and

after pneumatic rifle (A) and air pistol (B) tournament.

Figure 2. Average saliva K+ concentration before and

after the tournament for both disciplines separately and

jointly for all athletes.

Befor competition

mmol/l ± SE

After competition

mmol/l ± SE

Air rifle 51.15 ±1.74 50.54 ±2.33

Air pistol 52.30±2.30 55.0 ±4.41

Both 50.31±2.58 47.33 ±1.91

Table 1. Average concentrations of Na+ in the saliva of

player shooters collected before and after both

tournaments separately and jointly for all athletes.

Figure 3. Individual saliva protein concentration before

and after the air rifle (A) and air pistol (B) tournaments.

Figure 4. Average saliva protein concentration before

and after the tournaments separately for both

Figure 5. Individual sAA activity before and after the

air rifle (A) and air pistol (B) tournaments.

Figure 6. Average sAA activity before and after both

tournaments separately and jointly for all shooters.

The records of HR during the tournaments showed large

fluctuations with a period covering the shooting

performance (about 60 seconds). It seems that these

fluctuations coincided with the phases of shooter's

breathing. The differences in minimal and maximal

instantaneous HR in these periods reached 25-30 bpm

(Figure 7). Therefore as indexes of the sympathetic activity

of the ANS we accepted the rMSSD, maximal, average and

minimal HR, calculated within 5 min intervals. Heart rate

showed a characteristic increase till the middle of the

tournament and a gradual decrease to the end of the

tournament. The rMSSD index showed a mirror mode

dynamics (Figures 8 and 9). So we suggest that rMSSD

represents the most stable cardio vascular index of the

emotional stress level in the model used.

DISCUSSION

The concentrations of both saliva K+ and protein were in-

creased in all shooters. The sAA activity showed the same

dynamics with two exceptions only. The baseline of the

winner of the air rifle tournament (shooter number 4) is

lower than that of the other competitors and remains un-

changed after the tournament. This probably reflects the

good mental strength and conscious self-confidence of the

player. The sAA from the shooter ranked last (shooter

number 5) showed higher baseline in comparison to other

competitors and decreased after the tournament. Perhaps

this reflects the greater emotional stress in the begging of

the tournament, which decline rapidly after the initial poor

performance.

The changes in the biochemical parameters measured in

saliva in this experiment are in accordance with the

observations in emotional stress conditions, described in

the literature. This is an argument in favor of the idea that

such improvised tournaments could be used to study the

emotional stress in different sports.

HR showed a characteristic increase in the middle of the

tournament and a gradual decrease to the end. The rMSSD

index showed a mirror mode dynamics and we believe that

it is the most stabile HRV index of the level of emotional

stress in the model used (Figures 8 and 9). The analysis of

the dynamics of these parameters shows that the emotional

stress level in this model reaches the maximum most often

on the 20th min.

CONCLUSIONS

In this emotional stress model, the protein concentration

and amylase activity in saliva are sensitive markers. The

concentration of K+ in contrast to Na+ is a reliable marker

for assessing the level of emotional stress. HR fluctuations

during the shooting performance (about 60 seconds) make

difficult the use of HR, calculated for short periods of time

(less than 2 minutes) as indicators for ANS activation. As

indexes of stress during shooting competition we recom-

mend registration of following indexes: the minimal, aver-

age and maximal HR and rMSSD for 5-min periods.

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57

Petrov L., P. Atanasov, N. Zaekov ...

Figure 7. Five minutes (from 15th to 20th minute)

recording of instantaneous HR of the air rifle

Figure 8. rMSSD, minimal, average and maximal HR in

intervals of 5 minutes of the air rifle tournament winner

(shooter number 4).

Figure 9. rMSSD, minimal, average and maximal HR in

intervals of 5 minutes of the third ranked shooter in the

air rifle tournament (shooter number 2).

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1074-1080.

58

Physiological and non-invasive biochemical indexes in a model of ...

OLFACTORY BULBECTOMY INDUCES SHIFTS IN

LATERALIZATION OF LOCOMOTOR RESPONSES TO

VASOACTIVE INTESTINAL PEPTIDE

Ivanova M.2, S. Belcheva

1,3, I. Belcheva

1, N. Negrev

2, R. Tashev

1

1Institute of Neurobiology, Bulgarian Academy of Sciences,2 Department of Physiology and Pathophysiology, Medical University - Varna, 55,

3Faculty of Pre-School and Primary School Education, SU "Sv. Kl. Ohridsky" - Sofia

SUMMARY

The olfactory bulbectomy (OBX) is an animal model of depression that produces behavioral, physiological,

and neurochemical alterations resembling clinical depression. OBX rats typically exhibit hyperactivity in an

open field test. The effects of vasoactive intestinal peptide (VIP) in a dose of 100 ng microinjected unilaterally

into the hippocampal CA1 area on the locomoptor activity of sham-operated rats and bulbectomized rats

were examined. VIP injected into the left CA1 area suppressed the locomotor activity of sham-operated rats,

while the injection into the right CA1 area stimulated it. In the rats with OBX model of depression VIP de-

creased the number of movements when injected into the right CA1 area, while left-side administration fur-

ther stimulated locomotion. Our results suggest a shift in the lateralization of the locomotor effects of VIP

following bulbectomy which could be due to a reorganization of neuronal connections in the brain or to a

hemispheric asymmetry in the distribution of VIP or VIP receptors.

Key words: Vasoactive intestinal polypeptide, Olfactory bulbectomy, Hippocampus, Locomotor activity,

Lateralization, Rat

INTRODUCTION

Vasoactive Intestinal Peptide (VIP) is a 28 amino acids

polypeptide, which was first isolated from porcine small in-

testine (1). The neuropeptide is widely distributed in the

central and peripheral nervous system. VIP modulates

hippocampal synaptic transmission in a complex manner

involving multiple mechanisms (2). VIP has been sug-

gested to participate in the pathophysiology of neurological

and psychiatric disorders such as Parkinson's and Alzhei-

mer's diseases and depression. The reports on the lowered

plasma concentration of VIP in patients with anxiety and

depressive disorders as well as the lowered levels of VIP in

the cerebrospinal fluid of patients with depression suggest a

possible role of the peptide in the neurobiological

mechanisms of affective disorders (3,4,5).

The olfactory bulbectomy in rats is widely accepted as an an-

imal model of depression. The bilateral removal of bulbi

olfactorii produces a constellation of behavioral,

neurochemical and physiological alterations that resembles

human depressive disorders. It is considered that bulbectomy

causes a major dysfunction of the cortical-

hippocampal-amygdala circuit that underlies the behavioral

changes (6,7). The hippocampus is a brain structure with

high concentrations of VIP and VIP receptors. Together with

other structures of the limbic system it has been implicated in

the pathogenesis of major depressive disorder. Data are pres-

ent about the involvement of VIP in locomotion of rodents

but the role of the neuropertide in the hyperactivity exhibited

by OBX rats has not been examined.


The experiments were carried out on 36 male Wistar rats.

Experimental model of depression - bilateral olfactory

bulbectomy (OBX). Bilateral OBX was performed accord-

ing to the method described by Kelly et al., (6). Animals

were anesthetized with Calypsol (50 mg/kg i.p.) and placed

in a stereotaxic apparatus (Stoelting Co., USA). The surgi-

cal procedure involved drilling two burr holes 2 mm in di-

ameter at the points 8 mm anterior to bregma and 2 mm

from the midline on it's both sides (coordinates according to

the stereotaxic atlas of Pellegrino and Cushman (8). The

bulbs were aspirated with a stainless needle attached to a

water pump. After the surgery the rats were housed in

groups of two and were handled and weighed daily during

a 15 day period. The sham operation was performed in the

same way as in the case of olfactory bulbectomy without

the removal of the olfactory bulbs.

59



M. Ivanova, Dept. of Physiology and Pathophysiology,

Faculty of Medicine, Medical University of Varna

55 Marin Drinov St., Varna 9002, Bulgaria


Stereotaxic implantation and drug injection into

hippocampal CA1 area of OBX rats. After anesthesia

(Calypsol 50 mg/kg i.p.) the OBX rats were placed in the

stereotaxic apparatus and guide cannulae (right and left)

were implanted into CA1 hippocampal area according co-

ordinates to the stereotaxic atlas of Pellegrino and Cushman

(8). After surgery the animals were allowed 7 days to re-

cover before the behavioral studies. During the recovery

period the rats were handled daily.

Rats were microinjected into left or right hippocampal CA1

areas with VIP (100 ng) or with saline (1 ml). VIP (Sigma)

was dissolved ex tempore in saline and 1 ml of VIP solu-

tion (pH 7.4) was infused 5 minutes before the test for loco-

motor activity. Immediately prior to sacrifice rats were in-

jected with 1 ml 2% fast green dye through the injection

cannula. Brains were removed, and bulbectomy was veri-

fied macroscopically. Injection sites were verified anatomi-

cally in 25 mm coronal brain sections cut through the hip-

pocampus.

Test for locomotor activity. Locomotor activity was re-

corded in an Opto Varimex apparatus (Columbus Instru-

ments, USA) according to the method of Kohler and

Lorens (9). The apparatus records the number of photo

beam interruptions during the movements of the animal in

the experimental chamber. It provides selective counting of

the number of horizontal (ambulation) and vertical move-

ments (rearing) in arbitrary units (AU). The information

was recorded automatically for a 5 minute period of obser-

vation. The rats were placed in the central quadrant of the

activity monitor 5 min after the microinjection of VIP.

Statistical analysis. Data were processed by analysis of

variance (ANOVA). Separate ANOVA was used to pro-

cess the data obtained for the total number of movements

for a 5 minute period of VIP-treated sham-operated rats and

OBX rats. Where appropriate, ANOVA data were further

analysed by the t-test for post hoc group comparisons.

RESULTS

VIP (100 ng) administered into the left CA1 area of

sham-operated rats reduced the total number of horizontal

movements (t=4.63; P�0.001), while the microinjections

into the right CA1 area increased the total number of hori-

zontal movements (t=3.29; P�0.01) compared to the sa-

line-treated controls (Fig.1). VIP injected into the left CA1

area did not exert significant effect on the total number of

vertical movements; the microinjections into the right CA1

area significantly increased the total number of vertical

movements (t=6.57; P�0.001) compared to the saline

microinjections (Fig.2). VIP injected into the right CA1

area produced higher total number of both horizontal

movements (t=5.36; P�0,001) and vertical movements

(t=5.74; P�0.001) as compared to the left-side

microinjections (Fig.1, 2).

After microinjection of VIP into the right CA1 area OBX rats

showed adecrease in the total numberofhorizontal movements

(t = 3.03; P �0.01), as compared to the OBX saline-treated rats

(Fig.1). Microinjection of VIP into the left CA1 area did not af-

fect significantly horizontal activity of OBX rats (t=1.02; P -

N.S) (Fig.1).VIPinjected into the leftCA1areaofOBXrats in-

creased the total number of vertical movements (t=3.70; P�0.01) while upon administration into right CA1 area a decrease

in the number of vertical movements was observed (t=2.82; P�0.05) compared to the saline-treated OBX controls (Fig.2).

Right-sideVIPadministration inOBXratsproduced lower total

number of both horizontal movements (t=7.95; P�0.001) and

vertical movements (t=4.89; P�0.001) compared to the left

CA1 area (Fig.1,2).

60

Olfactory bulbectomy induces shifts in lateralization of ...

Fig 1. Effects of VIP (100 ng) injected unilaterally into

the hippocampal ÑÀ1 area of sham-operated rats and

OBX rats on the total number of horizontal movements

for a 5 minute period of observation. AU - arbitrary

units, **Ð £ 0.01; ***Ð £ 0.001-comparison to the

respective saline-injected controls. 000Ð ?

0.001-comparison of the number of movements after left

VIP injections vs. right VIP injections. Means (±S.E.M.)

are presented.

Fig 2. Effects of VIP (100 ng) injected unilaterally into

the hippocampal ÑÀ1 area of sham-operated rats and

OBX rats on the total number of vertical movements for

a 5 minute period of observation. AU - arbitrary units,

* Ð £ 0.05; ** Ð £ 0.01; ***Ð £ 0.001- comparison to

the respective saline-injected OBX controls.000Ð?

0.001-comparison of the number of movements after left

VIP injections vs. right VIP injections. Means (±

S.E.M.) are presented.

DISCUSSION

Òhe present study showed that olfactory bulbectomy in-

duced a shift in the lateralization of the locomotor effects of

VIP. Unilateral microinjection of VIP into hippocampal

CA1 area affected in an opposite way the locomotor activ-

ity of sham-operated rats and OBX rats, depending on the

side of injection. Left-side injection inhibited locomotion of

sham-operated rats while injection into the right CA1 stim-

ulated it. Hyperactivity (increased locomotor activity) in an

enclosed arena is a typical behavioural reaction of the OBX

rat; it is also accepted as an index of depressive-like behav-

ior in this animal model. VIP administration into the right

CA1 area inhibited the locomotor activity of OBX rats,

while VIP microinjection into the left CA1 area further

augmented the hyperactivity and aggravated the

depression-like state.

Literature data about central administration of VIP also

show opposite effects depending on the initial state of the

animal. VIP injected i.c.v. in rodents induced hyperactivity

(10,11). After lobectomy in rats, VIP (i.c.v.) showed a reg-

ulatory effect on locomotor activity and completely re-

stored the impairments in locomotion following the injury

(12). A correlation between locomotion and hippocampal

VIP was reported by Eilam et al. (13). Expression of VIP in

hippocampal rat neurons was increased after locomotor ac-

tivity, associated with an increase of VIP mRNA in both

the hippocampus and motor cortex.

The lateralized effect of VIP on locomotor activity of OBX

rats could be attributed to the hemispheric asymmetry in the

number of VIP interneurons or the density of the VIP re-

ceptors in the hippocampus; to the asymmetry in the pro-

jections from the hippocampus to other brain structures, in-

volved in the learning processes; as well as to asymmetry in

the neurotransmitter systems, with which VIP interacts.

Changes in the expression of neuropeptides in some brain

areas have been found following bulbectomy (7). We sup-

pose that the neurodegenerative changes in the hippocam-

pus after bulbectomy (14) and the following compensatory

neuronal reorganization could contribute to the opposite ef-

fects of VIP on locomotion. It is possible the asymmetry in

the expression of VIP or VIP receptors in the hippocampal

neurons to account for the observed shifts in the locomotor

effects of VIP after its administration into CA1 area.

CONCLUSION

VIP (100 ng) infused unilaterally into CA1 hippocampal

area of OBX rats produced opposite lateralized effects on the

locomotion of OBX rats and sham-operated rats. The shift in

the lateralization of the behavioral effect of VIP following

the olfactory bulbectomy could be due to a reorganization of

neuronal connections in the brain or to hemispheric asym-

metry in the distribution of VIP or VIP receptors.

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H e r n a n z , A . Plasma neuropeptides in affective

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olfactory bulbectomized rat as a model of depression:

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bulbectomized rat as model of depression. Neurosci

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and avoidance behavior following serotonin deple-

tion: A comparison of the effects of

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lesions. Pharmacol Biochem Behav. 1978; 8:223-233.

10. I t o h S , K a t s u u r a G , Y o s h i k a w a K .

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G l a z n e r G W , F o d e r a r o M A . Vasoactive

intestinal peptide: behavioral effects in the rat and

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latory effect of VIP on exploratory behavior of the rat

and on the neuromediator level in the normal brain

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Sechenova. 1989; 75:670–6.

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61

Ivanova M., S. Belcheva, I. Belcheva ...

LOW GRADE INFLAMMATION IN DIABETIC HYPERTENSIVE

INDIVIDUALS

Danovska M.1, M. Alexandrova

2

1Neurology Clinic, University Hospital "Dr G. Stranski" - Pleven,2Department of Biophysics, Medical University - Pleven

ABSTRACT

Individuals with hypertension and diabetes mellitus are at high risk of cerebrovascular and cardiovascular

morbidity and mortality. Such a combination of vascular risk factors has a multifactorial pathophysiology

but they are both recognized as pro-inflammatory conditions. The objective of the study was to test the hy-

pothesis that low grade systemic inflammation is present in hypertensive diabetic individuals. We examined 19

(8 male and 11 female) diabetic hypertensive individuals, median age 61 years, and 18 (7 male and 11 female)

healthy age-matched controls. Serum hs-CRP level (p=.000) and peripheral white blood cell count (p=0.049) were

found to be significantly higher in hypertensive diabetics than in control subjects. There is increasing evidence

that some markers of low-grade inflammation are associated with future risk of atherosclerotic complications

and may serve as predictors of stroke and myocardial infarction. Knowing the complex functional interrela-

tionship between traditional vascular risk factors and low-grade inflammation may help achieve primary

prophylaxis of cerebrovascular and cardiovascular events. Future studies should address the therapeutic po-

tential of inflammatory markers reduction for successful vascular protection in high-risk patients.

Key words: diabetes, hypertension, stroke, inflammation, CRP

INTRODUCTION

Arterial hypertension and diabetes mellitus play crucial role

in the etiology of atherosclerosis and atherosclerotic compli-

cations. The combination of arterial hypertension and ath-

erosclerosis as vascular risk factors accounts for a large pro-

portion of cardiovascular and cerebrovascular morbidity and

mortality. Recent advances on the role of inflammation in

mediating all stages of atherosclerosis provide important in-

formation about the complex interrelations between the vas-

cular risk factors and the complex mechanisms of

atherogenesis (5). Low grade inflammation indicated by the

levels of some inflammatory markers is an important predic-

tor of future coronary or cerebral vascular events, especially

in patients with arterial hypertension and diabetes mellitus,

both recognized as pro-inflammatory conditions (2). Despite

increasing experimental evidence that hs-CRP (high-sensi-

tivity C-reactive protein) level is a useful marker in the as-

sessment of vascular risk, its routine measurement in clinical

practice has not gained enough popularity yet.

The objective of the present study was to measure the levels of

some inflammatory markers indicating low grade inflamma-

tion in diabetic hypertensive individuals and to compare the

resultswith thoseobtained fromage-matchedcontrol subjects.


We studied 19 (8 male and 11 female) diabetic hyperten-

sive individuals, median age 63 (59-67) years (mean value

(25-th - 75-th percentile)) è 18 (7 male and 11 female),

age-matched healthy controls. In the study were not in-

cluded patients with acute or chronic infection, coexisting

liver, kidney or cancer diseases and those who had surgical

intervention. A fasting venous blood was collected and leu-

kocyte count was measured using standard biochemical

analysis. Concentrations of hs-CRP were measured by

immunoturbidimetric assay on Cobas Integra 400 with

CRP LT 300T (Roche Diagnostic).

All experiments were conducted in accordance with the

rules and regulations approved by the University Research

Ethics Committee. Inform consent was obtained by all the

patients or by their authorized relatives.

The statistical analysis was performed with the Statistical

Package of Social Sciences 19.0 (SPSS Inc., Chicago, IL).

As data were not normally distributed the significance of

differences between the patient groups was assessed with

the Mann-Whitney test. A value of p<0.05 was considered

statistically significant.

RESULTS

We found increased white blood cell count (Z=-1.963,

p=0.049) (Fig 1) and significantly higher levels of hs-ÑRP

63


(Z=-3.723, p=0.000) (Fig.2) in patients with arterial hyper-

tension and diabetes mellitus than in age-matched control

subjects (without history of diabetes and arterial

hypertension).

DISCUSSION

Inflammation plays a fundamental role in atherogenesis (5).

Evidence from experimental and clinical studies implicates

inflammatory mechanisms in the initiation and progression

of atherosclerosis associated with stroke and myocardial in-

farction (4). Furthermore, data exists that some inflamma-

tory parameters even modify the risk of atherosclerotic

complications regardless of presence of traditional risk fac-

tors (8). According to Saito et al (10) markers of inflamma-

tion such as CRP and leukocyte count are strongly associ-

ated with an increased risk of atherosclerotic complications

and might predict future myocardial infarction or stroke. At

the moment the complex interrelations between inflamma-

tory markers and some vascular risk factors like arterial hy-

pertension and diabetes are not completely understood (1),

and the sources of persistent low grade inflammation are

not well defined.

For example, Angiotensin II could be responsible in part

for the mediation of vascular inflammation, triggering oxi-

dative stress, up-regulation of pro-inflammatory transcrip-

tion factors that regulate the generation of inflammatory

mediators and lead to endothelial dysfunction and vascular

damage (11). Recently, the so-called acute phase reactants

have been studied as potential markers of persisting or

ongoing subclinical systemic vascular alterations (2).

Our findings suggest the existence of increased immune sta-

tus in diabetic hypertensive individuals as judged from en-

hanced white blood cell count (p=0.049) and significantly

higher serum hs-CRP levels (ð=0,000) as compared with the

control group. The obtained data are in accordance with the

results from experimental and clinical studies that have also

established the presence of low grade inflammation in pa-

tients with arterial hypertension and diabetes (7,11).

Vascular risk factors impair endothelium causing endothelial

dysfunction and trigger inflammation resulting in elevated

concentrations of CRP, chemokines and adhesion mole-

cules. However, it is still unknown whether these raised in-

flammatory parameters are only markers of low grade in-

flammation or active contributors to development and pro-

gression of atherosclerotic complications (3). Data have been

published that the nonspecific acute phase reactant CRP is

directly involved in the progression of vascular damage by

connecting and activating complement, inducing adhesion

molecules expression, mediating LDL capture by endothe-

lial macrophages and inducing accumulation of monocytes

in the arterial wall (3,9).

According to some published data, increased inflammatory

marker levels in diabetic hypertensive individuals are asso-

ciated with ongoing vascular pathology and predict in-

creased risk of vascular events (1,11).

Future prospective large population-based studies should

confirm the presence of increased immune state in hyper-

tensive diabetics. Conducting such studies will improve the

basic knowledge on the complex interrelations between

vascular risk factors and low grade inflammation responsi-

ble for progressive vascular damage. New scientific

achievements will answer to the question of how the in-

creased immune state could serve as additional marker for

cardiovascular and cerebrovascular risk prediction. Fur-

thermore, the potential involvement of inflammatory mark-

ers in the pathogenesis of vascular complications calls for

debate over use of anti-inflammatory treatment in the com-

plex therapeutic approach for primary prophylaxis of future

coronary and cerebral vascular events in high-risk patients

as diabetic hypertensive individuals.

CONCLUSIONS

Our study results show increased immune response in pa-

tients with arterial hypertension and diabetes mellitus. Future

studies should confirm the role of inflammatory markers in

64

Olfactory bulbectomy induces shifts in lateralization of ...

Fig.1. White blood cell count of hypertensive diabetics

and control subjects

Fig.2. Serum hs-CRP in hypertensive diabetics and

control subjects

vascular risk prediction and prevention of cerebrovascular

and cardiovascular morbidity and mortality.

REFERENCE

1. Anand A, Manikandan R, Begum T, Kumar

K, Akilandeswari G, Jer l ine M, Kumar P.

Hypertension, diabetes, metabolic syndrome, inflam-

mation and the risk of stroke. Int J Biol Med Res.

2011; 2(1): 429-432

2. Danesh J, Whincup P, Walker M, Lennon

L, Thomson A, Appleby P, Gal l imore R,

Pepys M. Low grade inflammation and coronary

heart disease: prospective study and updated

meta-analyses. BMJ 2000; vol 321:199-204.

3. Di Napoli M, Papa F. Inflammation, hemostatic

factors and antithrombotic agents in relation to

long-term risk of new cardiovascular events in

first-ever ischemic stroke patients. Stroke 2002;

33:1763-71.

4. Elkind M. Inflammatory mechanisms of stroke.

Stroke 2010; 41: S3-S8.

5. Libby P, Ridker P. Inflammation and atheroscle-

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The American Journal of Medicine 2004; 116, Suppl

1: 9-16

6. Libby P, Ridker P, Maser i A. Inflammation

and atherosclerosis. Circulation 2002; 105:1135.

7. Lind L. Circulating markers of inflammation and

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203-14.

8. Lindsberg P, Grau A. Inflammation and infec-

tions as risk factors for ischemic stroke. Stroke 2003;

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9. Ridker PM, Silver town JD. Inflammation, ñ-re-

active protein, and atherothrombosis. J Periodontol.

2008; 79 (8 Suppl);1544-51.

10. Sai to M, Ishimit tsu T, Minami J, Ono H,

Ohrui M, Matsuoka H. Relations of plasma

high-sensitivity C-reactive protein to traditional risk

factors. Atherosclerosis 2003; 167 (1) :73-79.

11. Savoia C, Schiffr in E. Vascular inflammation in

hypertension and diabetes: molecular mechanisms and

therapeutic interventions. Clinical Science 2007; 112:

375-384; Doi; 10.1042/CS20060247.

65

Danovska M., M. Alexandrova

EARLY VEP WAVES ARE AFFECTED STRONGER

BY GRATING LENGTH THAN WIDTH

Mihaylova M. , I. Hristov, K. Racheva, Ts. Totev, D. Mitov

Institute of Neurobiology, Bulgarian Academy of Sciences

SUMMARY

Visually evoked potentials (VEPs) were recorded at 3 spatial frequencies (SFs), 1.45, 2.9 and 5.8 c/deg while

grating lengths and widths varied. Stimulus contrast was 3 times above the detection threshold at each SF. In

most of cases the amplitude of the first negative VEP wave, N1, increased to a greater extends with increasing

stimulus length than with increasing stimulus width. However, such a difference between the effects of the stim-

ulus length and width on the amplitude of the first positive VEP-wave, P1, was not observed. The results ob-

tained are neurophysiological correlate of the psychophysically revealed difference in effect of increasing

grating length and width (Foley et. al, 2007, Vision Research, 47, 85-107), indicating that the underlying mecha-

nisms are arrays of slightly elongated receptive fields with lengths and widths inversely proportional to the SF.

Key words: Contrast, VEP, Spatial frequency, Receptive fields

INTRODUCTION

Many attempts have been applied to determine

psychophysically receptive fields form and size of human grat-

ing detecting mechanisms. To this aim Gabor patterns of vary-

ing size and shape were used and the effect of stimulus size on

the detection threshold was studied (for a review see 1).

Our psychophysical results (3) as well as the data obtained

by Foley et. al., 2007 (1) showed greater effect of gratings

length on the contrast detection threshold in comparison

with the effect of the width. Moreover, our data revealed

that the difference in the effects is stronger at higher than at

lower spatial frequencies.

The aim of the present study was to examine the effects of

stimulus size on the cortical visually evoked potentials

(VEPs) at various stimulus spatial frequencies (SFs) and to

search for a neurophysiological correlate of

psychophysically revealed difference in the effect of the

grating length and width on the detection threshold.

METHODS

Stimulation Vertical Gabor patterns of varying lengths and

widths were presented at 3 SFs, 1.45, 2.9 and 5.8 cycles deg-1.

At SFs of 1.45 and 2.9 cycles deg-1 the stimulus standard devi-

ation (�x and �y) varied between 0.146 and 2.33 deg. At the

highestSF,used in theexperiment,5.8cyclesdeg-1 , �x and �y

varied between 0.0833 and 2.33 deg. Stimulus contrast was 3

times above the detection threshold, measured for each subject

at each SF and at the smallest values of the grating length and

width. Stimulus duration was 100 ms. Stimuli were generated

on a black & white monitor (phosphor P4) by electronics de-

signed in our laboratory. The frame rate was 60 Hz, the spatial

resolution was 640x480 pxls, and the mean luminance - 100

cd/m2. Viewing was binocular, with natural pupils, from a dis-

tance of 114 cm, at which the screen subtended 11.6/8.7 deg of

visual angleat theeyes.Thestimuli alwaysappeared in thecen-

ter of the screen and small fixation lines were located along the

central horizontal line at the distance of 3x� from the centre.

VEP recording Stimuli of a given combination of SF, length

and width were presented in a block in which the

interstimulus interval was randomly varied between 1900

and 2700 ms. VEPs were recorded fromOz, O7 and O8. The

reference electrodes were positioned on both mastoids and

an Fp electrode served as a ground. The signal was ampli-

fied, band-pass filtered (0.3-70 Hz) and fed into a computer

at a sampling interval of 2 ms. Sweeps started 500 ms before

grating onset and lasted for 1500 ms. Four emmetropic

right-handed (5) observers participated in the experiment.


The three groups of traces were recorded at 1.45, 2.9 and

5.8 cycles deg-1 at various combinations of lengths and

widths. Early VEPs consist of negative-positive complex

which is characteristic for the responses to grating stimuli,

central fixation and registration at Oz (e.g. 2,4,6). It could

be seen that the N1 amplitude increases with increasing of

the stimulus size.

It might be seen at Fig. 2 that the stimulus length affects

VEP amplitude at Oz in a greater extent than the width.

When SF was 5.8 cycles deg-1, the VEP amplitude function

increased with length increase up to values of 0.29-0.58

67



M. Mihailova, Institute of Neurobiology, Bulgarian Academy of Sci-

ences, Acad. G. Bonchev Street, bl. 23, 1113 Sofia, Bulgaria,


deg. It might be also seen that increasing of the stimulus

width initially decreases N1 amplitude and than increases

it. At medium SF, 2.9 cycles deg-1, N1 amplitude in re-

sponse to small patterns increases with both stimulus length

and width Increase up to 0.58-1.17 deg. Contrary, for the

largest patterns N1 amplitude only slightly varies and even

decreases with increasing stimulus length and width. When

stimulus SF is low, 1.45 cycles deg-1, the response ampli-

68

Early vep waves are affected stronger by grating length than width

Fig. 1. Onset VEPs, recorded from Oz, at three SFs: 1.45 (left panel), 2.9 (middle panel) and 5.8 (right panel) cycles

deg-1 and various lengths and widths. Grating size is shown on the right of each trace. Subject LS.

69

Mihaylova M. , I. Hristov, K. Racheva ...

Fig. 2. VEP amplitudes of the earliest VEP wave recorded from Oz, N1, as functions of stimulus length, �y (the blue

lines and symbols) and stimulus width, �x (the red lines and symbols) at three SFs: 1.45 (left panel), 2.9 (middle panel)

and 5.8 (right panel) cycles deg-1. Subject LS.

tudes increase steadily up to the largest stimulus size avail-

able and show no signs of saturation.

VEP responses recorded at lateral occipital positions O7

and O8 (shown on Fig. 3) do not show significant differ-

70


Fig. 3. VEP amplitudes of N1, recorded at O8, as functions of stimulus length, �y (the blue lines and symbols) and

stimulus width, �x (the red lines and symbols) at three SFs: 1.45 (left panel), 2.9 (middle panel) and 5.8 (right panel)

cycles deg-1. Subject LS.

ence in effects of grating length and width on the VEP am-

plitude, except at 1.45 c/deg. This observation may imply

that the mechanism contributing to the different effect of

stimulus length and width is located rather in the striate cor-

tex than in the extrastiate visual cortex.

Contrary to N1, the second VEP wave, which is of positive po-

larity, P1, do not show significantly different effects of stimulus

length and width on its amplitude, as could be seen at Fig.4.

This observation may imply that the underlying mechanism is

involved predominantly in the initial part of the response.

71

Mihaylova M. , I. Hristov, K. Racheva ...

Fig. 4. VEP amplitudes of the second VEP wave, P1, recorded at Oz as functions of stimulus length, �y (the blue lines

and symbols) and stimulus width, �x (the red lines and symbols) at three SFs: 1.45 (left panel), 2.9 (middle panel) and

5.8 (right panel) cycles deg-1. Subject LS.

The stronger effect of stimulus length and width on the early

VEP wave amplitude obtained in the present experiment

support assumption (1) that the underlying mechanisms are

arrays of slightly elongated receptive fields. They are proba-

bly situated in the primary visual cortex, V1, and contribute

predominantly to the initial part of the VEP response.

The fact that the effect of the stimulus length and width on the

VEP amplitude is limited to different critical size at different

SFs might be interpreted as a consequence of the difference in

the size of the receptive fields tuned to different SFs.

REFERENCES

1. Foley JM, Varadharajan S, Koh ChC,

Far ias MCQ. Detection of Gabor patterns of differ-

ent sizes, shapes, phases and eccentricities. Vision Re-

search, 2007; 47: 85-107.

2. Jones, R. , & Keck, M. J. (1978). Visual evoked

response as a function of grating spatial frequency.

In6estigati6e Ophthalmology & Visual Science, 17,

652-659.

3. Mitov D, Totev Ts. Effects of grating width and

length on detection threshold. IX National Congress

of Bulgarian Society of physiological sciences, 9-11

November 2007, Blagoevgrad.

4. Musselwhi te , M. J. , & Jeffreys, D. A.

(1985). The influence of spatial frequency on the re-

action times and evoked potentials recorded to grating

pattern stimuli. Vision Research, 25, 1545-1555.

5. Oldf ield, R. C. (1971). The assessment and analy-

sis of handedness: The Edinburgh Inventory.

Neuropsychologia, 9, 97-113.

6. Vassi lev, A. , Stomonyakov, V. , &

Manahi lov, V. (1994). Spatial-frequency specific

contrast gain and flicker masking of human transient

VEP. Vision Research, 34, 863-872.

72


EVALUATION OF INDIVIDUAL, GENDER AND AGE DIFFERENCES

IN VISUAL MOTION PERCEPTION

Stefanova M.1, N. Bocheva

1, O. Georgieva

2

1Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia;2 Faculty of Mathematics and Informatics, Sofia University "St. Kl. Ohridski"

The aim of the present study is to explore the possibility to differentiate the age-related and the individual dif-

ferences in visual motion processing. Psychophysical experiments of motion direction discrimination using

the method of equivalent noise were performed. We applied two different analytical techniques: fuzzy cluster-

ing and mixed ANOVA to the data. The results suggest that the clustering methods allow better separation of

the gender and individual effects. Lowest sensitivity is observed for a group predominantly of older females,

while the highest sensitivity group contains mainly younger participants and few older males. The most dis-

tinct separation of the subjects in different groups based on their sensitivity to differences in motion direction

is observed in demanding tasks and difficult experimental conditions. The ANOVA provides a good descrip-

tion of the common trends in performance and the age-related effects. The combination of these analytical

methods offers new opportunities to use the data from the psychophysical studies to better describe the

changes in the cognitive processes in different experiment conditions and could be used as a potential diagnos-

tic tool for deviant behavior.

Key words: Motion discrimination, Ageing, Fuzzy clustering, Mixed ANOVA

INTRODUCTION

Many cognitive abilities decline with age, but ageing is ac-

companied by great variability within older population. Most

studies investigate the age-related changes in cognitive pro-

cesses by comparing the performance of participants from

two age groups (1,5) or by the correlation between different

behavioral measures and age (3). These analyses provide

valuable information about the factors that affect the age-re-

lated changes, but the individual differences among the sub-

jects are undermined. For this reason it is important to seek

for methods to evaluate the individual differences in order to

determine behavioral norms and to distinguish normal from

pathological ageing. The psychophysical studies have the

potential not only to describe human performance but also,

when combined with other data, to be used as an indicator

for degenerative processes.

The aims of the present study are to suggest new data ana-

lytic strategies to assess the individual differences between

the participants in psychophysical experiments and to de-

termine the most appropriate conditions for evaluation of

these differences. In order to achieve this goal we selected

four experiments from our dataset of studies on visual per-

ception of dynamic information. The experiments focus on

the age-related changes in the sensitivity to motion direc-

tion of dynamic noisy stimuli. This study is a first attempt to

apply clustering analysis to differentiate effects of age, gen-

der and individual differences on performance of

behavioral tasks.

METHODS

Subjects: Twelve younger subjects (mean age 19.5 yrs.,

range 16-24 yrs., six male) and 12 older subjects (mean age

73.9 yrs., range 66-82 yrs, four male) participated in the ex-

periments.

Stimuli: The stimuli consisted of 50 frame movie sequences

with spatially band-pass elements. They moved in circular

aperture with radius of 7.0 deg, positioned at the middle of

the computer screen. In Experiments 1 and 3 the moving el-

ements were 32, whereas in Experiments 2 and 4 they were

128. If any element left the aperture during its motion, it

re-appeared from the opposite side of the aperture in order

to keep the number constant. In Experiments 1 and 2 the

mean speed of motion was 6.64 deg/s, while in Experi-

ments 3 and the 4 the mean speed was 18.6 deg/sec. The

motion direction of each element was taken randomly from

a normal distribution of different spread. Six different val-

ues of the spread were used: 2°, 5°, 10°, 15°, 25°, and 35°.

They determined the level of external directional noise.

Procedure: The task of the observers was to indicate

whether the mean direction of motion appeared to the left

or to the right of vertical downward motion. An adaptive

QUEST (6) algorithm estimated the angular deviation of

the mean direction from the vertical to be presented on the

next trial.

Statistical analyses: A between-within subjects ANOVA

(mixed ANOVA) was applied to the log transformed

73



M. Stefanova, Sofia-1113, st. "Acad. G. Bonchev", bl.23, Institute of

Neurobiology


thresholds obtained in each experiment. A post-hoc Tukey

HSD test was applied to the results of ANOVA with fac-

tors: noise and subjects to divide the participants in

homogeneous groups.

Clustering algorithm: A fuzzy clustering technique that

finds not well separated data groups with vague and uncer-

tain boundaries was used. The clusters are described by

their centre. In the simplest case, this is a point in the data

space that is most representative for the cluster in probabil-

istic sense. Every point of the data space belongs to the dis-

tinct clusters with different degree of membership - a value

between 0 and 1, such that if the data is close to the cluster

centre, the membership degree is closer to 1.

The widespread Fuzzy-C-Means (FCM) algorithm (2) was

applied. It is an objective function-based algorithm with

clustering criteria defined by the Euclidean distance of the

data point from a cluster center, by a predefined number of

clusters (c) and degree of their overlap. As the number of

data N=24 in our study is relatively small, meaningful parti-

tion could be expected for clustering in no more than two or

three clusters.


A mixed model ANOVA with age and gender as be-

tween-group factors and dot number, speed and noise level

as within-group factors was applied to the data. The results

show significant effect of age (F(1,20)=6.84, p<0.05) due

to the higher discrimination thresholds and thus, to the

lower sensitivity to motion direction of older subjects. The

noise level had significant effect on the performance; the

thresholds increased with the increase in the noise levels

(F(5,16) = 20.92; p<0.05). The dot number also had signifi-

cant effect on the performance- (F(1,20)=12.5; p<0.05).

The sensitivity to the global direction of the moving stimu-

lus improves when the number of dots increases. The inter-

action between the speed of motion and the dot number

was significant (F(1,20)=9.19; p<0.05) due to the improved

sensitivity when the speed and the number of dots in-

creased. These results suggest that the sensitivity to motion

direction depends in different way on the spatial and the

temporal characteristics of the motion sequences, espe-

cially on the magnitude of speed. The interaction between

dot number and noise was also significant (F(5,16)=3.93;

p<0.05).

The lack of interaction between the age group and any of

the other experimental factors implies that the temporal and

the spatial characteristics of the moving patterns affect the

two age groups in a similar way, the only difference be-

tween the two groups being the lower sensitivity of the

older group. The insignificant interaction between the noise

level and the age group suggests that the reduced sensitivity

of the older observers is caused by an increase in the multi-

plicative noise in the visual system i.e. the noise that de-

pends on the strength of the signal (4). These results suggest

little effect of the gender on the sensitivity to motion direc-

tion, implying that based on their sensitivity to motion di-

rection, the subjects could be divided in two groups deter-

mined by their age. Figure 1 illustrates the effect of the

noise level on the mean thresholds in each experiment.

In the mixed ANOVA each subject serves as his/hers own

control under the assumption of equal variance of the ex-

perimental groups at all factor levels and therefore, the vari-

ance should be independent of the magnitude of the noise

level. To achieve this, we have transformed the discrimina-

tion thresholds by a logarithmic transformation. In the clus-

ter analysis such restrictions are not obligatory and we seek

groupings based on the performance of the subjects using

the untransformed data.

The diversity within the trivial groups based on age and the

discrimination thresholds of the subjects in the different ex-

perimental conditions could be investigated by partition in

three clusters.

The common trend in the data, observed in most of the ex-

perimental conditions, is the splitting of the older group

(Figure 2). This corresponds to the complexity of the age-

ing process and its dependence on multitude of factors that

leads to larger individual differences at older age. However

this tendency is not equally expressed and depends on the

spatial and motion parameters of the stimulation. When the

speed of motion is high (i.e. in Experiments 3 and 4) the

younger observers also show a tendency to split in two

groups in some conditions. The splitting of the data for ei-

ther age group, however, does not seem systematically

related to the noise level.

Characterization of the individual differences: To evaluate the

individual differences in task performance an ANOVA with

factors:noiseandsubject (regardedasa randomfactor),wasap-

plied to the discrimination thresholds of the subjects. A post hoc

Tukey HSD test was used to divide the observers in homoge-

neous groups based on their sensitivity to motion direction.

These groups include subjects of different age and gender.

However, the groups strongly overlap and we have no good

measure to affiliate a subject to a specific group. The minimal

number of homogenous groups we have obtained by the

post-hoc Tukey HSD test is seven - a large number for a group

of only 24 subjects that took part in our experiments. Hence, the

74

Evaluation of individual, gender and age differences in ...

Fig.1. Effect of the noise level (on the abscissa) on

differential threshold (tm). The error-bars represent one

standard error.

possibility to describe the individual differences between the

subjects by applying post-hoc tests is limited. Moreover, the

measurements for each subject are few, and the violation of the

ANOVA assumptions will have greater impact on the results.

For this reason, we have applied cluster analysis to the data.

Information about the individual differences between the

subjects is searched by clustering in a higher dimensional

data space, defined by the noise level values, independently

of the subjects' age and gender. When we have three clus-

ters centers, the participants are divided in a group of high

sensitivity (H) that corresponds to low thresholds values, a

group of medium (Md) sensitivity and a low sensitivity

group (L) that corresponds to high thresholds values. The

results show that:

a) The cluster H of the low tm values contains mainly

younger people and a few older ones;

b) The medium cluster Md associates female and

male older subjects and few young females;

c) The cluster L is formed predominantly by the older

females.

The level of association of a person to every cluster is pre-

sented in Figure 4. It could be seen that cluster H is more

compact than the others, which suggests small variation in

the sensitivity to motion direction of the members of this

cluster. As the others two groups predominantly associate

older people, this again confirms the higher variability of

the older group in comparison with the younger one. The

membership to a cluster is also better defined in Experi-

ments 3 and 4, where the speed of motion is high.

We have explored whether the noise level affects the dis-

tinctness of the clusters. The general observation is that the

clusters overlap more for small noise levels as well as for

Experiments 1 and 2, whereas they are more distinguish-

able for large levels of external noise and high speeds (i.e.

in Experiments 3 and 4). The clustering data suggest that

the level of external noise has little effect on the perfor-

mance for the participants in the high sensitivity group. The

cluster of the low sensitivity group depends strongly on the

external noise. In most experimental conditions single sub-

jects belong to this group, suggesting that they significantly

deviate in their performance from the rest of the subjects.

Experiment 4 allows the most distinct separation of the

subjects in different groups based on their sensitivity to mo-

tion direction. It provides the opportunity to classify reli-

ably new subjects to the existing groups and to treat the

grouping as a norm for characterizing people. The both the

speed of motion and the dot density in this experiment was

higher which imply that clustering data in these experimen-

tal conditions are related to the ability of the subjects to pool

the information about motion direction in space and time.

CONCLUSIONS

Our study is a first attempt to apply clustering algorithms to

differentiate the effects of age, gender and individual differ-

ences on performance of a behavioral task. The interpreta-

tion of the clustering results allows detecting the deviation

level of a subject from the respective age group. Whether

this is related to degenerative processes or not, could not be

determined only by the results of the present study; it re-

quires tracking the changes in the cognitive abilities of the

participants in longitudinal studies or a combination of the

75

Kolev N., L. Halacheva

Fig.2. Clustering results for c=3. Asterisks- cluster

centre; triangles – males; circles – females.

Fig. 3. The distance of the individual discrimination

thresholds from the cluster centres. Each point

represents the data of a single subject. The order of the

subjects in each plot is the same. Triangles – male;

circles – female; dashed line – older participants

data with other neuro-physiological measures of cognitive

aging. However, our approach provides opportunities to

use psychophysical methods for early diagnostics of the de-

terioration in the cognitive abilities of the individual with

age. The results of the cluster analysis suggest also, that it is

easier to detect the individual deviations in demanding

tasks and difficult experimental conditions. This makes

them a useful tool to characterize the process of ageing and

to seek association between the behavioral data and the

physiological changes in the brain.

ACKNOWLEDGEMENTS

This work was supported by grant TK01-200 of the Na-

tional Science Fund, Bulgaria.

REFERENCES

1. Berard, J .R. , Fung, J . , McFadyen, B.J . &

Lamontagne, A. , 2009. Aging affects the ability to

use optic flow in the control of heading during loco-

motion. Experimental Brain Research, 194(2):

183-190.

2. Bezdek, J . C. , 1981. Pattern Recognition with

Fuzzy Objective Function Algorithms. Plenum Press.

New York.

3. Bi l l ino, J . , Bremmer, F. , & Gegenfurtner ,

K. R. , 2008. Differential aging of motion processing

mechanisms: Evidence against general perceptual de-

cline. Vision Research, 48: 1254–1261.

4. Lu, Z.-L. , Dosher , B.A. 2009. Mechanisms of

Perceptual Learning. Learning and Perception, 1:

19–36.

5. Pi lz , K.S, Bennet t . P .J . & Sekuler , A. B. ,

2010. Effects of aging on biological motion discrimi-

nation. Vision Research, 50(2): 211-219

6. Watson, A. B. & Pell i , D. G. , 1983. QUEST: a

Bayesian adaptive psychometric method. Perception

& Psychophysics, 33(2): 113-20.

76

Evaluation of individual, gender and age differences in ...

SEX DIFFERENCES IN FUNCTIONAL ORGANIZATION

OF BIMANUAL SHOT-LIKE ISOMETRIC HANDGRIP


Department of Physiology and Pathophysiology, Medical University - Pleven

ABSTRACT

The aim of this study was to examine isometric force production during ballistic isometric handgrip in unilat-

eral (UL) and bilateral (BL) tasks in men and women. Ten right-handed young men and 12 young women

were studied. The subject was instructed after a command to perform a shot-like handgrip, raising maximal

force as fast as possible. Three series were performed as follows: 1) UL - right handgrip; 2) UL - left handgrip;

3) BL- handgrip. We measured peak force, time to peak of force and force rate. The BL/UL ratios for the right

(R) and left (L) hand and L/R ratios for the UL and BL tasks were calculated. The peak force and force rate

were smaller in women compared with men (p<0,01), indicating less forceful and powerful handgrip in

women. The L/R ratios for peak force and force rate ranged across all tasks and conditions from 0.71 to 0,75 in

both groups, suggesting that these measures indicate the handedness. The L/R ratios for time to peak of force

showed a functional symmetry in both groups. The BL/UL ratios for peak force were in men 0,89 and 0,89;

women - 0,91 and 0,92 for the R and L hand, respectively. These data suggested that symmetrical bilateral

strength deficit (BD) is present in men and women. These ratios for time force indicated asymmetrical BD in

women.

Key words: Handgrip, muscle force, bilateral deficit, sex differences

INTRODUCTION

Bilateral hand use occurs at least 24 percent of each day,

every day (5). With 26 muscles and 27 bones in each hand

and wrist structure (4), bimanual grasp force production is a

complex task. Many investigators have reported a reduc-

tion in maximal voluntary strength induced by simulta-

neous bilateral (BL) exertion as compared with the sum of

left and right unilateral (UL) limb actions (9,19). This "bi-

lateral limb deficit" (BD) has been observed in both upper

(17,20) and lower limbs (12,14). It was found to depend on

the type and speed of the muscle contraction (13), joint po-

sition (15), training (10) and age (8). The bilateral deficit of

voluntary force varied from 3 to 25% of maximal voluntary

contraction (2). The mechanism responsible for the bilat-

eral deficit is currently unknown. However, Jakobi and

Cafareli (11) found no bilateral deficit in the group of un-

trained young males which finding suggests that maturity

of the subjects investigated is important as well. Further-

more, a left/right asymmetry of bilateral deficit was first re-

ported (17) and thereafter was not confirmed (18) which

controversy might be due to the sample differences related

to age and training. Unfortunately, a little experimental in-

formation is available on sex differences of bilateral deficit

(16).

Our goal was to test the functional organization of maximal

force production during shot-like isometric handgrip in

young men and women. In addition to the traditionally used

measure peak of force we used as measures also the time to

peak of force and the rate of force.

METHODS

We examined isometric force production during shot-like

handgrip in unilateral and bilateral tasks. Ten healthy

right-handed males and 12 healthy right-handed females

were studied (Tabl.1). Handedness was determined by

questioning the subjects (1). The experimental subject

seated in an armchair with arms along the body, forearms

were flexed (90 deg elbow flexion) and semipronated and

77



N. Kolev, Dept. of Physiology and Pathophysiology, Medical Uni-

versity - Pleven, 1, Sv. Kliment Ohridski Str.

5800 Pleven, Bulgaria


Group nAge

(years)

Height

(cm)

Weight

(kg)BMI

Females 12 19,7 � 1,4163,2 �

6,857,3 � 7,1

21,49 �2,2

Males 10 20,3 � ��181,7 �

6,3

78,3 �

10,6

23,67 �2,5

Table 1. Group mean values � SD for age, sex, height,

weight and Body Mass Index.

wrists were supported. The subject was instructed after a

command to make a shot-like handgrip, raising maximal

force as fast as possible and to act only with forearm and

hand flexors and not to include elbow flexors and shoulder

muscles. Three series in random order were done as fol-

lows: 1) unilateral (UL) right handgrip; 2) UL left

handgrip; 3) bilateral (BL) handgrip.

We measured peak force and time to peak of force. The

force of rate was calculated (Fig. 1. Shot-like handgrip).

The BL/UL ratios for the right (R) and left (L) hand and the

L/R ratios for the UL and BL tasks were calculated.

An 1 - sample t test against 1 was used to determine if the

average ratio for each task was significantly different than

1.The main effects of the gander (men and women) as well

of hand (R and L) or task (UL and BL) were evaluated by

ANOVA. In each case significance was accepted at p <

0,05.


The group of the women showed significantly smaller peak

of force and force rate (Tabl. 2.) for all tasks and conditions

compared with men (p<0,01). These results indicate less

forceful and powerful handgrip contraction in females com-

pared with males that is in line with the findings that the cross

- sectional area of muscle and single fiber levels were larger

in men than in women (3,6). For time to peak of force, there

was no significant difference between the two sexes.

The L/R ratios (Fig. 2. Handedness - L/R ratios.) for peak

force and force rate ranged across all tasks and conditions

from 0,71 to 0,75 in both groups. They were significantly

lower than 1 (p<0,01), suggesting that these measures indi-

cate the handedness and similar levels of hemisphere domi-

nance in both sexes. The L/R ratios for time to peak of force

ranged across 1,0 to 1,04 in both groups. They did not differ

significantly from 1 suggesting a functional bilateral

symmetry for this measure.

The BL/UL ratios (Fig. 3. Bilateral deficit - BL/UL ratios.)

for peak force were in women - 0,91 and 0,92 and in men -

0,89 and 0,89, respectively for R and L hand. They were

significantly lower than 1 and showed a symmetrical bilat-

eral deficit (BD) in both sexses. There were no significant

differences between the groups. These data supported the

finding that no gander effect has been observed in the BD

during isometric elbow flexion (16). Also, it has been re-

ported that the degree of decrease of the maximal bilateral

grip strength was higher for the dominant hand in both

sexes (19). Our previous study (7) showed a symmetrical

BD in groups of male adults and adolescent oarsmen and

an asymmetrical BD in a group of untrained male adoles-

cents. The finding in the present study confirms our hy-

pothesis that the asymmetry of BD is not established phe-

nomenon (17,18), dependent on age and training. The

BL/UL ratios for time to peak of force in the group of the

women were significantly closer to 1 for L hand - 0,97 (NS)

compared to R hand - 0,94 (p<0,05) suggesting an asym-

metry of bilateral deficit in females. These ratios were in

78

Sex differences in functional organization of ...

GROUPPEAK FORCE (N) TIME TO PEAK FORCE (ms) FORCE RATE (N. s-1)

LUL LBL RUL RBL LUL LBL RUL RBL LUL LBL RUL RBL

Females196,6

� 14,5

180,2

� 13,7

260,8

� 16,6

239,3

� 17,2

197,3

� 7,0

189,3

± 7,1

197,0

� 9,2

184,9

� 8,9

994,3

� 59,1

949,0

� 55,8

1329,5

� 63,8

1290,2

± 54,2

Males267,6

� 18,2

240,7

� 20,0

370,6

� 32,3

331,5

� 32,6

196,9

� 6,4

181,8

� 8,9

190,2

� 9,1

180,2

� 8,3

1350

� 61,8

1309

� 63,1

1922,2

� 93

1810,7

� 106,4

Table 2. Group mean values � SE for Peak force, Time to peak force and Force rate

Fig.1

Fig.2

men significantly lower than 1 (p < 0,05) - 0,92 and 0,95,

respectively for R and L hand indicating a symmetrical BD

for this measure. The BL/UL ratios for the force rate did not

differ significantly from 1 in the group of the women - 0,97

and 0,96, respectively for R and L hand. These ratios in

men were significantly closer to 1 for the L hand - 0,97

(NS) compared to R hand 0,94 (p < 0,05) suggesting BD in

the dominant hand only.

CONCLUSIONS

The main finding of this study was that symmetrical bilateral

deficit for measure peak of force is present in both young

males and females and it is similar for both sexes. For the

time to peak force the bilateral deficit was symmetrical in the

men and asymmetrical in the women. For the rate of force bi-

lateral deficit was observed in the group of the men only, and

it was greater for the dominant hand. The results showed that

young men are stronger and more powerful than young

women for all tasks and conditions. There were no signifi-

cant differences for time to peak of force in both sexes. The

L/R ratios indicated that peak of force and rate of force are

indicative measures for the handedness. They suggested

equal levels of the handedness in both ganders.

REFERENCES

1. Annet M. A classification of hand preference by as-

sociation analisis. Br. J. Physiol., 1970; 61: 545-558.

2. Archont ides C. , J . A. Fazey. Inter-limb interac-

tions and constraints in the expression of maximum

force: A review, some implications and suggested

underlying mechanisms. J. Sports Sc.,1993;

11:145-158.

3. Brown R. E. , D .L. Edwards, J .M. Jakobi .

Sex differences in force steadiness in three positions

of the forearm. Eur. J. Appl. Physiol., 2010;

110:1251-1257.

4. Carmeli E. , H. Pat ish , R. Coleman. The ag-

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79


Fig.3

CHANGES IN THE PHYSICAL WORKING CAPACITY,

BODY COMPOSITION AND FAT METABOLISM

OF WOMEN ON A THREE-MONTH SPECIALIZED TRAINING

AND DIETARY PROGRAM

Angelova P.1, N. Boyadjiev

1, A. Ivanova

1, S. Muletarov

2

1 Department of Physiology;2 Department of Anatomy, Histology and Embryology, Medical University Plovdiv

ABSTRACT

A group of 15 healthy women (age 31.36±5.95 years, BMI 22.53±3.94) underwent a 3 month program with

specific training and diet, and took fat burning supplements for improving their physical working capacity

and for optimizing their body weight. A number of anthropometrical measurements (body mass, BMI, fat

mass, muscle mass), functional (VEpeak, Wpeak, VO2peak, HRpeak, O2pulse peak, recovery after

spiroergometry), metabolic (RQ, Wfat), blood (CHOL, HDL, TG) and sports tests were performed at the be-

ginning and at the end of the trial. In the third month the body fats were reduced from 25.76±8.86 to

25.27±8.38% (p<0.05), and the muscle mass was slightly increased. The functional examination established an

increase of the absolute (from 166.36±20.36 to 193.64±2.63 W, p<0.01), and relative (2.83±0.53 to 3.21±0.52

W/kg, p<0.05) work capacity. VO2peak (absolute and relative) and O2pulse peak (absolute and relative) were

also increased but not significantly. There was an improvement in heart rate recovery after step work load

(from grade 2.00±0.82 to grade 2.90±0.30, p<0.01). In the third month of the experiment the energy supply

was shifted to fat catabolism. The optimal loading for maximal adipolysis was decreased from 124.54±18.09 to

109.09±13.00 W (p<0.02). The sports tests determined an improvement in the physical efficiency and flexibil-

ity (from 14.73±4.92 to 16.59±3.95 cm, p<0.01) of the participants. In conclusion, in spite of the short duration

of the program, the specialized diet, supplements used and the training improved the capacity of fat burning,

the physical working capacity and the body composition of the female participants in the study.

Key words: exercise, physical working capacity, fat metabolism

INTRODUCTION

Contemporary people find themselves in a constant strife

for self-perfection and improvement of their lifestyle.

Threatening growth of obesity and related socially signifi-

cant diseases naturally lead to an ambition to optimize one's

body mass and increase the physical working capacity. Ef-

fective methods refer to a sustainable change of lifestyle,

proper diet and optimal physical activity. Aerobic training

programs have proven their efficacy both with young peo-

ple (improvement of working capacity) and with adults (9,

8,7,5). Controlled diet together with training sessions con-

tribute to improvement of anthropometric indices and

increase work capacity.

This study was aimed at investigating changes of certain

functional, anthropometric, clinical-and-laboratory values

and indicators of physical capacity following a three-month

specialized training and dietary program.


A group of 15 healthy women volunteers aged 31.36±5.95

with BMI 22.53±3.94 underwent a three-month program of

a specific motor and dietary regimen aimed at improving

physical capacity and optimizing body mass.

In the beginning and at the end of the program, anthropometric,

functional and clinical-and-laboratory examinations were per-

formed, as well as tests of aerobic capacity indicators.

TRAINING PROGRAM

Training sessions in Tae Bo (type of intensive aerobics with

martial art components) were carried out thrice-weekly with

a duration of 45-70 min according to the increasing physical

potential of the participants. Choreography consisted of se-

ries of punches and kicks, and aerobic steps set rhythmic mu-

sic. The basic complex at the start of the sessions was made

more and more complicated by adding more complex coor-

dination movements, and increasing music tempo, and dura-

tion, and also by changing the manner of performance from

81


Low impact to High impact, number of repetitions of aerobic

combinations and patterns (2, 3,4). Due to the high intensity

of performed combinations, in aerobics a mixed type of en-

ergy supply was predominant including both aerobic and

anaerobic paths for the production of ATP in muscles (6).

DIETARY PROGRAM

The participants were on a four meal food regimen providing

up to 2500 kcal/24 h, distributed in the following way: 1st

breakfast -20 % energy intake; 2nd breakfast -10 % ei.; lunch -

45 % ei.; dinner - 25 % ei. The last meal for the day was taken

at 18:30 h. Intake of salt, refined sugar, and cholesterol (<200

mg/24h)was reduced, and intake of fatswasup to 25-30 %, as

instructions for avoidance of fatty acids were followed. Given

the increased energy expenditure and the desire for enhance-

ment of muscle strength and endurance, a controlled protein

intake was introduced which was provided by protein blocks

and reduced to 1.5 g/êg/24 h.

All participants took L-carnitine (L-carnitine tartrate) and

Fat burner in dose 3x500 mg for both preparations (Neuber,

Austria). (Table 1)

The following anthropometric indices (TANITA BC-418,

Japan) were monitored twice: body mass (kg) and BMI

(kg/m2), percentage of body fats, percentage of water con-

tent, percentage of visceral fats, muscular and bone mass

(kg), 6 skin flaps: of m. triceps brachii, subscapular, iliac,

abdominal, of Õ-rib, and femoral (mm) measured by

caliperometry (Harpenden, Croatia).

Physical working capacity was also evaluated twice using

spiroergometry tests of the system SCHILLER AT104

SpiroErgo (Switzerland). The functional test began with syn-

chronization of measurement - without loading and with nor-

mal breathing for about 40 s, followed by a period of warm-

ing up for 1-3 minutes, and effective loading with a

step-by-step increase of 30W every two minutes.The recov-

ery period proceded with 10% of peak loading for a mini-

mum of five minutes. Spiroergometry tests were carried out

without a training session or heavy physical load during the

preceding day. Indices traced included peak ergometric

working capacity (Wpeak), relative peak working capacity

(Wpeak/kg), peak oxygen consumption (VO2peak), relative

peak oxygen consumption (VO2peak/kg), absolute and rela-

tive peak oxygen pulse (mlO2/bt/kg), maximum heart beat at

Wpeak (bt/min), and recovery of heart rhythm after

step-by-step loading, which was getting higher, from 0 by

30W, every 2 minutes until participant rejection.

RQ at rest and during peak loading was determined. Opti-

mal loading (W) and heart rhythm (bt/min) were also deter-

mined, where work was suported in energy terms mainly at

the expense of fat burning.

Clinical-and-laboratory examinations included initial and

final levels of cholesterol (mmol/l), HDL (mmol/l), triglyc-

erides (mmol/l), and blood sugar (mmol/l). OGTT was pre-

liminary performed for the purpose of monitoring the glu-

cose tolerance of the participants.

In the beginning and at the end of study standard

sports-and-pedagogic tests were carried out (pushups, ab-

dominal presses, rising dynamometry), as well as long

jump, and coordination and flexibility tests (1).

Results were presented as X±SD. A two-way ANOVA

t-test (STATISTICA v. 6.0, StatSoft, U.S.A.) was per-

formed to determine the authenticity of variance between

the value of indices which were measured twice. A vari-

ance at Ð<0.05 was assumed as significant.

RESULTS AND DISCUSSIONS

At the end of the period the percentage of body fats de-

creased from 25.76±8.86 to 25.27±8.38% (Ð<0.05), mus-

cular mass increased from 43.20±3,07 to 43.37±3.05kg

(Ð>0.05). Body mass and BMI also decreased, but with no

statistical significance.

82

Changes in the physical working capacity, body composition ...

Vitamin B1

(Thiaminmononitrate)0.80 mg

Vitamin B2 (Riboflavin) 0.88 mg

Vitamin B6

(Pyridoxinhydrochloride)1.35 mg

Vitamin B12 (Cyancobalamine

0.1%)150.0 µg

Folic acid 132.0 µg

Calcium-D-Pantothenate 3.69 mg

Nicotinamide 9.97 mg NE

Chronicum chelate 2.5% 555 µg

Inositol NF 12 230.77 mg

L-carnitinå tartrate 230.77 mg

L-Lysine 230.77 mg

L-Methionine 92.35 mg

Choline bitartrate 461.54 mg

Chitosan 230.77 mg

Table 1. Composition of daily dose of Fat burner

(Neuber, Austria).

Index BMI (kg/m2)

Muscular

mass (kg)Body fats (%)

Start 22.52±3.95 43.20±3.07 25.76±8.68

End 22.23±4.56 43.37±3.05 25.27±8.38

Variance

authenticityP>0.05 P>0.05 P<0.05

Table 2. Change of body composition prior to and after

completion of the program (X±SD).

Thus, an overall improvement of body composition after a

three-month program was demonstrated (Table 2).

Functional examinations showed an increase of absolute

peak working capacity (from 166.36±20.63 to

193.64±20.63W, Ð<0.01), and of a relative peak working

capacity (from 2.83±0.53 to 3.21±0.52W/kg, Ð<0.05).

Peak oxygen consumption (absolute and relative) also in-

creased, but with no statistical significance. The same ten-

dency was also observed with the absolute (mlO2/bt) and

relative oxygen (mlO2/bt/kg) pulse of the participants.

Maximum heart frequency at Wpeak increased from

167.27±20.03 to 172.27±44.30 (Ð>0.05) b p m. At the end

of the three-month program faster recovery of heart rate

was observed after step-by-step loading untill rejection

from 2.00±0.82 to 2.90±0.30 (Ð<0.01) according to a

three-stage scale. As a whole, functional tests showed an

increase of physical working capacity (Table 3).

Breathing coefficient at rest did not show any substantial

variance prior to and after completion of the period, though

at peak loading it decreased from 0.98±0.09 to 0.89±0.06

(Ð<0.01), which shows that in the third month a kind of ad-

aptation occurs and metabolism shifts to predominant supply

of energy at the expense of fat decomposition under the con-

ditions of aerobic loading. The indirect calorimetry per-

formed on each participant showed that maximum burning

of fat was achieved at a lower stage of loading at the end of

the period (124.54±18.09W up to 109.09±13.00W (Ð<0.02),

which was evidence of adaptation of mass metabolism to the

three-month training and dietary program (Table 4).

Initial average levels of cholesterol, HDL, triglycerides and

blood sugar remained within the reference limits after the

three-month program.

Sports-and-pedagogic tests carried out in the beginning and

at the end of the training period showed improvement of the

indices of physical ability, enhancement of rising strength

from 100.18±12.01 to 107.50±9.50 kgm (Ð<0.05) and better

flexibility from 14.73±4.92 to 16.59±3.95 ñm (Ð<0.01).

CONCLUSION

The three-month training and dietary program had a posi-

tive effect on the indices of aerobic capacity of the body, re-

duced the fat mass and improved the working capacity.

REFERENCES

1. Åâðîôèò. Åâðîïåéñêè òåñòîâå çà ôèçè÷åñêà

ãîäíîñò. Êîìèòåò çà ðàçâèòèå íà ñïîðòà ïðè

Ñúâåòà íà Åâðîïà. Òåñòîâå çà ó÷àùèãå. Ñ. 1990.

2. Èâëåâ Ì. Ï., Àåðîáèêà-òåîðèÿ è ìåòîäèêà

ïðîâåäåííèÿ çàíÿòèé. Ìîñêâà, 2002.

3. Ìèíåâà Ì., Ì. Òðúí÷èêîâà. Ïðàêòè÷åñêî

ðúêîâîäñòâî ïî àåðîáíà ãèìíàñòèêà. Ñîôèÿ, 2006.

4. Íåñòîðîâà Ä. Ðàçðàáîòâàíå ìîäåë íà ó÷åáíè

ïðîãðàìè ïî àåðîáèêà çà ñðåäíàòà ñòåïåí íà

îáùîîáðàçîâàòåëíîòî ó÷èëèùå. Ñîôèÿ, 2006.

5. Ahmed Abdul Allah El Roby. The effect of a tae bo

exercise program on physical fitness and some kines-

thetic perceptions for university level basketball play-

ers in Egypt. World Journal of Sport Sciences 3 (2):

107-112, 2010.

6. Kikuchi H. and H. Sasaki. A study on the physical

characteristics of aerobic gymnastics junior competi-

tors. Bull Hokkaido Assasi Gauken Coll, 2005,

43:21-30.

7. Loretta DiPietro, James Dziura, Catherine W. Yeckel,

and P. Darrell Neufer. Exercise and improved insulin

sensitivity in older woman: evidence of the enduring

benefits of higher intensity training. J Apply Physiol,

100: 142-149, 2006.

8. Martins et al. Effects of aerobic and strength-based

training on metabolic health indicators in older adults.

Lipids in Health and Disease 2010, 9:76.

9. Noakes T. Lore of Running. Fourth Edition. Human

kinetics. Cape Town. 2003.

83

Angelova P., N. Boyadjiev, A. Ivanova ...

IndexVO2peak/kg

(mlO2/min/kg)Wpeak/kg

(W/kg)

Relative peak

oxygen pulse

(mlO2/bt/kg)

Start 31.64±9.37 2.83±0.53 0.91±0.05

End 35.83±6.03 3.21±0.52 0.21±0.03

Variance

authenticityp>0.05 p<0.05 p>0.05

Table 3. Change of physical working capacity prior to

and after the program (X±SD).

Index RQpeak

Optimum

load for fat

burning (W)

Maximum fat

burning

(kcal/h)

Start 0.98±0.09 124.54±18.09 255.46±109.78

End 0.89±0.06 109.09±13.00 326.36±43.42

Variance

authenticityP<0.01 P<0.02 P>0.05

Table 4. Change of indices of fat metabolism prior to

and after the program (X±SD).

THE EFFECTS OF PACED BREATHING ON SPECTRAL

PARAMETERS OF HEART RATE VARIABILITY IN ATHLETES AND

UNTRAINED CONTROLS

Somlev P.

Department of Physiology and Biochemistry, National Sports Academy "Vassil Levski", Sofia

ABSTRACT

Respiration influences the high frequency power (HFP) of heart rate variability. When assessing autonomic

cardiac regulation in athletes with spectral analysis of HRV it is recommended to use controlled breathing

rate. Studies evaluating the changes in HRV induced by paced breathing in trained and untrained men are

scarce. The aim of this investigation was to assess the effects of paced breathing on HRV indices in athletes and

untrained men. METHODS: Ten trained athletes (age=±years) and eleven (age=±years) untrained controls

participated in the study. RR intervals of each subject were recorded for 5 min during spontaneous breathing

and during breathing at rate of 0.25 Hz. From RR intervals the standard spectral HRV parameters were ob-

tained - low frequency (LF) power, high frequency (HF) power, both in absolute (ms2) and normalized units

(n.u.) and LF/HF ratio. RESULTS: In sedentary controls paced breathing induced significant decrease in LF

(ms2), LF (n.u.), and LF/HF and increase in HF (n.u.) in comparison with spontaneous breathing. In athletes

paced breathing induced significant decrease only in LF (ms2) and LF/HF. There was no difference in HRV

between two groups during spontaneous breathing. During breathing at 0.25 Hz LF (n.u.) and LF/HF were

significantly lower, and HF (n.u.) was significantly greater in athletes than in untrained. CONCLUSIONS:

The influence of paced breathing on HRV is less pronounced in athletes. The paced breathing at rate of 0.25

Hz emphasizes the differences in HRV indices between trained and untrained men.

Key words: heart rate variability, paced breathing, athletes

INTRODUCTION

Spectral analysis of heart rate variability (HRV) is a

noninvasive tool for assessing the modulating influence of

the autonomic nervous system (ANS) on the activity of the

sinus node (18). Two main spectral HRV indices are com-

monly used: high frequency (HF) - the range between 0.15

and 0.40 Hz, considered a marker of parasympathetic activ-

ity; low frequency (LF) - the range between 0.04 and 0.15

Hz, witch reflects both sympathetic and parasympathetic

cardiac control (Task, 1996).

In recent years there is an increasing interest in using HRV

to study the effects of training on cardiac autonomic regula-

tion (2,8).

Respiration significantly affects cardiac autonomic regula-

tion through the mechanism of respiratory sinus arrhythmia

(3). The frequency of breathing has particularly pro-

nounced effect on high frequency power (HFP) of HRV (9,

15). If respiratory rate is below 12 breaths/min (i.e. <0.2

Hz) HF and LF can overlap (5,10). In connection with this

phenomenon there is recommendation to standardize

breathing at 15 breaths/min when using HRV to assess au-

tonomic activity in athletes, who may have low respiratory

rate (17). In the available literature of recent years, studies

evaluating the differences in HRV between trained and un-

trained men during controlled breathing are scarce. The aim

of this study is to compare the effects of paced breathing on

the HRV in athletes and untrained individuals.


Òen athletes (aged 23±3.86 years, weight 77.40±7.59 kg, height

181.35±7.25 cm) and eleven sedentary controls (age

21.55±1.29 years, weight 73.91±11.70 kg, height 179.68±5.14

cm) were included in the study after providing written consent.

The athletes were representatives of sports characterized by en-

durance training (athletics, cross country skiing, orienteering,

biathlon, canoeing). At the the time of the study they all partici-

pated in training sessions. None of the sedentary subjects was

engaged in any form of sporting activity.

After 20 min in supine rest RR intervals of each subject

were recorded with Polar S810i heart rate monitor (Polar

Electro, Finland) with a accuracy of 1 ms (20). For each

subject were made two 5-minute recordings - during spon-

taneous breathing and during paced breathing with fre-

quency 0.25 Hz (i.e. 15 breaths/min). The subjects con-

85



P. Somlev, Dept. of Physiology and Biochemistry, National Sports

Academy "Vassil Levski", 1700, Sofia, Studentski grad,


trolled their respiratory rate with the help of a computer

generated audio signal.

From RR intervals the standard HRV parameters were ob-

tained with Kubios HRV 2.0 software (Biosignal Analysis

and Medical Imaging Group, Department of Physics, Uni-

versity of Kuopio; Kuopio, Finland). Frequency domain

parameters were determined by spectral analysis using fast

Fourier transform after detrending and interpolation at rate

of 4 Hz of RR series. The power spectrum indices included:

LF (low frequency, 0.04 Hz-0.15 Hz) and HF (high fre-

quency, 0.15-0.40 Hz), both expressed in absolute (ms2)

and in normalized units (n.u.). LF/HF ratio was also calcu-

lated. Mean HR (1/min) was also determined.

Simultaneously RR intervals were recorded with Suunto

t6c heart rate monitor. The data were downloaded from the

monitor via usb interface in the Suunto Training Manager

2.3 software (Suunto, Finland), which calculates respira-

tory rate with an accuracy of 93% through a specific pro-

cessing algorithm (6).

For each subject the mean breathing rate (Mean BR) was

calculated for the same 5-minute period of RR measure-

ment, which is used to determine HRV indices during

spontaneous breathing condition. The software is also used

to check whether subjects really accomplished paced

breathing with required frequency of 0.25 Hz.

Aerobic fitness (VO2max) was determined indirectly with

Astrand cycle ergometer test (1).

Independent t-test was used to compare the HRV parame-

ters between the two groups during spontaneous and paced

breathing. To assess the effect of change in respiratory rate

on HRV, the parameters were compared within each group

by paired t-test. The statistical significance threshold was

set at p<0.05.

RESULTS

Athletes had a significantly higher VO2max than untrained

subjects - 57.55±11.54 ml.kg-1.min-1 vs. 35.80±7.78

ml.kg-1.min-1 (p<0.001). During spontaneous breathing

Mean BR in untrained individuals was 13.5±1.8 breaths/min

and in athletes - 12.5±2.1 breaths/min. There is no statistical

difference in Mean BR between two groups. As summarized

in Table 1 during spontaneous breathing frequency HRV pa-

rameters showed no statistical difference between both group,

but Mean HR was significantly lower in athletes.

During paced breathing with 0.25 Hz frequency the difference

between the Mean HR in trained and untrained remained (Ta-

ble 2). The differences in LF (n.u.), HF (n.u.) and LF/HF be-

tween the two groups are statistically significant (Table 2).

86

The effects of paced breathing on spectral parameters of ...

Athletes Untrained

Mean HR (1/min) 57.08±6.63 70.29±8.33*

LF (ms2) 1233.59±873.18 1105.13±795.30

HF (ms2) 1564.22±754.84 1006.02±770.32

LF (n.u.) 42.73±16.38 53.48±7.90

HF (n.u.) 57.27±16.38 46.52±7.90

LF/HF 0.88±0.52 1.20±0.34

Table 1. Mean HR and spectral indices in athletes and

untrained during spontaneous breathing; * - significant

difference between the compared parameters (p<0.05).

spontaneous

breathingpaced breathing

Mean HR (1/min) 70.29±8.33 72.44±8.31*

LF (ms2) 1105.13±795.30 417.29± 82.15*

HF (ms2) 1006.02±770.32 711.59±559.12

LF (n.u.) 53.48±7.90 38.51±6.57**

HF (n.u.) 46.52±7.90 61.49±6.57**

LF/HF 1.20±0.34 0.64±0.18*

Table 3. Mean HR and spectral indices during

spontaneous and paced breathing in untrained; * -

significant difference between the compared parameters

(p<0.05), ** - (p<0.001).

Athletes Untrained

Mean HR (1/min) 57.59±6.83 72.44±8.31**

LF (ms2) 480.86± 24.85 417.29± 82.15

HF (ms2) 1294.51±964.82 711.59± 559.12

LF (n.u.) 24.79±13.10 38.51±6.57*

HF (n.u.) 75.21±13.10 61.49±6.57*

LF/HF 0.37±0.26 0.64±0.18*

Table 2. Mean HR and spectral indices in athletes and

untrained during paced breathing; * - significant

difference between the compared parameters (p<0.05),

** - (p<0.001).

spontaneous

breathingpaced breathing

Mean HR (1/min) 57.08±6.63 57.59±6.83

LF (ms2) 1233.59±873.18 480.86± 24.85*

HF (ms2) 1564.22±754.84 1294.51±964.82

LF (n.u.) 42.73±16.38 24.79±13.10

HF (n.u.) 57.27±16.38 75.21±13.10

LF/HF 0.88±0.52 0.37±0.26*

Table 4. Mean HR and spectral indices during

spontaneous and paced breathing in athletes;

* - significant difference between the compared

parameters (p<0.05).

In the group of untrained subjects the paced breathing causes

significant increase in Mean HR and HF (n.u.) and signifi-

cant decrease in LF (ms2), LF (n.u.) and LF/HF (Table 3).

In the group of athletes the breathing rate of 0.25 Hz did not

cause a change of Mean HR, as well as in high-frequency spec-

tral indices-HF(ms2)andHF(n.u.) (Table4).There isastatisti-

cally significant decrease in LF (ms2) and LF/HF during paced

breathing in comparison with spontaneous breathing.

DISCUSSION

The marked difference in indirectly determined VO2max

between the two groups showed significantly better aerobic

capacity in athletes, developed under the influence of train-

ing. Training, especially in endurance sports, induces both

structural and functional changes in the athletes' heart (14,

19). Bradycardia at rest is the most common

electrocardiographic change in the activity of the sinus

node, as it is observed in up to 91% of endurance athletes

(7,19). Usually there is inverse relationship between exer-

cise capacity and the heart rate and bradycardia is seen as

an indicator of a certain level of training (4,7). The differ-

ence in heart rate between the two groups underlines the

difference in training status.

During 0.25 Hz breathing the differences in LF (n.u.), HF

(n.u.) and LF/HF ratio between the two groups became statis-

tically significant. LF (n.u.) can be used as an index of sympa-

thetic modulation of the heart rate (11). The greater value of

HF (n.u.) and smaller value of LF (n.u.) in trained subjects

compared with untrained show that athletes have a more pro-

nounced vagal influences on heart rate, combined with de-

crease of sympathetic tone. The difference in LF/HF ratio

highlights the shift in sympathovagal balance in athletes.

These results can be explained by the fact that the paced

breathing concentrates high-frequency rhythms in the area of

0.25 Hz and allows better separation of HF and LF, as found

in other studies with controlled breathing (13). Paced breath-

ing emphasizes the differences in autonomic cardiac regula-

tion between subjects with different level of training.

In sedentary subjects controlled breathing causes significant

changes in spectral characteristics of HRV - the increase in

HF (n.u.) and the reduction of LF (ms2), LF (n.u.) and LF/HF

ratio are signs of the shift of autonomic balance towards en-

hancement of parasympathetic effects on heart rate. This

parasympathetic enhancement is also result of the concentra-

tion of high-frequency oscillations around 0.25 Hz.

The comparisons of HRV indices during spontaneous and

paced breathing in the group of untrained individuals con-

firm the data from the literature that the breathing pattern

affects HRV and especially HF (5,9,12,15,16).

In the group of athletes it is remarkable the lack of signifi-

cant change in HF (ms2) and HF (n.u.). This is an evidence

of adaptation of cardiac autonomic regulation occurred in

trained subjects, expressed in markedly increased parasym-

pathetic influences on heart rate at rest - since the vagal tone

has been already increased it could not be expected to fur-

ther increase under the influence of paced breathing. The

decrease of LF/HF ratio also shows the dominance of para-

sympathetic effects in this group, and a tendency towards

decrease of sympathetic influences expressed in the reduc-

tion of LF (n.u.) during paced breathing. Since LF (ms2) re-

flects both sympathetic and parasympathetic influences on

heart rate, there is a significant reduction of this parameter

during 0.25 Hz breathing.

CONCLUSIONS

The study indicates that frequency indices of HRV show

differences in autonomic cardiac regulation between ath-

letes and untrained subjects, which are more pronounced

during paced breathing at 0.25 Hz. The influence of paced

breathing on HRV is smaller in trained individuals, sug-

gesting the presence of enhanced cardiac parasympathetic

control in comparison with untrained subjects. In cross-sec-

tional studies with HRV, when comparing athletes and sed-

entary men it is preferable to record RR intervals for spec-

tral analysis during both spontaneous and paced breathing.

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2010, No. 4, 779-86.

88

The effects of paced breathing on spectral parameters of ...

HEART RATE VARIABILITY AT REST IN ELITE AND FORMER

SOCCER PLAYERS

Somlev P., G. Uzunova, E. Pavlova


ABSTRACT

The aim of this investigation was to assess the differences in HRV parameters between elite soccer players and

soccer players, who have discontinued their training. METHODS: Eleven elite soccer players (age=21.45±3.8

years) from elite professional club and fourteen former soccer players (age=21.79±2.46 years) participated in

the study. RR intervals of each subject were recorded for 5 min in supine rest. From RR intervals the standard

HRV parameters were obtained - SDNN, RMSSD, pNN50% , low frequency (LF) power, high frequency (HF)

power, both in absolute (ms2) and normalized units (n.u.) and LF/HF ratio. Aerobic fitness (VO2max) was de-

termined indirectly with submaximal cycle ergometer test. RESULTS: VO2max was greater in elite soccer

players. SDNN, RMSSD, pNN50% and HF power were significantly higher in elite soccer players than in for-

mer players. LF (n.u.) and LF/HF ratio were significantly lower in elite players than in former players. Elite

soccer players presented also a marked bradycardia. CONCLUSIONS: HRV parameters, which reflect vagal

influences on heart rate, are more pronounced in elite soccer player. The results suggest that there is a para-

sympathetic predominance in elite soccer players in comparison with soccer players, who have discontinued

their training.

Key words: heart rate variability, soccer players, training

INTRODUCTION

Heart rate variability (HRV) is a non-invasive tool to ana-

lyze the role of the autonomic nervous system in the cardiac

regulation, based on the measurement of instantaneous

variations of RR intervals from the electrocardiogram (17).

Changes in the HRV parameters reflect the parasympa-

thetic and sympathetic influences on heart rate.

In the last years HRV is being used to assess the effect of

exercise training on cardiac function and autonomic control

(3). Most of the cross-sectional studies compare sedentary

individuals and endurance-trained athletes (1).

Studies with HRV, evaluating athletes with different train-

ing status from sports with aerobic and anaerobic training

are scarce.

Soccer is an intermittent sport, in which is important the de-

velopment of both aerobic and anaerobic systems (2).

There are only few studies, in which HRV is used to evalu-

ate soccer players e.g. (4,12).

The aim of this investigation was to assess the differences

in HRV parameters between elite soccer players and soccer

players, who have discontinued their training.


Eleven elite soccer players (age=21.45±3.8 years,

weight=78.55±7.7 kg, height=183.14± 4.18 cm) from elite

professional club and fourteen former soccer players

(age=21.79±2.46 years, weight=73.43±6.66 kg,

height=178.25±6.25 cm) participated in the study. The elite

soccer players were studied in january before training

camp. The former players had stopped their soccer training

for a period of 4 months to 5 years, but played soccer for

educational and/or recreational purposes.

After 20 min in supine rest RR intervals of each subject

were recorded for 5 min with Polar S810i heart rate monitor

(Polar Electro, Finland) with a accuracy of 1 ms (18). From

RR intervals the standard HRV parameters were obtained

with Kubios HRV 2.0 software (Biosignal Analysis and

Medical Imaging Group, Department of Physics, Univer-

sity of Kuopio; Kuopio, Finland).

Time domain parameters included Mean RR (ms) - the

mean of RR intervals, SDNN (ms) - the standard deviation

of normal-to-normal RR intervals, RMSSD (ms) - the

square root of the mean squared differences of successive

RR intervals and pNN50% - the percentage of intervals dif-

fering by more than 50 ms from preceding interval. Mean

HR (1/min) was also determined.

SDNN reflects all the cyclic components responsible for

variability, RMSSD and pNN50% are markers of parasym-

pathetic modulations of RR intervals (6,17).

89



P. Somlev, Dept. of Physiology and Biochemistry, National Sports

Academy "Vassil Levski", 1700, Sofia, Studentski grad,


Frequency domain parameters were determined by spectral

analysis using fast Fourier transform method after

detrending and interpolation at rate of 4 Hz of RR series.

The power spectrum indices included: LF (low frequency,

0.04 Hz-0.15 Hz) and HF (high frequency, 0.15-0.40 Hz),

both expressed in absolute (ms2) and in normalized units

(n.u.). LF/HF ratio was also calculated.

HF (ms2) and HF (n.u.) are markers of vagal contribution to

heart rate regulation, while LF (ms2) reflects both sympa-

thetic and vagal influences (17). LF/HF ratio is an estimate

of sympathovagal balance.

Aerobic fitness (VO2max) was determined indirectly with

submaximal cycle ergometer test (5).

Statistical analysis included independent t-test and

Mann-Whitney test. The statistical significance threshold

was set at p<0.05. Data are presented as mean±standart de-

viation (Mean±SD).

RESULTS

VO2max in elite soccer players was higher than in former

players (Table 1).

Elite soccer players had a significantly lower resting heart

rate than former players (Table 2). Mean RR, SDNN,

RMSSD and pNN50% were greater in elite soccer players

compared with former players (Table 2).

Analysis of the frequency domain parameters (Table 3) re-

vealed that elite soccer players had a significantly higher

LF (ms2), HF (ms2) and HF (n.u.) than former players. LF

(n.u) and LF/HF ratio were greater in former players than in

elite players.

DISCUSSION

The higher value of VO2max of elite players reflects their

better aerobic capabilities developed under the influence of

training. The resting bradycardia, demonstrated by elite

players, suggests a large degree of cardiac adaptation to

training (13). Accordingly, the duration of RR intervals was

greater in elite players, which corresponds to the pro-

nounced bradycardia in these athletes.

The lower SDNN in the former players shows significantly

reduced overall HRV in comparison with elite athletes.

RMSSD and pNN50% are vagal-related HRV indices. The

higher values of these parameters suggest increased para-

sympathetic tone in elite players. These results are similar

to data obtained by other authors, who compared time do-

main indices in trained and untrained individuals (9,11).

Several studies have shown differences between trained

and untrained individuals in time domain of HRV, but not

in frequency parameters (8,10). Sacknoff et al. (14) re-

ported increased SDNN and pNN50% but reduced spectral

components in athletes compared with control subjects.

In our study HF (ms2) and HF (n.u.) were significantly

higher in elite players than in the former players. These data

indicate more pronounced parasympathetic effects on car-

diac regulation in elite players in comparison with former

players.

Low frequencies in normalized units, LF (nu) show less

marked sympathetic cardiac influences in elite players, be-

cause those parameter can be interpreted as an index of

sympathetic modulation of the heart rate (7).

The higher value of LF (ms2) in elite players probably dem-

onstrates the greater involvement of parasympathetic influ-

ences since this parameter represents both sympathetic and

vagal activities. This is also reflected in the LF/HF ratio,

which is significantly lower in elite players.

The differences in frequency domain parameters between

elite and former players are similar to data found in various

works, which compared trained and untrained subjects and

showed greater HF and lower LF in athletes (15,16).

The results of our study show that both time domain and

frequency domain indices, which reflect parasympathetic

influences in heart rate regulation, are greater in elite, i.e.

well trained soccer players than in former athletes.

90

Heart rate variability at rest in elite and former soccer players

VO2max (ml.min-1.kg-

1)

Elite players 54.12± 5.88

Former players 45.57± 6*

Table 1. VO2max (Mean±SD); *- significant difference

(p<0.05).

Elite players Former players

Mean HR (1/min) 53.28±5.38 68.00±9.38*

Mean RR (ms) 1142.61 ±106.09 900.98±129.05*

SDNN (ms) 80.47±31.30 43.47±7.80*

RMSSD (ms) 76.82±37.14 34.47±8.66*

Table 2. Mean HR and time domain HRV parameters;

*- significant difference (p<0.05).

Elite players Former players

LF (ms2) 1576.32±1117.47 731.19±200.40*

HF (ms2) 2417.47±1791.17 457.25±187.49*

LF (n.u.) 42.56±16.11 61.91±10.21*

HF (n.u.) 57.44±16.11 38.09±10.21*

Table 3. Frequency domain HRV parameters; *-

significant difference (p<0.05).

CONLUSIONS

The study shows that HRV parameters, which reflect vagal

effects on heart rate, are greater in elite players than in for-

mer players. These results are similar to findings of other

authors, comparing trained athletes and sedentary subjects.

The present findings suggest that there is a parasympathetic

predominance at rest in elite soccer players in comparison

with soccer players, who have discontinued their training.

The differences in HRV indices could reflect the influence

of different training status on autonomic cardiac regulation.

REFERENCES

1. Aubert , A.E. , B. Seps, and F. Beckers . Heart

rate variability in athletes. Sports Med., 33, 2003, No.

12, 889-919.

2. B a n g s b o , J . , M . M o h r , a n d P .

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7. Lahir i , M.K. , P.J . Kannankeri l , and J.J .

Goldberger . Assessment of autonomic function in

cardiovascular disease: physiological basis and prog-

nostic implications. J Am. Coll. Cardiol., 51, 2008,

No. 18, 1725-33.

8. Mart inel l i , F .S. , M.P. Chacon-Mikahi l ,

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Soares, and P. Lago. (Is autonomic control of the

heart rate at rest altered by detraining? A study of

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N. Spon: London, 1990, 371-426.

14. Sacknoff , D.M., G.W. Gleim, N.

Stachenfeld, and N.L. Coplan. Effect of ath-

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127, 1994, No. 5, 1275-8.

15. Shin, K. , H. Minamitani , S. Onishi , H.

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1482-90.

17. Task Force. Heart rate variability: standards of mea-

surement, physiological interpretation and clinical

use. Circulation., 93, 1996, No. 5, 1043-65.

18. Weippert , M., M. Kumar, S. Kreuzfeld, D.

Arndt , A. Rieger , and R. Stol l . Comparison of

three mobile devices for measuring R-R intervals and

heart rate variability: Polar S810i, Suunto t6 and an

ambulatory ECG system. Eur. J. Appl. Physiol., 109,

2010, No. 4, 779-86.

91

Somlev P., G. Uzunova, E. Pavlova

CONTRACTILE RESPONSES OF THE RAT UTERINE SMOOTH

MUSCLE TO INFLUENCES WITH ANGIOTENSIN II AND

VASOPRESSIN

Georgiev Ts., P. Hadzhibozheva, A. Tolekova

Department of Physiology, pathophysiology and pharmacology, Medical Faculty,

Trakia University - Stara Zagora

ABSTRACT

The purpose of this study was to compare the registered Angiotensin II (Ang II) and Arginine - Vasopressin

(AVP) - provoked contractions in experiments in vitro on muscle strips from rat uterus. Female non-pregnant

mature Wistar rats, weighting 250-300g, were used. Longitudinal strips from uterine horns were prepared

and influenced by Ang II and AVP. The recorded force-vs.-time curves were analyzed including calculation of

amplitudes, area under the curve (AUC) of the smooth muscle contraction, as well as defining of time-parame-

ters. Ang II and AVP applied in a dose of 10-6

M caused powerful tonic contractions with approximately equal

amplitudes. AVP-induced responses were several times greater than those of Ang II. The recording of

AVP-mediated contractions was stopped on the 30th

minute because they showed slow tendency to decrease.

The experiment demonstrates the significance of the investigated peptides for the uterine function and pro-

vides a direction for further research work on AVP- and Ang II - mediated contractions.

Key words: smooth muscle contraction, uterus, peptides, hyperactivity, in vitro experiments

INTRODUCTION

The disorders in uterine contractile activity are in the basis

of a number of conditions such as preterm deliveries, dys-

functional labour, post-partum hemorrhage, primary

dysmenorrhea etc., which affect the normal rhythm and

function of the whole organism, and often lead to fatal

complications for the individual (9). A better understanding

of the mechanisms that lead to myometrial contractility and

the humoral factors responsible for the regulation of the

uterine tone is of an essential importance for the prophylac-

tic and treatment of these contractile uterine disorders.

Ang II and AVP are peptides, which role is generally asso-

ciated with regulation of the smooth muscle tone. They act

in a similar pattern, by activating the inositol-triphosphate

signal pathway.

It has been reported that in the uteri of a number of species,

local production of Ang II and the enzymes for its synthesis

are present. Besides the proven contractile effect of Ang II

on the uterine arteries, research in this area showed that

myometrium is also sensitive to the effect of this

octapeptide (7).

There is substantial evidence for the involvement of AVP

in conditions of uterine hyperactivity. Even more, it has

been shown that the human myometrium is more sensitive

to AVP than to oxytocin (2).

The purpose of this study was to investigate and analyze the

contractile responses of the uterine smooth muscle to influ-

ences with Ang II and AVP.


Sample preparation

The experiments were performed in the uterus smooth

muscles isolated from adult non pregnant Wistar rats,

weighing 200-250 g. The animals were anesthetized with

Nembutal 50mg/kg i.p. and exsanguinated. The experi-

ments were carried out in accordance with the national reg-

ulations, the European Council Directive - 86/609/EEC,

and Directive 2003/65/EC of the European Parliament,

concerning the protection of animals used for scientific and

other experimental purposes.

Abdominal cavity was opened and the uterus was dissected

out and immediately placed in cold Krebs solution (3 °C),

containing the following composition (in mmol):

NaCl-118.0, KCl-4.74, NaHCO3-25.0, MgSO4-1.2,

CaCl2-2.0, KH2PO4-1.2 and glucose 11.0. The surrounding

tissue was dissected and sections from uterine horns (ap-

proximately 10 mm long) were prepared.

The two ends of each preparation were tied with ligatures.

The distal end was connected to the organ holder; the prox-

imal end was stretched and attached to a mechano-electri-

cal transducer FSG-01 (Experimetria Ltd., Hungary) via a

hook. The preparations were placed in organ baths

TSZ-04/01, containing Krebs solution, pH 7.4, continu-

93




ously bubbled with Carbogen (95% O2, 5%CO2). The or-

gan baths were mounted in parallel above an enclosed wa-

ter bath, maintaining the solution temperature at 37 °C.

Preparations were placed under an initial tension (preload)

of 1 g and allowed to equilibrate for at least 75 min (three

periods: 15 min, 45 min and 15 min and two washes with

Krebs solution between them). After the equilibration pe-

riod, preparations were influenced by Ang II and AVP in a

dose of 1 mol (10-6 M).

Recording of mechanical activity

Mechanical activity was digitized and recorded by using

S.P.E.L. ISOSYS Advanced Software (Experimetria Ltd.,

Hungary). The conversion of the data for later analysis was

performed with KORELIA-Processing and the analysis

and graphic processing-with KORELIA-Dynamics

computer programs (10).

Chemicals and drugs

Ang II (Sigma-Aldrich) and AVP (Sigma-Aldrich) were

solubilized in bidistillated water. All reagents for prepara-

tion of Krebs solution were purchased from Sigma-Aldrich.

Data analysis and statistical processing

The recorded force-vs.-time curves were analyzed includ-

ing calculation of amplitudes, area under the curve (AUC)

of the smooth muscle contraction, as well as defining of

time-parameters (Fig. 1): half-contraction time (Thc), con-

traction time (Tc), half-relaxation time (Thr), contraction

plus half-relaxation time (Tchr). The duration of interval for

analysis of tonic contraction was defined from the begin-

ning of contraction, until the amplitude fell to 50%.

Fmax - maximal force of the smooth muscle contraction

(SMC)

Fmax/2 - half of maximal force of the SMC

Thc - time interval between the start of the SMC and Fmax/2

Tc - time interval between the start of the SMC and Fmax

Thr - time interval between Fmax and Fmax/2

Tchr - time between the start of the SMC and Fmax/2

Obtained data were processed by the statistical program

Statistica 6.1, StaSoft, Inc. and presented as mean ± stan-

dard deviation. A P-value less than or equal to 0.05 was

considered to be statistically significant.

RESULTS

Ang II at concentration of 1 µmol induced tonic contraction

with maximum amplitude of 6.00 ± 0.22 g (n = 8) and an

integral force of muscle contraction of 1150.00 ± 614.70 gs

(Fig. 2).

AVP applied in the same concentration as Ang II induced

tonic contractions with amplitude of 6.61 ± 0.39 g (n = 8)

which was no significantly different from amplitude of Ang

II - mediated contractions (P>0.05). The integral force of

AVP-provoked muscle response was 7245.00 ±901.00 gs

and there was a statistical significant difference (P<0.05)

with AUC of Ang II - mediated contractions (Fig. 3).

The duration of the AVP-induced responses was several

times greater than those of Ang II (Fig. 4) and the recording

of AVP-mediated contractions was stopped on the 30th

minute without achievement of Tchr parameter.

The calculated time-parameters are given on Fig. 5.

*P<0.05 vs. Thc of AVP-mediated contraction. **P<0.05

vs. Tc of AVP-mediated contraction. #P<0.05 vs. Tchr of

AVP-mediated contraction.

94

Contractile responses of the rat uterine smooth muscle to ...

Fig. 1. Scheme of time-parameters analysis.

Fig. 2. Original recording of Ang II - mediated

contraction of rat uterus

Fig. 3. Original recording of AVP - mediated

contraction of rat uterus

Fig. 4.Graphical comparison of the duration of the

smooth-muscle contractions caused by Ang II and AVP

DISCUSSION

Our experiment confirmed the contractile effect of these

two peptides on the myometrium, which is in accordance

with the results of other authors working on the same issues

(1,3,7).

The contractions induced by both peptides have similar

amplitudes, but they are with different duration and charac-

teristics. The registered AVP - provoked uterine responses

were found to have a sustained oscillating character. When

analyzed by mathematical modeling such contractions

were recognized as underdamped process - the system tries

to establish a stable level different from the baseline (11).

The differences in the developed contractions may be due

to split of the classical or inclusion of additional

transductional pathway for each of the studied peptides.

Both of them have several main groups of receptors. The

receptors for Ang II are AT1 and AT2 (4), while the

receptors for AVP are V1a, V1b and V2 (8).

To establish the importance of these receptors for the uter-

ine muscle contraction will be the subject of our next exper-

iments. However, several interesting facts immerge:

First - the constrictor effect of Ang II is associated with

AT1 receptors, but the uterus is one of the few organs (with

a. uterina inferior and the kidney arteries) where AT2 re-

ceptors are predominant (7). AT2 receptors are mainly re-

garded to oppose the effects of AT1 and cause dilation,

blood pressure reduction, nitric oxide production (6), (7).

Perhaps the significantly shorter phase of contraction and

relaxation was due to their activation under the influence of

Ang II in the uterus.

Second - the constrictor effect of AVP is realized by V1a

receptors. With regard to the contractile response of the

myometrium, however, there are statements that the result-

ing contraction from the AVP influence is due to activation

of other receptors, different from the mentioned above (1).

Some authors go even further and argue that AVP accom-

plish its effect on uterine musculature by OT receptors,

which have big similarity with V1 receptors (3).

Considering that both peptides are released from supraoptic

nuclei in the hypothalamus and that they have a powerful con-

tractile effect on the smooth muscle, it is appropriate to search

a closer connection between them in preparing the uterus for

pregnancy and labour. Probably these two peptides act syn-

chronously which potentiate their own effects (5).

Studies on rats show that AVP is more potent uterotonic

agent than OT in non pregnant condition (2) and during

parturition OT predominantly promotes uterine contrac-

tions, while AVP is more important for vasoconstriction,

thus reducing the bleeding after delivery (3), (5).

CONCLUSION

The study of Ang II - and AVP - mediated uterine contrac-

tions contributes considerably for the revealing of the

mechanisms that generate and modulate uterine activity.

This could be beneficial for a better understanding and con-

trol of myometrial dysfunction.

Acknowledgements:

This study was supported by Grant DDVU - 02 - 24/2010

from the National Science Fund, Sofia, Bulgaria

REFERENCES

1. Anouar A. , M. Clerget , T .Durroux, C.

Barber is , G. Germain. Comparison of

vasopressin and oxytocin receptors in the rat uterus

and vascular tissue. Eur J Pharmacol, 1996, 11;

308(1):87-96.

2. Bossmar T. , N. Osman, E. Zi lahi , M. Haj ,

N. Nowotny, J . Conlon. Expression of the

oxytocin gene, but not the vasopressin gene, in the

rat uterus during pregnancy: influence of oestradiol

and progesterone. J Endocrinol, 2007, 193(1):121-6.

3. Chan W., N. Wo, M. Manning. The role of

oxytocin receptors and vasopressin V1a receptors in

uterine contractions in rats: implications for tocolytic

therapy with oxytocin antagonists. Am J Obstet

Gynecol, 1996, 175(5):1331-5.

4. De Gasparo M., K. Cat t , T. Inagami, J .

Wright , T. Unger . International union of pharma-

cology. XXIII. The angiotensin II receptors.

Pharmacol Rev, 2000, 52(3):415-72.

5. Douglas A. , S. Scul l ion, I . Antoni jevic , D.

Brown, J . Russel l , G. Leng. Uterine contractile

activity stimulates supraoptic neurons in term preg-

nant rats via a noradrenergic pathway. Endocrinology,

2001;142(2):633-44.

6. Hannan R. , E. Davis , R. Widdop. Functional

role of angiotensin II AT2 receptor in modulation of

AT1 receptor-mediated contraction in rat uterine ar-

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Pharmacol, 2003; 140(5):987-95.

7. Keski l Z. , M. Bayram, Z. Ercan, R.

Türker . The contribution of nitric oxide and

endothelins to angiotensin: II. Evoked responses in

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Georgiev Ts., P. Hadzhibozheva, A. Tolekova

Fig. 5. Analysis of the time-parameters of

smooth-muscle contractions of rat uterus after

application of Ang II and AVP in dose of 1 µmol.

the rat isolated uterus smooth muscle. Gen

Pharmacol, 1999; 33(4):307-12.

8. Petersen M. The effect of vasopressin and related

compounds at V1a and V2 receptors in animal models

relevant to human disease. Basic Clin Pharmacol

Toxicol, 2006; 99(2):96-103.

9. Wray S. Insights into the uterus. Exp Physiol,

2007; 92(4):621-31.

10. Yankov K. Preprocessing of Experimental Data in

Korelia Software. Trakia Journal of Sciences, 2010;

8, Suppl. 3: 41-48.

11. Yankov K. Recognition and function association of

experimental data. Proc. of the Int. Conference on In-

formation Technologies (InfoTech-2009),

Constantine and Elena resort, sept.17-20, 2009,

Varna, Bulgaria, 131-141.

96

Contractile responses of the rat uterine smooth muscle to ...

CARDIOVASCULAR STRESS AND MYOCARDIAL PROTECTION (I)

Kolarova R.


ABSTRACT

Hemodynamic and oxidative stress injuries are characteristic for cardiovascular diseases and the role of the

endogenous and exogenous antioxidant systems as cardioprotective mechanisms are quite interesting. The dy-

namics of antioxidant action of Vit. E on some functional and metabolic changes in the myocardium of hyper-

tensive rats was studied. The following methods were applied: a model of vasorenal hypertension; treatment

of the experimental animals with the antioxidant Vit. E; tracing of changes in arterial blood pressure, the ratio

of grams of myocardial weight/100 g body weight and activity of some "key enzymes" of cardiac metabolism -

of glycolysis, pentosomonophosphate shunt, cycle of Krebs, as well as enzymes of energetic metabolism.

Throughout the experimental periods (3rd

, 15th

and 30th

day) a progressively developing hypertension was es-

tablished. Glycolytic enzymes showed no essential deviations in their activity, whereas the activity of

pentosomonophosphate shunt was increased significantly at all experimental terms. A considerable increase

in the myocardial weight and values of the enzyme activity close to those of the control animals were estab-

lished in the groups of Vit. E-treated animals with experimental hypertension. The results obtained are char-

acteristic for the protective effects of Vit. E in the course of arterial hypertension regarding some metabolic

changes in the myocardium. The dominating role of the pentose monophosphate shunt as an anti-stress reac-

tion becomes prominent in this kind of hemodynamic injury of the myocardium. The combined treatment of

the experimental animals with Vit. E leads to cardioprotection, probablyby limiting of lipid peroxidation.

Key words: experimental hypertension, myocardial hypertrophy, myocardial enzyme activity, Vit. E

INTRODUCTION

Haemodynamic stress is one of the most common mecha-

nisms of injury not only of heart but also of the vascular

system. The causes for this stress can be different: increased

levels of catecholamines in the blood, treatment with

sympathomimetics, diverse forms of hypertension.

Haemodynamic stress could be acute, chronic, continuous

or interrupted (10). The changes in haemodynamics ulti-

mately lead to both pressure and volume overload to the

myocardium (17,19) with correspondingly severe conse-

quences later (1,4,5,6,11,13,18).

Regarding the role of endogenous antioxidant systems, it is

considered (12) that myocardial hypertrophy in experimen-

tal conditions provokes an increase of the cell antioxidant

reserve, which reduces the oxidative stress. Other authors

(2,3,9) have reported a protective effect of antioxidants in

patients with hypertension and still reversible vascular

damage, pointing out the potential therapeutic value of

long-term treatment with vitamin E in modulating or pre-

venting the pathogenesis of heart failure. It was found (8)

that the supplementation with a combination of vitamin C

and E among basketball players enhanced their body

antioxidant defense system by reducing the free radicals.

Having in mind the data from recent years for possible in-

volvement of free radicals in the pathogenesis of myocar-

dial hypertrophy and hypertension (7,12,14,15,16), the

question how free radical neutralization by the antioxidants

will reflect on the myocardial metabolism arises, since the

myocardial injury ultimately depends on metabolic

disorders.

In view of above data, we aimed to study the effect of anti-

oxidant Vit. E in dynamics on the activity of "key" enzymes

of myocardial metabolism in conditions of experimental

hypertension.


1. Experimental animals.

Male Wistar rats with an initial body weight of 168 ±9 g

were used.

2. Groups:

a) a control group - healthy, unoperated animals

(n=6)

b) a group with experimental hypertension, untreated

(n=7)

c) a group with experimental hypertension, treated

with the solvent of vitamin E (n=18)

d) a group with experimental hypertension, treated

with vitamin E (n=20)

3. Experimental period.

Studies were performed on the 3rd, 15th and 30th day

after the hypertension creation.

4. Model of experimental hypertension.

97


A vasorenal hypertension was induced according to

method of H. Selye, modified by R. Kolarova. In breif -

the aortic stenosis was performed between the both

renal arteries and in this way the left kidney became a

source of pressure substances, which determined

permanent hypertension and myocardial hypertrophy.

5. Vitamin E treatment.

A part of the animals with experimental hypertension

were treated with fat-soluble vitamin E (made in Japan)

at a dose of 10 mg/kg b.w., applied by an intraperitoneal

injection every day from the surgery till the animal

killing.

6. Systolic blood pressure measurement.

The systolic blood pressure was measured directly

through a catheter inserted into a. carotis communis.

Animals were given Nembutal (Abbott) at a dose of

40mg/kg b.w to induce anesthesia prior surgery. A

multi-channel polygraph Biomedika C3 was used, after

that the animals were decapitated.

7. Material harvesting.

Immediately after animal decapitation, the hearts were

removed, weighed and a part of the left ventricle was

taken for enzymatic determinations. The myocardial

weight/100g ratio was calculated using the heart weight

and body weight.

8. Enzyme activity measurement.

Parts of the left ventricle were homogenized using a

Potter type homogenizer, then the resulting

homogenate was centrifuged in a refrigerated

centrifuge and a series of dilutions from the supernatant

were prepared for measurement of the enzymes

activities. The activity of the following enzymes:

GL-6-PDH (glucose-6-phosphate dehydrogenase), PK

(pyruvate kinase), ICDH (isocitrate dehydrogenase),

MDH (malate dehydrogenase), CK (creatine kinase)

and CK-MB (isoenzyme of creatine kinase) was

determined using Boehringer enzyme test kids.

Enzyme activity was expressed as U/g protein. Total

protein was determined by the method of Lowry.


1. Blood pressure and myocardial weight.

A progressively developed hypertension and increased

myocardium / 100 g b.w. ratio was found in the group

of rats operated after Selye method. In the group of

hypertonic animals treated with Vitamin E compared to

untreated group a slight but statistically significant

(P<0.05) decrease of the blood pressure was detected

only on the 30th day of the experimental period (Fig.1).

A downward trend on the 15th day and statistically

significant reduction in the myocardium / 100 g b.w.

ratio on the 30th day of experiment (P<0.05) was also

established (Fig.2) .

2. Changes in enzymatic activity of the myocardium.

The changes are presented on Fig.3, Fig.4, Fig.5, Fig.6,

Fig.7, Fig.8.

The Gl-6-PDH activity (Fig.3) in the untreated hyperten-

sive rats showed a typical feature - an increase in every

tested period (due to increased energy and plastic require-

98

Cardiovascular stress and myocardial protection (I)

Fig. 1. Changes in arterial blood pressure in rats with

experimental hypertension and hypertensive rats treated

with Vit. E.

Fig. 2. Changes in the ratio of myocardium/100 g b.w.

(R) in rats with experimental hypertension and

hypertensive rats treated with Vit. E.

Fig. 3. Changes in the enzyme activity of GL-6-PDH

(glucose-6-phosphate dehydrogenase) in rats with

experimental hypertension and hypertensive rats treated

with Vit. E.

ments in myocardial hypertrophy, found in our previous

studies). Upon Vit. E treatment the enzyme activity signifi-

cantly decreased compared with that of hypertensive rats,

but does not reach the control level. This change corre-

sponds to the reduction of the ratio g myocard/100 g. b.w.

and reflects the reduced energy and plastic requirements by

heart.

Demonstrative changes of PK-activity (Fig.4) are regis-

tered during the experimental period (an expressed increase

in the activity of the enzyme on the 3rd day and a mani-

fested decrease of the activity on the 15th day and again in-

crease of the PK-activity on the 30th day. It should be noted

that Vit. E treatment prevents all these changes.

The activity of Krebs cycle enzymes (ICDH / Fig.5/ and

MDH / Fig.6) - in hypertensive rats showed a dramatic

drop. When hypertensive animals were treated with Vit.E,

this supplementation prevented the decrease significantly

with exception of the 30th day (for both enzymes), which

clearly demonstrated that the oxidation processes in this pe-

riod cannot be stabilized.

The reduced activity of CK (Fig.7) in untreated hyperten-

sive rats on the 3rd and 15th day probably reflected the in-

creased energy consumption of the myocardium subjected

to compensatory hyperfunction, while in regard to the sharp

increase in activity of the enzyme on the 30th day we as-

sumed that it could be compensatory due to reduced activ-

ity of other metabolic pathways. In Vit. E treated hyperten-

99

Kolarova R.

Fig. 4. Changes in the enzyme activity of PK (pyruvate

kinase) in rats with experimental hypertension and


Fig. 5. Changes in the enzyme activity of ICDH

(isocitrate dehydrogenase) in rats with experimental

hypertension and hypertensive rats treated with Vit. E.

Fig. 6. Changes in the enzyme activity of MDH (malate

dehydrogenase) in rats with experimental hypertension

and hypertensive rats treated with Vit. E.

Fig. 7. Changes in the enzyme activity of CK (creatine

kinase) in rats with experimental hypertension and


Fig. 8. Changes in the enzyme activity of CK-MB

(isoenzyme of creatine kinase) in rats with experimental

hypertension and hypertensive rats treated with Vit. E.

sive rats only a trend towards normalization of enzyme ac-

tivity was observed in all experimental periods.

The changes in the CK-MB-activity, which is highly selec-

tive for the heart muscle, are particularly typical of the un-

treated hypertensive rats. They showed a progressive in-

crease throughout the entire experimental period, which is

indicative of myocardial ischemic damage. In Vit. E treated

hypertensive rats the CK-MB-activity was falling, as on the

15th day reached the control level.

The obtained results about the changes in the activity of

some key enzymes studied in hypertensive rats treated with

Vit. E are evidence for the marked protective effect of the

antioxidant Vit. E on myocardial metabolism.

CONCLUSIONS

The myocardium/100 g b.w. ratio was increased in the rats

with experimental hypertension and it was reduced in hy-

pertensive rats treated with Vit. E at the end of the experi-

mental period (on the 30th day after surgery); in the rats with

experimental hypertension the pentose monophosphate

shunt activity was significantly increased, CPK- and

CK-MB activity was also increased; in hypertensive rats

treated with Vit. E the pentose monophosphate shunt activ-

ity was reduced in all tested periods, the PK-acivity (a rep-

resentative of glycolysis) deviations were reduced also, the

changes in the Krebs cycle enzymes activity were an ex-

pression of improvement in the oxidative processes,

CPK-activity tended to normalize and CK-MB activity in

all tested periods of the experimental period was lower - an

indication for reduction of ischemic heart damage. The

changes in the enzyme activities studied in hypertensive

rats treated with Vit. E are an expression of the cardio-pro-

tective effects of Vit. E.

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1. Chandola T, Bri t ton A, Brunner E, Heming-

way H, Malik M, Kumari M et al , Work Stress

and coronary heart disease: what are the mechanisms?

Eur Heart J 2008; 29, 640-648.

2. Dhal la AK, Hil l MF & Singal PK. Role of ox-

idative stress in transition of hypertrophy to heart fail-

ure. J Am Coll Cardiol 1996; 28, 506-514.

3. Dhal la NS, Elmoselhi AB, Hata T &

Makino N. Status of myocardial antioxidants in

ischemia-reperfusion injury. Cardiovasc Res 2000;

47, 446-456.

4. De Rosa S, Cir i l lo P, Pagl ia A, Sasso L, Di

Palma & Chiar iel lo M. Reactive oxygen species

and antioxidants in the pathophysiology of cardiovas-

cular disease: does the actual knowledge justify a

clinical approach? Curr Vasc Pharmacol 2010; 8,

259-275.

5. Forogos R. Does Stress Really Cause Heart Dis-

ease? 2008.

6. I to H, Tori i M & Suzuki T. Comparative study

on free radical injury in the endothelium of SHR and

WKY aorta. Clin Exp Pharmacol Physiol Suppl 1995;

22, S157-159.

7. Nakamura K, Fushimi K, Kouchi H, Mihara

K, Miyazaki M, Ohe T et al , Inhibitory effects

of antioxidants on neonatal rat cardiac myocyte hy-

pertrophy induced by tumor necrosis factor-alpha and

angiotensin II. Circulation 1998; 98, 794-799.

8. Naziroglu M, Kil inc F, Uguz AC, Cel ik O,

Bal R, Butterworth PJ et al , Oral vitamin C and

E combination modulates blood lipid peroxidation

and antioxidant vitamin levels in maximal exercising

basketball players. Cell Biochem Func 2010; 28,

300-305.

9. Parik T, All ikmets K, Teesalu R & Zilmer

M. Evidence for oxidative stress in essential hyper-

tension: perspective for antioxidant therapy. J

Cardiovasc Risk; 3, 49-54.

10. Perr ino C, Naga Prasad SV, Mao L, Noma

T, Yan Z, Kim HS et al . Intermittent pressure

overload triggers hypertrophy-independent cardiac

dysfunction and vascular rarefaction. J Clin Inves

2006; 116, 1547-1560.

11. Sharma AB, Sun J, Howard LL, Wil l iams

AG, JR & Mallet RT. Oxidative stress reversibly

inactivates myocardial enzymes during cardiac arrest.

Am J Physiol Heart Circ Physiol 2007; 292,

H198-206.

12. Singh N, Dhal la AK, Seneviratne C &

Singal PK. Oxidative stress and heart failure. Mol

Cell Biochem 1995; 147, 77-81.

13. Stopler M. Stress; 2010 [Accessed 1/28/2010].

14. Taddei S, Virdis A, Ghiadoni L & Salvet t i

A. Endothelial dysfunction in hypertension: fact or

fancy? J Cardiovasc Pharmacol 1998; 32 Suppl 3,

S41-7.

15. Taddei S, Virdis A, Ghiadoni L, Sudano I

& Salvet t i A. Endothelial dysfunction in hyperten-

sion. J Cardiovasc Pharmacol 2001; 38 Suppl 2,

S11-14.

16. Taddei S, Virdis A, Ghiadoni L, Versar i D

& Salvet t i A. Endothelium, aging, and hyperten-

sion. Curr Hypertens Rep 2006; 8, 84-89.

17. Touyz RM. Advancement in hypertension

pathogenesis: some new concepts. Curr Opin Nephrol

Hypertens 2011; 20, 105-106.

18. Victor VM., Rocha M, Sola E, Banuls C,

Gardia-Malpart ida K & Hernandez-Mijares

A. Oxidative stress, endothelial dysfunction and ath-

erosclerosis. Curr Pharm Des 2009; 15, 2988-3002.

19. Wojciechowski P, Jur ic D, Louis XL,

Thadapi l ly SJ, Yu L, Taylor C et al .

Resveratrol arrests and regresses the development of

pressure overload- but not volume overload- induced

cardiac hypertrophy in rats. J Nutr 2010; 140,

962-968.

100

Cardiovascular stress and myocardial protection (I)

ANTIOXIDANT PREVENTION IN EXPERIMENTAL

HYPERTENSION: HISTOMORPHOLOGICAL CHANGES (II)

Kolarova R.1, Z. Gendzhev

2

1Department of Physiology and Biochemistry, National Sports Academy "Vassil Levski", Sofia;2Chemical Pharmaceutical Research Institute, Sofia

ABSTRACT

The dynamics of antioxidant action of Vit. E (for 30 days) on some histomorphological changes in the

myocardium of rats with experimental hypertension was studied. The following methods were applied: a

model of coarctation (vasorenal) hypertension after the method of H. Selye, modified by R. Kolarova; treat-

ment of the experimental animals with the antioxidant Vit. E (10 mg/kg) b.w., i.p.; tracing of changes in arte-

rial blood pressure, the ratio of grams of myocardial mass/100 g body mass and histomorphological study. A

progressive rise of arterial blood pressure and a statistically significant increase in the ratio of grams of myo-

cardial mass/100 g body mass were found in the course of the experimental period. In the group of hyperten-

sive rats untreated with the antioxidant typical histomorphological injuries were observed: focal or diffuse

myolysis of cardiomyocytes, without vascular injuries on the 3rd

day, myocardial hypertrophy, accompanied

with focal or diffuse myocardiofibrosis and coronarofibrosis on the 15th

day, which were strongly manifested

on the 30th

day of the experimental period. In the hypertensive rats treated with Vit. E the injuries were signif-

icantly less expressed as compared to the untreated animals in all experimental stages. Mostly necrotic and fi-

brous changes as well as coronarofibrosis and coronarosclerosis in the myocardium at the later term - 30th

day

were reduced. These results indicate the positive prophylactic role of Vit. E on the cardiac tissue in circum-

stances of hemodynamic stress in experimental arterial hyprtension.

Key words: experimental hypertension, myocardial hypertrophy, cardiac histomorphological changes, Vit. E

INTRODUCTION

It is known that in stress provoked heart damage destructive

disorders of cell membrane structures occur - a result of the

free radicals attacks. There are extensive clinical data about

both early (arrhythmias and sudden cardiac death) and late

(mostly coronary) heart damage (1,3,4,13,18). The distur-

bance of normal adrenergic mechanism in a number of

stress conditions gives rise to so-called lipid triad of

biomembranes damage. The activation of lipid

peroxidation (LPO) lead to severe disorders in

myocardiocytes structure (1,12). Many authors provide

data (some of them still hypothetical) about the role of free

radicals in pathogenesis of myocardial hypertrophy

(10,11), in induction of endothelial dysfunction (15,16) as

well as in development of heart failure (6,7). On the other

hand, in recent years, the data (mostly from the clinical

practice) about the cardioprotective effects of antioxidants

(2,5,6,12,14,17) increase. Another group of authors (9) has

ascertained the protective effect of vitamin E on the integ-

rity and function of mitochondria in cross-striated muscle,

whereas in regard to heart, such data are scanty.

Having in mind the data on the involvement of free radicals

in the genesis of hypertension and myocardial hypertrophy,

we aimed to study in dynamics the effect of the antioxidant

vitamin E on histomorphological damage in rat heart, sub-

jected to experimental hypertension.


1. Experimental animals.

In the experiments 51 male Wistar rats with an initial

body weight of 168± 9g were used.

2. Groups:

a) a control group - healthy, unoperated (n=6)

b) a group with experimental hypertension, untreated

(n=7)

c) a group with experimental hypertension, treated

with the solvent of vitamin E (n=18)

d) a group with experimental hypertension, treated

with vitamin E (n=20)

3. Experimental period.

Studies were conducted on the 3rd, 15th and 30th day

after the hypertension induction.

4. Model of hypertension

A vasorenal hypertension was induced according to the

method of Selye, modified by Kolarova. In breif - the

aortic stenosis was performed between the both renal

arteries and in this way the left kidney became a source

of pressure substances, which determined permanent

hypertension and myocardial hypertrophy.

101


5. Treatment with vitamin E.

A part of the animals with experimental hypertension

were treated with fat-soluble vitamin E (made in Japan)

at a dose of 10 mg/kg b.w. applied by an intraperitoneal

injection every day from the day of surgery till the

animal killing.

6. Systolic blood pressure measurement.

The systolic blood pressure was measured directly

through a catheter inserted into a. carotis communis.

Animals were given Nembutal (Abbott) at a dose of 40

mg/kg b.w to induce anesthesia prior surgery. A

multi-channel polygraph Biomedika C3 was used.

After than the animals were decapitated.

7. Material harvesting.

Immediately after animal decapitation, the hearts were

removed, weighed and a part of the left ventricle was

taken for histomorphological analysis. The myocardial

weight/100g b.w. ratio was calculated.

8. Histomorphological analysis.

After fixation with 3% formalin (neutral) the sections

were prepared and stained with hemalaun-eosin.

RESULTS

1. Blood pressure and myocardial mass.

In the rats operated after the method of Selye a

progressively developed hypertension and increased

myocardium/100g b.w. ratio were found. In the

hypertensive animals, treated with vitamin E, compared

to those of the untreated group, a slight but statistically

significant (P <0.05) decrease in blood pressure was

102

Antioxidant prevention in experimental hypertension ...

3rd

day from the begging

of the hypertension

15th


of the hypertension

30th


of the hypertension

Myocardial

damage

Solvent

n=5

Vitamin E

n=5

Solvent

n=8

Vitamin E

n=7

Untreated

n=7

Solvent

n=5

Vitamin E

n=8

Without special

feature

Mild

Severe

2

1

2

-

2

3

3

1

4

2

4

1

3

1

3

1

-

4

2

5

1

Mild - only eosinophilic

myositis and / or 1-3 small foci

of myolysis

Severe - more than 3 foci of

myolysis

Mild - only muscular hypertrophy

and / or 1-3 small foci of myo- or

arteriofibrosis

Severe - more than 3 foci of myo- or

arteriofibrosis or diffuse myofibrosis

Mild - only muscular hypertrophy and / or

1-3 small foci of myo- or arteriofibrosis

Severe - more than 3 foci of myo- or

arteriofibrosis and / or necrosis

Table 1. Degree of myocardial injury in hypertensive rats untreated, treated with the solvent of vitamin E or treated

with vitamin E

Fig. 1. Rat myocardium on the 3rd day of hypertension

induction. Diffuse myolysis of the left ventricular wall.

Activated endothelial cells of capillaries and small

number of fibroblasts were infiltrated the muscle fibers,

underwent myolysis. XE stain, zoom 80x.

Fig. 2. Rat myocardium on the 3rd day of hypertension

induction. Contractile stripes with light fields between

them /possibly due to rupture of protofibers/ in

myocardium area, unaffected directly by the myolysis.

XE stain, zoom 200x.

detected only on the 30th day of the experimental

period. It is also established a downward trend (on the

15th day of the experiment) and statistically significant

reduction (on the 30th day of the experiment) in the

myocardium/100 g b.w. ratio (P <0.05).

2. Histomorphological data - The histomorphological data

were presented on Table 1

On the 3rd day of hypertension, when haemodynamic stress

was particularly great in both groups of animals - untreated

and treated with vitamin E, severe heart damage was ob-

served (Fig.1, 2). On the 15th and 30th day of the experiment

(in contrast to the 3rd day) the histomorphological changes

in the myocardium of the hypertensive rats treated with vi-

tamin E differed significantly from those of untreated rats,

in particular: in the animals, treated with vitamin E there

was a pronounced myocardial hypertrophy, but the other

disabilities were poorly expressed or absent. Above all, the

necrotic and fibrotic changes were reduced - the focal and

diffuse myocardial fibrosis as well as coronary fibrosis and

coronary sclerosis. The histomorphological changes in un-

treated and treated hypertensive animals are presented on

Figures 1,2,3,4,5, and 6.

DISCUSSION

The fact that myocardial hypertrophy, coronary fibrosis and

myocardial fibrosis (as well as some other metabolic pa-

rameters, tested in our previous study, performed in the

same experimental setup) were influenced by the antioxi-

dant vitamin E, suggests its positive effect on the heart.

In regard to the cardioprotective effect of vitamin E we can

assume (based on data of other authors - 6,8,12,14,17), that

through inhibition of free-radical processes their harmful

effects on the myocardium reduce.

Having in mind that the genesis of myocardial hypertrophy

and myocardial and vascular lesions in hypertension, in-

duced after Selye, is a complex process (pressor overload,

catecholamines, RAS), our results give us reason to accept

the possible participation of another factor - the free radical

processes in this model of hypertension.

The possible mechanism of the protective effect of vitamin

E against cardiovascular damage in experimental hyperten-

103

Kolarova R., Z. Gendzhev

Fig. 3. Rat myocardium on the 15th day of hypertension

induction. Numerous foci of myocardial fibrosis covered

by hypertrophied muscle fibers in the left ventricular

wall. XE stain, zoom 80x.


induction; the hypertensive rats were treated with

vitamin E. In the left ventricular wall the muscle fibers

were strongly hypertrophied without any other damage.

XE stain, zoom 200x.


induction. Necrotic rings spots of the left ventricular

wall. Hypertrophied muscle fibers were found nearby.

XE stain, zoom 80x.

Fig. 6. Rat myocardium on the 30th day of the beginning

of hypertension the hypertensive rats were treated with

vitamin E. Pronounced hypertrophy of myocardial cells

without focal impairment. Part of the coronary artery

with minimal sclerotic changes. XE stain, zoom 80 x 20.

sion, induced according to the method of Selye, in our view

could be presented as follows (Fig. 7).

CONCLUSIONS

1. In the hypertensive rats, treated with vitamin E a slight

decrease in systolic blood pressure of the 30th day of

the experiment was detected.

2. In the hypertensive animals, treated with vitamin E a

reduction of the myocardium/100 g b.w. ratio on the

30th day of surgery was observed.

3. In this experimental model the treatment of the

hypertensive rats with vitamin E significantly protected

the myocardium from severe structural damage

(myocardial fibrosis, coronary fibrosis and coronary

sclerosis).

REFERENCES

1. Ìååðñîí ÔÇ, Ïøåííèêîâà ÌÃ. Àäàïòàöèÿ ê

ñòðåññîðíûì ñèòóàöèÿì è ôèçè÷åñêèì íàãðóçêàì;

Ìîñêâà, Måäèöèíà, 1988.

2. Benderi t ter M, Maingon P, Abadie C,

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Makino N. Status of myocardial antioxidants in

ischemia-reperfusion injury. Cardiovasc Res 2000;

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RR. Novel mechanisms in the treatment of heart fail-

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Sci Sports Exerc 2007; 39, 1544-1553.

9. Magalhaes J , Ferreira R, Neuparth MJ,

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min E prevents hypobaric hypoxia-induced mitochon-

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giotensin II. Circulation 1998; 98, 794-799.

11. Singh N, Dhal la AK, Seneviratne C &

Singal PK. Oxidative stress and heart failure. Mol

Cell Biochem 1995; 147, 77-81.

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Coldi tz GA, Rosner B & Willet t WC. Vita-

min E consumption and the risk of coronary disease

in women. N Engl J Med 1993; 328, 1444-1449.

13. Stopler M. Stress 2010; [Accessed 1/28/2010].

14. Schiffr in EL. Antioxidants in hypertension and

cardiovascular disease. Mol Interv 2010; 10, 354-362.

15. Taddei S, Virdis A, Ghiadoni L, Sudano I

& Salvet t i A. Endothelial dysfunction in hyperten-

sion. J Cardiovasc Pharmacol 2001; 38 Suppl 2,

S11-14.

16. Taddei S, Virdis A, Ghiadoni L, Versar i D

& Salvet t i A. Endothelium, aging, and hyperten-

sion. Curr Hypertens Rep 2006; 8, 84-89.

17. Thurich T, Berei ter-Hahn J, Schneider M &

Zimmer G. Cardioprotective effects of

dihydrolipoic acid and tocopherol in right heart hy-

pertrophy during oxidative stress.

Arzneimittelforschung 1998; 48, 13-21.

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104

Antioxidant prevention in experimental hypertension ...

EXPERIMENTAL HEMODINAMIC STRESS AND ITS INFLUENCE

FROM PHYSICAL EXERCISE, HYPOKINESIA AND ANTIOXIDANTS

Kolarova R.

Department of Physiology and Biochemistry, National Sports Academy "V. Levski", Sofia

ABSTRACT

Main myocardial adaptive reaction in different kinds of cardiac overload is its hypertrophy. In this respect,

the effect of chronic hemodynamic stress (experimental vasorenal hypertension for 30 days) in three groups of

Wistar rats - group 1 - animals with previous training by swimming, group 2 - animals with previous immobi-

lization and group 3 - with previous treatment by antioxidant was investigated. Changes in arterial blood

pressure; the ratio of grams of myocardial weight/100 g body weight (R); activity of some enzymes of cardiac

metabolism - of glycolysis (LDH, a-HBDH, PK), pentosomonophosphate shunt (Gl-6-PDH); GOT, GPT

(transaminases); as well as CK (of energetic metabolism) were studied. The negative effect of hypokynesia was

demonstrated - a reduced ability of the myocardium for adaptation to new hemodynamic overload (arterial

hypertension). On the other hand, under the influence of physical training by swimming, myocardial meta-

bolic rearrangement in rats rationalizing the cardiac reaction to a new pressure overload was observed. A

cardioprotective effect of antioxidants in hypertensive rats was also found.

Key words: physical exercise, hypokinesia, Vit. E, experimental hypertension, myocardial metabolism

INTRODUCTION

Hemodynamic stress on the cardiovascular system could be

of diverse origin. It may induce development of diastolic

dysfunction, damage of beta-adrenoreceptor mechanisms

and vascular structural changes. Often these injuries are the

cause of certain types of arrhythmias and sudden cardiac

death (3,9,12,14,16).

Main pathogenic mechanisms in the development of

stress-provoked damages are changes in membrane perme-

ability, activation of lipid peroxidation, calcium overload in

cardiomyocytes (1,6,12,13).

In regard of these disorders two questions arise: about en-

ergy provision of the myocardium and about the participa-

tion of individual metabolic pathways at distinct overloads

of the heart muscle. Under conditions of increased require-

ments towards the heart, as a result of various chronic

causes, the major adaptive response of the myocardium is

its hypertrophy. Moreover, the different conditions for the

occurrence of myocardial hypertrophy (MH) act as specific

"trigger" factors and determine certain specificity of the

metabolic changes in various forms of cardiac hypertrophy.

On the other hand, it is interesting whether the preliminary

functional status of myocardium, completely determined

by the metabolic processes within, will affect a new heart

overload, occurred subsequently. In this connection the aim

of the present study is to investigate the influence of prior

held physical training and pre-immobilization

(hypokinesia) on subsequently induced experimental hy-

pertension (a model of chronic hemodynamic stress), and

along with this, to investigate the role of antioxidant vita-

min E in hypertension prevention. For this purpose, in the

indicated experimental conditions, we monitored the

changes in the activities of certain "key" enzymes of

myocardial metabolism.


Experimental animals

Male Wistar rats with an initial body weight of 180 ±9 g.

Groups:

� 1 - control - healthy, unoperated rats (10)

� 2 - rats with experimental hypertension, untreated (7)

� 3 - rats with physical training held prior to the

experiment through swimming for 30 days +

subsequent induction of hypertension (12)

� 4 - rats with pre-immobilization for 45 days +

subsequent induction of hypertension (7)

� 5 - rats with experimental hypertension + antioxidant (9).

Experimental period

Studies were conducted on the 30th day after the induction

of hypertension.

Method of physical training: through swimming

(submaximal test) for 30 days.

Method of immobilization: the animals were housed in nar-

row plastic cages for 45 days.

Model of hypertension: according to the method of H.

Selye, modified by R. Kolarova (stenosis of the aorta be-

tween the renal arteries).

105


Treatment with the antioxidant vitamin E: the hypertensive ani-

mals receivedvitaminE10mg/kgb.w., i.p.daily, for30days.

Measurement of the enzyme activities

After several steps - material harvesting from the left ventricle,

weighing, determination of the coefficient R, homogenization

of myocardial tissue with subsequent centrifugation and ap-

propriate dilutions, the enzyme activities (U/g protein) were

determined. The activities of the following enzymes

Gl-6-PDH, PK, LDH, a-HBDH, GOT, GPT, CK were mea-

sured. The test reagents were purchased from Boehringer Ltd.

(the biochemical analyses in details are presented in our previ-

ous publications). The g myocard/100g b.w. ratio (R) was cal-

culated. The systolic blood pressure was measured directly us-

ing polygraph "Biomedica" C3. Statistics - The data were ana-

lyzed by the method of variational analysis.


The results are presented on Figures 1-9.

The systolic blood pressure in hypertensive animals (2nd

group), as well as in those combined with subsequent hyper-

tension (3rd, 4th and 5th groups) was statistically significantly

higher than that in control animals / p (1st-2nd) <0.001/, p

(1st-3rd) <0.001, p (1st-4th) <0.001, p (1st-5th) <0.001.

The increased blood pressure in these groups correlated

with growth rate of the coefficient R, suggesting the devel-

opment of myocardial hypertrophy /p (1st- 2nd <0.001 and p

(1st-3rd) <0.001, p (1st-4th) <0.05, p (1st-5th) < 0.01/.

Enzyme activities - more typical were the following

changes:

106

Experimental hemodinamic stress and its influence from ...

Fig. 1. Changes in arterial blood pressure.

Fig. 2. Changes in the ratio of myocardium/100 g b.w. (R).

Fig. 3. Changes in the enzyme activity of PK (pyruvate

kinase).

Fig. 4. Changes in the enzyme activity of LDH (lactate

dehydrogenase).

Fig. 5. Changes in the enzyme activity of alpha-HBDH

(alpha-hydroxi-butirate dehydrogenase).

Fig. 6. Changes in the enzyme activity of GL-6-PDH

(glucose-6-phosphate dehydrogenase).

Glycolytic enzymes - PK (Fig. 3) and LDH (Fig. 4): a

higher activity of PK in group 2 /p (1st-2nd) <0.01/, and

group 3 /p (1st-3rd) <0.001/, but decreased activity of this

enzyme in group 4 /p (1st- 4th) <0.05 / was detected. In con-

trast, in regard to LDH the opposite changes were observed

- a tendency of decreased activity in groups 2 and 3, and

statistically significantly increased activity in group 4 /p

(1st-4th) <0.001/.

Alpha-HBDH (Fig. 5) - its activity did not show statisti-

cally significant changes in the experimental groups com-

pared with the control.

Gl-6-P-DH (Fig. 6) - the most typical change in this en-

zyme was its reduced activity in the hypertensive rats,

treated with vitamin E (group 5) compared with untreated

hypertensive animals (group 2) with clearly expressed sta-

tistical accuracy, p (2nd-5th) <0.001, as this value tended to

approach the control value.

CK (Fig.7) - the changes in creatine kinase activity in all

groups were particularly demonstrative compared with the

control. There was a pronounced decrease in groups 2, 3

and 4, as in the three groups p <0.001 compared to the con-

trol animals, while in rats treated with antioxidant the activ-

ity is also reduced, but still showed some tendency to ap-

proach the control value - p (1st-5th) <0.01.

The transaminases GOT (Figure 8) and GPT (Figure 9)

showed similar changes in their activities in the distinct

groups. It was noteworthy that the highest values of both

enzymes were measured in the immobilized rats with sub-

sequent hypertension-for GOT the p (1st-4th) <0.001, and

for GPT - p (1st-4th) <0.05. In the hypertensive animals

(group 2) the activities of both enzymes were also increased

in comparison with the control values, which was more

pronounced in regard to GOT.

The resulting changes in myocardial enzyme activity in the

groups could be interpreted as a result of the impact of the

additionally induced hemodynamic stress (hypertension)

on the heart of experimental animals under different condi-

tions - previously conducted physical training (by swim-

ming) and pre-immobilization (hypokinesia), and antioxi-

dant treatment of the hypertensive rats. It is possible that

these changes were due to the subsequent severe overload

of the heart muscle thought hypertension moreover on the

background of these preliminary impacts.

The increase in blood pressure and the increase of the coef-

ficient R could be explain with the developing myocardial

hypertrophy (MH), as in the swimming group the factors

for MH development were the following - physical training

plus hypertension. In some our previous studies, we ob-

served in Wistar rats with induced immobilization stress

only, statistically significant changes in both blood pressure

and coefficient R The changes in regard to these two in-

dexes in this group clearly were due to the hypertension,

induced later.

The increased activity of pyruvate kinase (PK-catalyzed re-

action from the 10th step of glycolysis) in groups 2 and 3

suggested activation of glycolytic exchange in

hemodynamic stress, confirming the potential power of

glycolysis as a compensatory mechanism of the

myocardium under conditions of increased demands on

him (1). In these same two groups a tendency of fall in

LDH was observed, which is in favor of aerobic metabo-

lism within them, whereas in group 4 with pre-immobiliza-

tion the LDH activity was strongly increased in comparison

with other groups - most likely an indicator of compensa-

tory activated anaerobic glycolysis. It is noteworthy that

Gl-6-PDH - an enzyme with a powerful compensatory role

in chronic overload of the myocardium particularly, was

with highly increased activity only in the group of hyper-

tensive animals, while in the other groups its activity did

not differ significantly from the control value. The fact, that

in trained animals this enzyme showed only an upward

trend, could be linked with the opinion of other authors

107

Kolarova R.

Fig. 7. Changes in the enzyme activity of CK (creatine

kinase).

Fig. 8. Changes in the enzyme activity of GOT

(glutamate oxalacetic transaminase).

Fig. 9. Changes in the enzyme activity of GPT

(glutamate pyruvate transaminase).

(4,10) for the protective role of physical exercise on the

heart. An important result was that in the hypertensive rats

treated with vitamin E a significant decrease in

Gl-6-PDH-activity was found /p (2nd-5th) <0.001/, which is

consistent with our results obtained in another experimental

design and using a synthetic antioxidant (7). The significant

decline of CK-activity in all groups most likely was due to

increased energy consumption because of rapid overload of

the myocardium. Only in group 5 this enzyme showed a

weak tendency to approach the control level, which still

speaks in favor of the protective effect of vitamin E. In rats

with prior immobilization, except the above results on

LDH, the transaminases were increased significantly,

which was probably a compensatory response to increased

protein synthesis, which correlated with the increase of the

coefficient R - an indicator for the MH development. Ac-

cording to some authors, there are probably other, still hy-

pothetical mechanisms to stimulate protein synthesis and

survival of cells (2,17). There is also evidence that the

hypokinesia, except induction of oxidative stress, induces

also antioxidant response with individual variability (8,11).

CONCLUSION

The results show that the negative effect of hypokinesia

consisted in reducing the level of certain metabolic pro-

cesses in the myocardium, which determinate its reduced

ability to adapt in the subsequent considerable overload -

hypertension. On the other hand, the physical exercise

through impact on important exchange pathways enables

optimal response of the heart under conditions of increased

requirements. The results also speak in favor of the

cardioprotective effects of antioxidants.

REFERENCES

1. Ìååðñîí ÔÇ, Ïøåííèêîâà ÌÃ. Àäàïòàöèÿ ê

ñòðåññîðíûì ñèòóàöèÿì è ôèçè÷åñêèì íàãðóçêàì;

Ìîñêâà, Måäèöèíà, 1988.

2. Cat taruzza M, Hecker M. A new twist to the

complex response of vascular cells to oxidative stress.

Circ Res 2008; 102, 273-274.


Palma & Chiar iel lo M. Reactive oxygen species

and antioxidants in the pathophysiology of cardiovas-

cular disease: does the actual knowledge justify a

clinical approach? Curr Vasc Pharmacol 2010; 8,

259-275.

4. Emter CA, McCune SA, Sparagna GC,

Radin MJ & Moore RL. Low-intensity exercise

training delays onset of decompensated heart failure

in spontaneously hypertensive heart failure rats. Am J

Physiol Heart Circ Physiol 2005; 289, H2030-8.

5. Gey KF, Puska P, Jordan P & Moser UK.

Inverse correlation between plasma vitamin E and

mortality from ischemic heart disease in cross-cultural

epidemiology. Am J Clin Nutr 1991; 53, 326S-334S.

6. Hamil ton KL. Antioxidants and cardioprotection.

Med Sci Sports Exerc 2007; 39, 1544-1553.

7. Kolarova R. Myocardial catecholamine stress and

antioxidant protection. Sci. Res. J. of South-West Univ

2008; 1, 33-37.

8. Margari t is I , Rousseau AS, Marini JF &

Chopard A. Does antioxidant system adaptive re-

sponse alleviate related oxidative damage with long

term bed rest? Clin Biochem 2009; 42, 371-379.

9. Perr ino C, Naga Prasad SV, Mao L, Noma

T, Yan Z, Kim HS et al . Intermittent Pressure

overload triggers hypertrophy-independent cardiac

dysfunction and vascular rarefaction. J Clin Inves

2006; 116, 1547-1560.

10. Puffer JC. Exercise and heart disease. Clin Corner-

stone 2001; 3, 1-9.

11. Sahin E & Gumuslu S. Immobilization stress in

rat tissues: alterations in protein, lipid peroxidation

and antioxidant defense system. Comp Biochem

Physiol Toxicol Pharmacol 2007; 144, 342.

12. Sam F, Kerstet ter DL, Pimental DR,

Mulukut la S, Tabaee A, Bris tow MR et al . ,

Increased reactive oxygen species production and

functional alterations in antioxidant enzymes in hu-

man failing myocardium. J Card Fail 2005; 11,

473-480.

13. Schiffr in EL. Antioxidants in hypertension and

cardiovascular disease. Mol Interv 2010; 10, 354-362.

14. Sharma AB, Sun J, Howard LL, Wil l iams

AG, JR & Mallet RT. Oxidative stress reversibly

inactivates myocardial enzymes during cardiac arrest.

Am J Physiol Heart Circ Physiol 2007; 292,

H198-206.

15. Stampfer MJ, Hennekens CH, Manson JE,

Coldi tz GA, Rosner B & Willet t WC. Vita-

min E consumption and the risk of coronary disease

in women. N Engl J Med 1993; 328, 1444-9.

16. Touyz RM. Advancement in hypertension

pathogenesis: some new concepts. Curr Opin Nephrol

Hypertens 2011; 20, 105-106.

17. Wong CM, Cheema AK, Zhang L, Suzuki

YJ. Protein carbonylation as a novel Mechanism in

redox signaling. Circ Res 2008; 102, 310-318.

108

Experimental hemodinamic stress and its influence from ...

AGE-RELATED EFFECTS ON THE SENSITIVITY TO GLOBAL

MOTION DIRECTION DETERMINED BY THE METHOD

OF CLASSIFICATION IMAGES

Stefanov S.1, K. Alexiev

2, N. Bocheva

1, B. Genova

1

1Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia; 2Institute of Information and

Communication Technologies, Bulgarian Academy of Sciences, Sofia

SUMMARY

The method of classification images was used to evaluate whether the perceptual template applied by subjects

in the discrimination of global motion direction changes with ageing. The stimuli were 25-frame motion se-

quences of signal and noise band-limited elements. The motion direction of the signal dots was randomly se-

lected from a uniform distribution with range of 60° and mean ±10° from the vertical direction. The noise dots

moved in random directions taken uniformly in the range 0°-360°. The signal-to-noise ratio was determined in

a preliminary experiment separately for each observer to ensure 75% correct responses. The task of the ob-

servers was to discriminate whether the mean direction of motion was to the left or to the right of the vertical.

Nine younger (mean age 19.8 yrs) and nine older subjects (mean age 72 yrs) participated in the study. Classifi-

cation images were calculated from the directional distribution of all potential dot trajectories in the motion

sequences. The results show that the older observers have higher internal noise and they need higher sig-

nal-to-noise ratio in order to successfully perform the task. The calculated perceptual templates show similar

directional tuning but different temporal evolution for the two age groups. These findings are discussed in re-

lation to the age-related changes in center-surround suppression in dynamic information processing.

Key words: motion direction discrimination, classification images, internal noise, directional tuning, temporal

integration

INTRODUCTION

Motion information is essential for various everyday tasks

like navigation, figure-ground segregation, collision avoid-

ance, determination of spatial layout and the three-dimen-

sional shape of the objects, etc. Several studies have dem-

onstrated visual deficits in motion processing with age (e.g.

13,14,16,3,4). Kavcic et al (7) suggest that age-related

changes in spatio-temporal integration are the main factor

contributing to the impaired perception of optic flow in el-

derly and in Alzheimer patients. Differences in perfor-

mance might occur, however, not only due to perceptual

factors but to changes in attention and the to the impaired

attentional capacities of the elderly people (9), their inabil-

ity to scale attention to the scope of relevant stimuli (5) or to

differences in the decision strategies used in task

performance.

The aim of the present study was to evaluate the age-related

changes in the cognitive processes in a task related to inte-

gration of motion information. The experimental design

used in the study resembles the classification image para-

digm (e.g. 1,2,10 for a recent review). In a typical experi-

ment of this type the observers classify signals in the pres-

ence of additive noise. The characteristics of the noise pat-

terns for the correct and the incorrect trials are analyzed in

order to reveal the perceptual "template" used for solving

the task e.g. to infer the stimulus features that determine the

perceptual decisions. Our stimuli could be described as a

signal modulated by noise in the presence of background

noise. This stimulation increases the perceptual load and al-

lows not only to evaluate the perceptual abilities of the sub-

jects, but also to reveal differences in performance due to

attentional processes and different decision strategies.


Nine older (mean age 73.9, range 66-82 years) and nine

younger ( mean age 19.5; range 16-24 year) observers took

part in the study. All participants have normal or corrected

to normal vision and have passed eye examination. None of

them reported having any major health problems.

The stimuli were motion sequences of 48 moving elements.

Each motion sequence contained 25 frames. The moving

elements were spots with intensity profile that varied as a

Laplacian of Gaussian. The mean speed of motion was 3.67

deg/s. The moving elements were divided in two groups.

109



S. Stefanov, Institute of Neurobiology, Bulgarian Academy of Sci-

ences, 23 Acad. G. Bonchev St. Sofia Bulgaria 1113


In the first, the direction of motion was restricted to a range

of 60? and the speed varied within 50-150% from the mean

speed. Each of these elements randomly changes direction

on every frame. The range of directions was centered either

at ±10° from the vertical downward direction. These ele-

ments will be labeled as the main set. The other group of el-

ements moved with constant direction chosen randomly in

the range 0°-360° with a constant speed of 3.67 deg/s. They

will be referred as supplementary set. The number of ele-

ments in each set was determined in preliminary experi-

ment to ensure an accuracy of 75% in motion discriminate

for each observer. The moving elements were presented in

a circular aperture with radius of 7 deg of arc. Table 1 pres-

ents the number of elements in the main set needed to en-

sure similar performance for the two age groups.

The stimuli were presented on a computer screen (21" Dell

Trinitron with Nvidia Quadro 900XGL graphic board) in a

1280x1024 resolution mode. A chin-rest was used to main-

tain a fixed distance to the screen. The refresh rate of the

monitor was 85 Hz. The observation was binocular.

The task of the observers was to indicate whether the mean

direction of motion was to the left or to the right of the verti-

cal. To respond the younger observers used two buttons of

a computer mouse, while the older observers gave an oral

response and it was recorded by the experimenter. Each ob-

server participated in 10 experimental sessions of 500 trials

(5000 trials in total). The random seed for stimulus genera-

tion was recorded in order to reproduce the motion se-

quences for further analysis. The last 500 trials were exact

repetition of the trials used in the previous session and were

used to evaluate the internal noise of the two age groups.


First, we estimated the proportion of cases when the ob-

servers gave the same response to the identical stimuli ses-

sions 9 and 10 i.e. the proportion of coincidence. If the level

of internal noise in the visual system is low or negligible,

the responses to the identical stimuli should be the same.

Fig. 1 shows the effect of age on this measure of internal

noise; Fig. 2 shows the proportion of correct responses ob-

tained for each observer for the whole experimental set. It is

clear that the level of internal noise is higher in the older

groups and that without feedback the performance of the

older observers deteriorates from the initial level.

The classification images methodology is based on present-

ing a signal perturbed by noise. On every trial the noise

characteristics could either help or hinder the signal detec-

tion because they will correlate with the template used by

the observer to perform the task. Therefore, the methodol-

ogy relies on studying the collection of noise patterns clas-

sified by subject's responses. In our stimuli the signal is the

uniform distribution of directions in the main set and in the

supplementary sets as they both has fixed range and mean.

On every trial the distribution of the directions varied de-

pending on the number of elements in the main and the sup-

plementary set and the process of random generation, but

110

Age-related effects on the sensitivity to ...

Fig. 1. The proportion of coincidence of subject’s

responses on equivalent stimuli

Fig. 2. The proportion of correct responses for all

experimental session

Fig. 3. Example of the different decision template for

the stimuli moving left (solid line) and right (dash line)

their distributions remained the same. Thus, we can regard

as noise patterns the random deviations of the motion

directions in the stimulus from the predefined uniform

distributions.

To evaluate the motion direction of an element we deter-

mined its closest neighbor on the next frame. This selection

rule corresponds to the preference of the visual system to-

wards slow motions (e.g. 15). We estimated the distribution

of the directions of the elements and determined the differ-

ence in frequencies of the simulated distributions and their

realization on every frame. For this purpose we divided the

range of 360° in 45 bins of directions. These were the noise

patterns we classified by the subjects' response and by the

stimulus type. All subsequent analyses were performed

separately on the noise patterns obtained for each subject.

Here we present the results related to the directional per-

ceptual template used by the observers to discriminate the

motion directions i.e. the template averaged by time. This

decision was based on the physiological data for the tempo-

ral dynamic of directional selectivity in MT (e.g. 12) that

show that changes in the receptive field structure and pref-

erence of the neurons in MT occur over the first hundred

milliseconds. Moreover, Neri & Levi (11) have shown an

independence of the temporal and spatial parameters in dis-

crimination motion direction using classification images.

We used a generalized additive model (as suggested by 8)

with a probit link to evaluate the contribution of each direc-

tional bin on the discrimination performance. The analysis

was applied to the noise patterns taking into account

whether the stimulus moved on average to the left or to the

right. This approach allows to obtain a smooth classifica-

tion image and to take into account the dependencies

introduced by the observer's choices.

We tested two models -a linear template model that as-

sumes that the decision is determined by the correlation of

the noise pattern and the perceptual template, and a sec-

ond-order model that assumes the existence of

nonlinearities in the decision processes. For this case addi-

tional terms were added by squaring the directional infor-

mation of the noise patterns.

The results show that all subjects used different templates

for the left and for the right stimuli. The non-linear model

describes better the performance and significantly increas-

ing the explained deviance. The significant contribution of

the non-linear terms might be related to center-surround ef-

fects in processing directional information by the neurons

in MT (12), to temporal effects or to the correlation be-

tween the neurons with similar directional preferences (6).

Our task requires comparison between the perceived direc-

tion and an internal standard. The internal standard will

vary in location due to the internal noise. Therefore, the

perceptual template might be the difference between the

distribution of locations of this standard and the distribution

of the signal. This would suggest that the observers were

able to separate the elements from the main set from those

of the supplementary set i.e. that the observers are sensitive

to the motion differences between the two sets. For exam-

ple, the observers might separate the two sets by the differ-

ences in velocity or in temporal dynamics. However, an-

other possible discrimination strategy would be to compare

the mean direction of motion of all moving elements to the

internal standard. These two cases would differ by the posi-

tion of the peaks in the perceptual template. Fig. 3 shows

examples of these strategies from the two age groups. It

should be mentioned that there is a significant variability in

both groups. It is related to the differences in sensitivity and

to differences in strategies used by the observers.

CONCLUSIONS

The present study aims to evaluate whether differences in

the sensitivity to global motion direction affect the decision

strategy of the observers. The performance of the task to

discriminate the motion direction of a noisy motion se-

quence with respect to an internal standard depends both on

the external and the internal noise in the stimulus. The re-

sults confirm the higher level of internal noise of the older

observers. However, no significant differences were ob-

served in the decision strategy used by the two age groups.

The data suggest that to make a decision, the observers pool

all motion directions and compare their mean with an inter-

nal standard. The second-order terms significantly contrib-

ute to the perceptual template used to discriminate the

global motion direction of dynamic patterns. Further

analyses are needed to evaluate the temporal dynamics in

this process.

REFERENCES

1. Ahumada, A. J . , Jr . Perceptual classification im-

ages from Vernier acuity masked by noise. Percep-

tion, 25, (1996). ECVP Abstract Suppl.

2. Ahumada, A. J . , Jr . Classification image weights

and internal noise level estimation. J. Vis.,2, 2002,

121-131

3. Bennet t , P. J . , R. Sekuler , A.B. Sekuler .

The effects of aging on motion detection and direc-

tion identification. Vision Res, 47, 2007, 799-809

4. Bil l ino, J . , F . Bremmer, K. R. Gegenfurtner.

Differential aging of motion processing mechanisms:

Evidence against general perceptual decline. Vision

Res., 48, 2008, 1254-1261

111

Stefanov S., K. Alexiev, N. Bocheva ...

Older participants Younger participants

Signal dots number Signal dots number

2 15 B 8

3 31 C 26

4 20 D 29

7 26 K 24

Table 1. Number of the signal elements needed to

ensure 75% correct motion direction discrimination

5. Greenwood, P.M, R. Parasuraman. The scal-

ing of spatial attention in visual search and its modifi-

cation in healthy aging. Percept. Psychophys.,66,

2004, 3-22

6. Huang, X. , S.G. Lisberger . Noise Correlations

in cortical area MT and their potential impact on

trial-by-trial variation in the direction and speed of

smooth pursuit eye movements. J. Neurophysiol.,

101, 2009, 3012-3030

7. Kavcic , V, W. Vaughn, C.J . Duffy . Distinct

visual motion processing impairments in aging and

Alzheimer's disease. Vision Res., 51, 2011, 386-95.

8. Knoblauch, K. , L.T. Maloney. Estimating

classification images with generalized linear and addi-

tive models. J. Vis., 8, 2008, 1-19

9. Mapstone, M., K. Dickerson, C. J . Duffy .

Distinct mechanisms of impairment in cognitive age-

ing and Alzheimer's disease. Brain, 131, 2008,

1618-1629

10. Murray, R. F. Classification images: a review. J.

Vis., 11(5), 2011,1-25

11. Neri , P. , D. M. Levi . Temporal dynamics of di-

rectional selectivity in human vision. J. Vis., 8(1),

2008, 1-11

12. Perge, J .A. , B. G. Borghuis , R. J . E. Bours ,

M. J . M. Lankheet , R. J . A. van Wezel . Dy-

namics of directional selectivity in MT receptive field

centre and surround. European J. Neurosci., 22, 2005,

2049-2058

13. Scialfa , C. T. , D. W. Kline, B. J . Lyman.

Age-differences in target identification as a function

of retinal location and noise-level-Examination of the

useful field of view. Psych. Aging, 2, 1987, 14-19

14. Snowden R. J. , E. Kavanagh. Motion percep-

tion in the ageing visual system: minimum motion,

motion coherence and speed discrimination thresh-

olds. Perception, 35, 2006, 9-24

15. Weiss , Y. , E.H. Adelson. Slow and smooth: a

Bayesian theory for the combination of local motion

signals in human vision. A.I. Memo No. 1624., 1998

Cambridge, MA: MIT.

16. Wood J. M. , M. A. Bullmore. Changes in the

lower displacement limit for motion with age.

Ophthal. Physiol.l Optics, 15,199, 36.

ACKNOWLEDGEMENT

This research is supported by Grant TK01/200-2009 of the

National Science Fund, Bulgaria

112

Age-related effects on the sensitivity to ...

SPECIFICITY IN BREATHING REGULATION DURING PHYSICAL

EXERCISE IN TAEKWONDO NATIONAL ATHLETES

Tzvetkov S.

NSA - Department of Sports medicine, NSA - Sofia

ABSTRACT

Maximal oxygen consumption (VÎ2max) is a parameter which depends on breathing response, oxygen carry-

ing capacity of blood, cardiac output and intensity of metabolic processes. According to some scientific

sources, up to 11% of the body total VÎ2 during heavy exercise can be used by the respiratory muscles. In

Taekwondo training specific breathing patterns are essential and fundamental factors for successful competi-

tive performance. The purpose of the study was to compare the breathing pattern and maximal respiratory

parameters of national level Taekwondo athletes and of non-athlete students and their effect on VÎ2max regis-

tered during incremental exercise test. The results demonstrated that significantly higher values for

VO2max/kg were registered in the athlete group and these higher values were not accompanied by correspond-

ing differences in maximal speed, maximal heart rate and maximal lactate concentration. It was also found

that Taekwondo athletes reacted to exercise with moderate and high intensity with markedly less expressed

increase of pulmonary ventilation (VE) compared to the student group, this increase being predominantly due

to a significant increase of tidal volume (VT) with less pronounced increase in breathing frequency (BF). As a

conclusion, we can summarize that the use of specific breathing exercises in the training process of

Taekwondo athletes improves the regulation of their external breathing and ensures optimal ventilatory re-

sponse, characterized by increase in VE during exercise with moderate and heavy intensity as a result of opti-

mal increase predominantly of VT with less increase of BF.

Key words: ventilatory response, maximal oxygen consumption, maximal incremental exercise test,

taekwondo athletes

INTRODUCTION

It is widely acknowledged that maximal oxygen consump-

tion (VÎ2max) is a key parameter for assessment of athletes'

maximal functional capacity (6). Researches have also found

that VÎ2max is an integral parameter depending to a large ex-

tent on breathing response, oxygen transport capacity of arte-

rial blood, cardiac output and intensity of cellular metabolic

processes (8). According to J. Wilmore and D. Costill

(2006), up to 11% of the body total VÎ2 and 15% of the car-

diac output during heavy exercise can be used by the respira-

tory muscles. Some authors even comment that the oxygen

cost of the maximal exercise ventilation may reach up to

18% of the maximal oxygen consumption (5). For that rea-

son there is considerable interest within the scientific com-

munity in the effect of specific respiratory muscle training

and specific breathing regulation on the performance of re-

spiratory muscles and on total exercise performance (2). Fur-

thermore, there isn't yet a common scientific consensus on

the problemand discussions on the effect of respiratory train-

ing on exercise performance parameters continue.

It is also well known that in Martial Arts appropriate

breathing and particularly specific breathing patterns are

essential and fundamental factors for concentration and

successful performance. Therefore athletes involved in

Eastern martial arts disciplines (such as Taekwondo) dedi-

cate significant time during training sessions for practicing

specific breathing techniques. Moreover, a leading ap-

proach in Taekwondo training is the belief that during per-

formance of a Taekwondo form, with harmony of breath-

ing and correct breathing pattern, one can feel rhythmical

movement at every moment of execution of the

Taekwondo sport technique. According to the main breath-

ing instructions in the breathing model most commonly

used in Taekwondo "the Taekwondo athletes have to

breath slowly and imagine the air they are breathing as it

fills their entire abdomen, concentrating and when they ex-

hale, they can imagine the energy moves to any part of their

body while the air is being released through their nose" (7).

In accordance with these instructions, athletes practicing

this sport purposely use specific breathing techniques for

improvement of external breathing regulation during

exercises which are typical for this kind of sport.

The purpose of the study was to compare the main charac-

teristics of the exercise breathing pattern and some key

maximal respiratory parameters of national level

113



S. Tzvetkov, NSA – Department of Sports medicine,

St. grad, 1700 Sofia, Bulgaria


Taekwondo athletes with those of non-athlete students and

their potential effect on maximal oxygen consumption reg-

istered during maximal incremental exercise test.

METHODS

The subjects of the study were 10 high-level national male

athletes (main group - MG) from the Bulgarian national

team of Taekwondo, Olympic version - WTF, and 8 male

non-athlete students from the National Sport Academy

(control group - CG). All subjects were healthy, injury-free

and non-smokers with the following physical characteris-

tics: main group: age - 23.90±5.13 years; height -

178±7.59 cm; weight - 71.54±8.25 kg; BMI - 22.67±1.76

and control group: age - 22.78±1.45 years; height -

179±8.32 cm; weight - 74.10±7.54 kg; BMI - 23.03±1.94.

Before participating in the study, each subject was familiar-

ized with the experimental procedure and informed of the

risks associated with the testing protocol. All participants

gave their written voluntary informed consent. To avoid

external influence on the tested functional parameters, 2

days prior testing all athletes from the main group carried

out their training at low intensity. The subjects from both

groups were tested on separate consecutive days within 1

week. After a 5-minute warm-up at low speed, which al-

lowed the subjects to adapt to exercise, they performed a

maximal incremental test on a treadmill (Quasar 4.0 Med;

HP Cosmos, Germany). An exercise stepwise model was

applied, with initial speed of 6 km/h, increased by 1.2 km/h

every 90 sec, with constant elevation of 2.5%, until objec-

tive voluntary exhaustion was achieved (3). Blood lactate

concentration was measured immediately prior the test and

during the recovery period (2nd; 4th; 7th; 10th; 12th min)

through analysis of arterialized capillary blood. The follow-

ing criteria were used to verify maximal exertion: a plateau

in oxygen consumption dynamics; RERmax>1.10; regis-

tered maximal heart rate close to the maximal heart rate

predicted from the subject's age; physical inability to con-

tinue the test (8). Gas exchange was measured throughout

the test using a metabolic system Oxycon Pro (Erich Jaeger

GmBH & Co Wuerzburg, Germany). Before each test, the

gas-analyzers were calibrated using ambient air (20.9% O2

and 0.04% CO2) and calibration gas (0.00% O2 and 4.83%

CO2). In all tested subjects we registered the following

functional and hematological parameters: maximal

ergometric work capacity (expressed as the maximal speed

achieved on the treadmill, Smax); maximal oxygen con-

sumption (VO2max); maximal pulmonary ventilation

(VÅmax); maximal breathing frequency (BFmax); maximal

tidal volume (VTmax); maximal heart rate (HRmax); maximal

lactate concentration (Lamax). Relative values were calcu-

lated to ignore the influence of body weight in the analysis

of the maximal oxygen consumption. They were expressed

as ratio of absolute values to body weight of the respective

athlete (VO2max/kg). Student's T criterion was used for eval-

uation of the statistical results with standard level of signifi-

cance (SPSS 14.0; p<0.05). The graphical presentation of

results was realized with specialized software

(GraphPadPrism 5.0).

RESULTS

The statistical analysis of the results of the tested subjects from

both groups demonstrated that, despite the lack of a significant

difference in physical characteristics, significantlyhigheraver-

age values for VO2max/kg were registered in national level

Taekwondo athletes compared to the subjects fromthe control

group (VO2max/kg (MG) (56.99±5.94) - VO2max/kg (CG)

(49.23±1.89)= 7.76ml/kgmin, p<0.05). It was also established

that these higher VO2max/kg values were not accompanied by

statistically significant differences in the registered values of

Smax, HRmax and Lamax (Smax (MG) (14.54±1.53) - Smax (CG)

(13.21±0.91) = 1.33 km/h, p>0.05; HRmax (MG) (194.3±8.12)

114

Specificity in breathing regulation during physical exercise in ...

Fig. 1. Dynamic of pulmonary ventilation in subjects

from both experimental groups (main group: n = 10;

control group: n = 8).

Fig. 2. Dynamic of tidal volume in subjects from both

experimental groups (main group: n = 10; control

group: n = 8).

Fig. 3.Dynamic of breathing frequency in subjects from

both experimental groups (main group: n = 10; control

group: n = 8).

- HRmax (CG) (197.1±10.41)=-2.80 beats/min, p>0.05; Lamax

(MG) (12.61±1.93) - Lamax (CG) (13.87±2.11)=

-1.26mmol/l, p>0.05).

At the same time, statistically significant difference between

the groups was found for the main maximal respiratory pa-

rameters: VEmax (MG) (130.7±13.68) - VEmax (CG)

(146.8±17.43)=-16.1 l/min, p<0.05; VÒmax (MG) (2.74±0.42)

- VÒmax (CG) (2.11 ±0.35) = 0.63 l, p<0.05; BFmax (MG) (48.4

±4.71) - BFmax (CG) (62.6 ±8.19) = -14.2 min-1, p<0.05;

VEmax/VO2max (MG) (32.06±3.28) - VEmax/VO2max (CG)

(40.24±5.67)=-8.18, p<0.05. The registered significantly

lower VEmax and BFmax values in Taekwondo athletes moti-

vatedus toanalyze indetail thedynamicof thestudied respira-

tory parameters during the incremental exercise tests in both

groups. It was found that during the test all Taekwondo ath-

letes reacted to exercise with moderate and high intensity with

markedly less expressed increase of pulmonary ventilation

(VE) compared to the control group (Fig. 1), this increase be-

ing predominantly due to a significant increase of tidal volume

(VT) with less pronounced increase in breathing frequency

(BF) - Fig. 2 and Fig. 3.

DISCUSSION

According to P. Macklem and J. Mead (1988), the effect of

VE during exercise depends on actual alveolar ventilation

(VA) which is determined by VE, physiological dead space

(VDS) and VT. This dependence is expressed with the fol-

lowing equation: VA = VE (1 - VDS/VT). Therefore as a

matter of principle a person can achieve the optimal VA if he

has high VE and low VDS/VT. Thus, the ratio VDS/VT

would be low if the breathing pattern ensures optimal low

VDS and optimal high VT. For that reason it is generally ac-

cepted that optimal ventilatory response is observed when

the breathing pattern during exercise is characterized with

predominant increase of VT in relatively constant BF (6).

This form of ventilatory response pattern is characteristic of

individuals who entrain their breathing cadence to a some

constant unit consistent with specifics of physical activity.

Such breathing pattern is observed in the subjects from the

main group resulting most probably fromthe routine practice

of specific breathing techniques (Fig. 2, Fig. 3). From an-

other point of view, it is established that during exercise at

moderate and high intensity in cases when VE (respectively

VEmax) is typically inordinately high for the work rate per-

formed, increase of VDS/VT is observed resulting in limited

efficacy of the ventilatory response (8). Furthermore, ac-

cording to some authors the inordinately high VEmax and

the associated inefficient ventilatory mechanical work,

which could be also related to inadequate pulmonary blood

flow and perfusion, would result in lower VO2max (1,6). In

accordance with these scientific positions, the presented

graphical analysis of the exercise breathing patterns of the

tested subjects (Fig. 1, Fig. 2, Fig. 3) and the lack of statisti-

cally significant difference in Smax, HRmax and Lamax between

the groups, we can assume that the registered higher

VO2max/kg values (VO2max/kg (MG)= 56.99±5.94ml/kg min,

VO2max/kg (CG)=49.23±1.89ml/kg min) in Taekwondo ath-

letes are probably due to a more efficient ventilatory re-

sponse pattern and lower losses as a result of an optimal

maximal ventilatory mechanical work (VEmax

(MG)=130.7±13.68l; VEmax (CG)=146.8±17.43 l). The ob-

served significantly higher breathing economy in the main

group compared to the non-athlete students group

(VEmax/VO2max (MG) - VEmax/VO2max (CG) = - 8.18, p<0.05)

is an additional confirmation for this hypothesis. At the same

time, we should stress that according to some authors the in-

creased breathing economy in breathing patterns with pre-

dominant increase of VT should not be accepted as an abso-

lute physiological rule, since breathing with excessively high

VT is associated with disproportionately large increase in the

elastic work of breathing, especially during the exercises

with heavy intensity, leading to significant increase in the

cost of total ventilatory mechanical work (4).

CONCLUSION

Taking into consideration the data obtained in this study

and the opinion of W. McArdle et al. (2006) that "deeper

breathing provides more effective alveolar ventilation than

a similar minute ventilation achieved through an increased

breathing rate" we can summarize that the purposeful use

of specific breathing exercises in the training process of

Taekwondo athletes improves the regulation of their exter-

nal breathing and ensures optimal ventilatory response,

characterized by increase in pulmonary ventilation during

exercise with moderate and heavy intensity as a result of

optimal increase predominantly of tidal volume with less

increase of breathing frequency.

REFERENCES

1. Caruana-Montaldo, B. , Gleeson, K. ,

Zwil l ich, C. The control of breathing in clinical

practice. Chest, 2000, 117, 1, 205-225.

2. Gigl iot t i , F . , Binazzi , B. , Scano, G. Does

training of respiratory muscles affect exercise perfor-

mance in healthy subjects? Respiratory medicine,

2006, 100, 2, 1117-1120.

3. I l iev, I . A method for complex testing of high-class

athletes. Sofia, BSFS, 1974, 56-79.

4. Macklem, P. , Mead, J . Handbook of Physiology:

The Respiratory System. Philadelphia, An American

Physiological Society Book, Lippincott Williams &

Wilkins, 1988, 605-629.

5. Marks D. The oxygen cost of ventilation and its ef-

fect on the VO2 plateau. K�ln, VDM Verlag Dr

Muler, 2009, 41-51.

6. McArdle, W., Katch, F. , Katch, V. Exercise

Physiology: Energy, Nutrition and Human Perfor-

mance, Philadelphia, Lippincott Williams & Wilkins,

6th edition, 2006, 260-275.

7. Savoie , G. Taekwondo: A technical manual.

Berkeley, California, North Atlantic Books, 2009,

43-67.

115

Tzvetkov S.

8. Wasserman, K. , Hansen, J . , Sue, D. et al .

Principles of exercise testing and interpretation. Phila-

delphia, Lippincott Williams & Wilkins, 4th ed., 2005,

77-106.

9. Wilmore, J . , Cost i l l , D. Physiology of sport and

exercise. Champaign, Human Kinetics, 3rd ed., 2006,

242-264.

116

Specificity in breathing regulation during physical exercise in ...

THE STRENGTH-DURATION PROPERTIES IN SIMULATED

DEMYELINATING NEUROPATHIES DEPEND ON THE MYELIN

SHEATH AQUEOUS LAYERS

Krustev S. M.1, N. Negrev

1, D. I. Stephanova

2

1Medical University - Varna; 2Institute of Biophysics and Biomedical Engineering,

Bulgarian Academy of Sciences - Sofia

ABSTRACT

BACKGROUND: The strength-duration properties (time constants and rheobases) reflect the membrane

properties in body excitable structures. OBJECTIVE: To expand our studies on the membrane properties,

the effect of the myelin sheath aqueous layers on the strength-duration properties in simulated by us

demyelinating neuropathies is compared. METHODS: Using a multi-layered model of the myelin sheath of

human motor nerve fibre, the strength-duration time constants and rheobases are calculated and compared

in simulated normal case and in three cases of progressively greater demyelinations [such as internodal sys-

tematic demyelinations (ISDs), paranodal systematic demyelinations (PSDs) and paranodal internodal sys-

tematic demyelinations (PISDs)] without/with aqueous layers within the myelin sheath. Two cases of each

demyelinated type are mild and one is severe. RESULTS: When the aqueous layers are taken into account, the

strength-duration properties are improved in the ISDs and in the PSDs, except for the rheobases in the ISDs

where they worsen. However, the effect of the aqueous layers on these properties is neutralized when the

demyelinations are heterogeneous such as in the PISDs. CONCLUSIONS: Previously, it has been proven by

us that the ISDs are specific indicators of Charcot-Marie-Tooth disease type 1A (CMT1A) whereas the PSDs

and the PISDs are specific indicators of chronic inflammatory demyelinating polyneuropathy (CIDP) and its

subtypes. The present study shows that the strength-duration properties in patients with CMT1A and CIDP

depend on the myelin sheath aqueous layers.

Key words: myelin sheath aqueous layers, strength-duration time constant, demyelinating neuropathies,

computational neuroscience

INTRODUCTION

The typical picture which we have from electron micros-

copy of fixed myelin sheath is rather misleading. The my-

elin lamellae are not tightly compacted according to X-ray

and neutron diffraction (5,6), so that up to half the volume

fraction of myelin is taken up by water with salts (2,14).

Recently, it has been observed that the peripheral nerves of

patients with Charcot-Marie-Tooth disease type 1A

(CMT1A) and chronic inflammatory demyelinating

polyneuropathy (CIDP) have significant abnormalities in

their multiple membrane excitability properties such as

threshold electrotonus, strength-duration time constant,

rheobasic current and recovery cycle (4,12,13) measured

by a non-invasive threshold tracking technique (3). The

CMT1A is the most common form of hereditary neuropa-

thy. Its hallmark is uniform demyelination, which results in

slowing of conduction in motor and sensory nerves (1,11).

The CIDP as a peripheral nerve disorder is one of several

chronic demyelinating neuropathies that are believed to

have autoimmune etiology (7). There are subtypes of

chronic demyelinating neuropathies (8,10) that are broadly

classified under the term of chronic inflammatory

demyelinating polyneuropathy.

The present study compare the effect of the aqueous layers

on the previously calculated strength-duration properties,

reflecting the adaptive processes in simulated

demyelinating neuropathies such as CMT1A, CIDP and its

subtypes (9,18,19).

METHODS

The electrogenesis of human motor nerve fibres can now

be studied successfully using a multi-layered model of the

fibres, which is thoroughly described and discussed earlier

(15). The myelin sheath as a series of 150 aqueous and 150

lipid layers was first presented in this model. The same

multi-layered model has been used by us (9,18,19) to simu-

late normal case and three types of progressively greater

117



D.I. Stephanova, Institute of Biophysics and Biomedical Engineer-

ing, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., Bl. 21,

Sofia 1113, Bulgaria.


internodal, paranodal and paranodal internodal

demyelinations, each of them sys tem atic (Fig.1).

The internodal sys tem atic demyelination (ISD) is as so ci -

ated with a cor re spond ing loss of the my elin lamellae with -

out/with their aque ous lay ers away from the axolemma.

The paranodal sys tem atic demyelination (PSD) is as so ci -

ated with a cor re spond ing loss of the my elin end bulbs

away from the axolemma with out/with my elin sheath

aque ous lay ers. This case is equiv a lent to the case of the re -

duc tion of the paranodal seal re sis tance. The paranodal

internodal demyelination (PISD) is as so ci ated with a cor re -

spond ing loosing or lift ing of the my elin end bulbs and my -

elin lamellae with out/with their aque ous lay ers away from

the axolemma. The char ac ter is tic pa ram e ters de fin ing the

com pared cases are given in Ta ble 1, for the reader's con ve -

nience. Two cases of each demyelinated type are mild

(70%, 80%), and one is se vere. The 70% (ISD1, PSD1,

PISD1) and 80% (ISD2, PSD2, PISD2) re duc tion val ues

(of the my elin lamellae or of the paranodal seal re sis tance,

or of the paranodal seal re sis tance and my elin lamellae, re -

spec tively) uni formly along the fi bre length are not suf fi -

cient to de velop a con duc tion block and such

demyelinations are re garded as mild. The 93% (ISD3),

90% (PSD3) and 82% (PISD3) re duc tion val ues are the

first de gree of the demyelinated sub types for achiev ing the

con duc tion block and such demyelinations are re garded as

se vere.

The strength-du ra tion time con stant is based on the pas sive

(RC) fi bre mem brane pa ram e ters in par al lel, ac cord ing to

the clas si cal the ory. The strength-du ra tion and charge-du -

ra tion curves are ob tained fol low ing a thresh old stim u lus at

dif fer ent du ra tion. The thresh old stim u lus du ra tion is in -

creased in 0.025 ms steps from 0.025 ms to 1 ms, in or der to

ob tain the strength-du ra tion curves. A poly no mial func tion

of de gree 2 (trans fer stan dard pa rab ola), which re lates

thresh old charge (Q) to stim u lus du ra tion (t), pro vides an

ac cu rate fit of the data: Q = a2[t2+(a1/a2).t +a0/a2], where a0,

a1, a2 are the poly no mial co ef fi cients. The strength-du ra tion

time con stant is de fined as the ab so lute value of the small -

est square root of the func tion (i.e. only one of the di rect in -

ter cepts of the re gres sion curve on the du ra tion axis has a

bio phys i cal sense and only this di rect in ter cept is shown on

the given be low fig ures). The rheobasic cur rent is de fined

as the fi nal de creased thresh old value, af ter which the po -

ten tial gen er a tion can not be ob tained with an in crease in the

stim u lus du ra tion.

RESULTS

The strength-du ra tion curves (Fig. 2) and charge-du ra tion

curves (Fig. 3) in the nor mal (n) and ISDs (a), PSDs (b),

PISDs (c) cases of the hu man mo tor nerve fibres with out

(Figs. 2, 3 - left col umns) and with (Figs. 2, 3 - right col -

umns) aque ous lay ers are shown. His to grams are also used

118

The strength-duration properties in ...

Fig. 1. Diagram of human motor nerve fibres from the7th to the 14th nodes in the normal, ISD, PSD and PISDcases. The reduction of the myelin lamellae (ISD) or ofthe myelin end bulbs (PSD) or simultaneously of themyelin lamellae and myelin ends bulbs (PISD)without/with myelin sheath aqueous layers is uniformalong the fibre length.

Table 1. Membrane parameter values characteristic forhuman motor nerve fibres without and with aqueouslayers in the normal and in the ISDs, PSDs, PISDscases, when the demyelinations are mild (ISD1, PSD,PISD1 – 70%; ISD2, PSD2, PISD2 – 80%) and severe(ISD3– 93%, PSD3 – 90%, PISD – 82%), respectively.N (number of myelin lamellae); Rmy (myelin resistance);Cmy (myelin capacitance); Raql (aqueous-layeredlongitudinal resistance), Raqr (aqueous-layered radialresistance), Rpa (periaxonal resistance), Rpn (paranodal seal resistance).

119

Krustev S. M., N. Negrev, D. I. Stephanova

Fig. 2. Comparison between the strength-duration curves of human motor nerve fibres in the normal (n), ISDs (a),

PSDs (b), PISDs (c) cases without (left column) and with (right column) aqueous layers within the myelin sheath.

120


Fig. 3. Comparison between the charge-duration curves of human motor nerve fibres in the normal (n), ISDs (a), PSDs

(b), PISDs (c) cases without (left column) and with (right column) aqueous layers within the myelin sheath.

121


Fig. 4. Comparison of the strength-duration time constants (left column) and rheobasic currents (right column)

without (hatched bars) and with (unhatched bars) aqueous layers within the myelin sheath.

to provide a better illustration of the strength-duration time

constants and rheobasic currents (Fig. 4). The threshold

currents are significantly higher in all investigated system-

atically demyelinated cases without/with aqueous layers

than in the normal ones in the different strength-duration

curves. There is an inverse relationship between the

strength-duration time constants (longer/shorter) which are

0.583/0.497, 0.757/0.592, 1.149/0.736 ms for the ISD1,

ISD2 and ISD3 cases without/with aqueous layers, respec-

tively. There is also an inverse relationship between the

time constants, however, they are shorter/longer

(0.093/0.099, 0.065/0.068, 0.064/0.068 ms) for the PSD1,

PSD2 and PSD3 cases without/with aqueous layers, re-

spectively. An overlap of the strength-duration time con-

stants is obtained for the PISDs cases without /with aque-

ous layers. They are substantially shorter 0.211, 0.202,

0.201 ms for the PISD1, PISD2 and PISD3, respectively,

than for the normal one. The results show that the aqueous

layers do not modulate the normal strength-duration time

constant and rheobasic current, which are 0.291 ms and

0.388 nA, respectively. For the ISD1, ISD2 and ISD3

cases, the rheobasic currents increase in both cases with-

out/with myelin sheath aqueous layers and are 0.568/0.605,

0.709/0.805, 1.444/1.718 nA, respectively. However, they

are increased/decreased 0.842/0.793, 1.149/1.084,

1.741/1.654 nA for the PSD1, PSD2 and PSD3 cases with-

out/with aqueous layers, respectively. For both cases with-

out/with aqueous layers, the rheobasic currents signifi-

cantly increase in the PISDs cases with the increase of the

degree of demyelination. The rheobases are 1.020, 1.487,

1.616 nA for the PISD1, PISD2 and PISD3, respectively.

DISCUSSION

The present study shows that the aqueous layers within the

internodally and paranodally systematically demyelinated

sheaths modulate the strength-duration properties of the hu-

man motor nerve fibres. However, the aqueous layers do

not modulate these properties, when the systematic

demyelinations are mixed, i.e. paranodal internodal

(PISDs). The time constants are longer/shorter for the

ISDs/PSDs, respectively without aqueous layers, than the

normal one. The addition of the aqueous layers improves

the strength-duration time constants for both demyelinated

types. However, these time constants are changed in totally

opposite directions, because of the different electrogenesis

of both demyelinations. The slopes of the charge-duration

curves in the ISDs and in the PSDs cases are in opposite di-

rections, because of the different changed RC membrane

parameters. According to the classical theory, there is an in-

verse relationship between the time constant and rheobasic

current. Compared to the normal case, the study shows that

this is valid for the PSDs when the aqueous layers are taken

into account. The ISDs are regarded as specific indicators

of CMT1A, whereas the PSDs and PISDs are regarded as

specific indicators of CIDP and its subtypes (16,17). Con-

sequently, our present study confirms that the systematic

demyelinations without/with aqueous layers may provide

important information on the pathophysiological

mechanisms underlying hereditary and chronic

demyelinating neuropathies.

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123


PSYCHOPHYSIOLOGICAL EVALUATION OF EMOTIONS DUE TO

THE COMMUNICATION IN SOCIAL NETWORKS

Minchev Zl.1, P. Gatev

2

1Joint Training Simulation and Analysis Center, Institute of Information and Communication

Technologies, 2Department of Cognitive Psychophysiology, Institute of Neurobiology,


ABSTRACT

The emerging social networks like Facebook and Twitter with their huge popularity produce a strong impact

to our life. The study of human emotions by means of bioelectrical signals and psychometrics has a long his-

tory, but the data for an influence of Web-based social networks is still scarce and specific methods are lack-

ing. We tried to assemble an ad-hoc methodology for exploring emotions of a Facebook pilot group of users

implementing time-frequency spectral analysis (based on S-transform) and rhythm dynamics characteristics

representation of a complex set of recorded bioelectrical signals with parallel psychometric tests. The spectral

nature of the studied signals suggests correlation with the emotions of the Facebook users. The obtained initial

results have shown that the assembled methodology for bioelectrical signals' recording and analysis might be

a valuable tool for the study of impact of social networks on human emotions and facilitation of IT develop-

ments assessment and control in the information society.

Key words: human emotions, bioelectricity, social networks, spectral analysis, rhythm dynamics

INTRODUCTION

In today's world social interaction is increasingly per-

formed via social networks like Facebook and Twitter re-

lated to the huge popularity of modern information technol-

ogies (15). However, there is a paucity of studies of human

emotions and behavior related to this communication pro-

cess as well as the hidden threats behind it.

There are two kinds of theories for studying human emo-

tions because of the different understandings of the nature

of emotions and their relation to the emotions' stimuli (6):

(i) cognitive based (3,7,13) and (ii) somatic factors based

(4,11,14). Due to the complex nature of the emotions there

are and two approaches for their exploration: (iii) discrete

(assuming the existence of a number of basic emotions that

are universally displayed and recognized, like: happiness,

sadness, anger, fear, disgust and surprise (2), (5), whilst

other emotions are expressed as combination of these basic

ones) and (iv) continuous (assuming a possibility for

multivariate measurement of emotions, like e.g. the

arousal-valence model (12).

The present study uses a cognitive based model and contin-

uous approach to evaluate a methodological framework for

studies of emotions in the context of IT threats identifica-

tion for Facebook social network users.


We have studied 18 healthy volunteers (15 men and 3

women) aged between 15-18 years. We assembled an

ad-hoc methodology for their evaluation as Facebook us-

ers, which includes: (i) habits and purposes in using the so-

cial network Facebook; (ii) Eysenk's personality question-

naire (both available at web page of TK 02/60:

www.cleverstance.com, "Links" section); (iii) 3 minutes

monitoring of Facebook navigation of two profiles: (a) the

one of the popular singer Shakira and (b) the profile of the

scientific project "Cortical Regulation of the Quiet Stance

during Sensory Conflict", Grant TK 02/60 of the NSF in-

cluding popular 39 photos for the standing balance

problems.

Here it should be noted that (iii) has been performed by an

own software tool "Social Network Activities Recorder" -

SNAR v. 1.0 (developed in Borland Delphi® 2008 soft-

ware environment) for user screen activity recording that

encompasses: mouse clicks and X-Y screen position coor-

dinates together with the background changes snapshots

(the studied subjects have been instructed to operate with

the mouse excluding any keyboard activities for simplifica-

tion of the study). These experiments has been performed

on personal computers during the Cyber security training

course at XI Summer School of Mathematics and Informa-

125



Zl. Minchev,

Joint Training Simulation and Analysis Center, Institute of Informa-

tion and Communication Technologies, Bulgarian Academy of Sci-

ences, Sofia 1113, acad. Georgi Bonchev Str., Bl. 25A, room 116,

Tel.: + 359 2 979 6631,

å-mail: [email protected]

tics, August 17-19, 2011 in collaboration with the High

School Institute of Mathematics and Informatics, Institute

of Mathematics and Informatics - Bulgarian Academy of

Sciences.

Additionally, a psychophysiological monitoring of 5 male

volunteer, aged between 26-42 years has been performed

afterward at the laboratory of the Department of Cognitive

Psychophysiology at the Institute of Neurobiology, Bulgar-

ian Academy of Sciences, with Mitsar 202 equipment re-

cording: EEG (following 10-20 system - F3, Fz, F4, C3,

Cz, C4, P3, Pz, P4), ECG (leads from the hands wrists) and

EMG (by bipolar leadings-off from the mimic muscles: m.

orbicularis oculi and m. corrugator superscilii) activities

during the performance of the tasks (i) - (iii). Finally, only

one 42 years experimental subject has been selected as a

stable, extrovert representative Facebook user. All data was

digitized in 500 Hz sampling frequency.

This experimental monitoring has been performed on HP

6730s laptop machine with Intel Pentium Dual-Core Pro-

cessor T4200, 3072 MB RAM, 320 GB SATA HDD, ATI

Radeon HD 3430 video card with 256 MB dedicated video

memory and with WXGA BrightView 15.4" screen. The

mimic of face has been also recorded via the build-in web

camera and WebCam Max®, v.7.5.3.2 software.

The subject's instruction was to operate with the mouse ex-

cluding any keyboard activities for simplicity of the study.

The SNAR v. 1.0 software has been accomplished for the

mouse clicks capturing, by using a short (1 ms duration and

1 kHz frequency) sound signal (produced as a result of left

mouse button click) via the standard audio output of the

experimental setup

All experiments have been performed in the context of vali-

dating the Facebook information flows threats model pub-

lished in (10) and developed in the framework of EU Net-

work of Excellence in Managing Threats and Vulnerabili-

ties for the Future Internet: Europe for the World - SySSec.

This model determines as a critical threat for the Facebook

users "Friends" and as hidden threats: "Photos_Videos",

"Profile", "Wall" and "Other".

The records of the physiological monitoring have been pro-

cessed by using Matlab® R2011a. All records have been

initially filtered with zero-phase shift digital filter using

Chebishev IIR 50 Hz notch filter with slope -18 dB/oct and

passband ripple 0.5 dB, Wn=[47,52] Hz. Additionally,

Butterworth band pass IIR filter with slope -18 dB/oct, and

passband ripple = 0 dB has been also used, as follows:

Wn=[2, 42] Hz for the EEG, Wn=[1, 150] Hz for the EMG

and ECG.

Finally, time-frequency spectral analysis (using S- trans-

form and the approach presented in (9)) was performed.

RESULTS

Regarding the participants' habits and purposes in using the

social network Facebook almost all users (about 90%) are

reporting Facebook usage for communication with real

people and on-line stay for more than 12 hours daily, hav-

ing more than 100 friendships. Only 10% of the studied

participants report Facebook usage for entertainment

and/or other purposes.

The generalized results of the Facebook psychological

analysis and item usage validation according to (i) - (iii)

tasks from the previous paragraph are shown for 18 healthy

volunteers on Figure 1. As it is clear from Figure 1 (left),

the stable and extroverts participants are encompassing al-

most all of the Facebook users stuff. On Figure 1 (right) the

item "Wall" has 90% coincidence between model's pre-

dicted and really used items, whilst item "Friends" and

"Other" have the lowest coincidence.

The pilot results of the polyphysiographic monitoring dem-

onstrate that changes in the heart rate variability coincide

with the S-transform variability of m. corrugator superscilii

and/or m. orbicularis oculi (Figure 2 A). These results con-

126

Psychophysiological evaluation of emotions due to ...

Fig.1. Eysenck’s personality test generalized results expressed in percentages of the sample (left) and Facebook item

usage (right) expressed in percentages of the coincidence between model predicted and really used items.

firmed that heart rate variability can be connected with dif-

ferent kind of emotions (8).

The EEG S-transform of F3, C3 P3 vs F4, C4, P4 leads dur-

ing 7 second epochs showed typical frontal area left-right

asymmetry (1) as well as centro-parietal cortex left-right

asymmetry.

DISCUSSION

The psychometric results showed that extroversion and sta-

bility of the studied Facebook users are correlated posi-

tively with the intensity of social networks usage. The re-

sults of the threats coincidence of Facebook social network

for the "Wall" item and the differences for "Friends" and

"Other" items, suggesting that the obtained differences

could be generators of hidden threats, which was not

initially expected.

The pilot results of the polyphysiographic monitoring have

demonstrated promising initial base for evaluation of the

experts' models threats' foresees and psychometric mea-

surements using bioelectrical signals like: EEG, EMG and

ECG dynamic changes monitoring.

127

Minchev Zl., P. Gatev

Fig.2. A: parallel changes in EMG envelope S-transform of m. corrugator superscilii (top), m. orbicularis oculi

(middle) and hear rate variability (bottom) during 3 minutes gallery items navigation of Facebook profile for standing

balance problems; B: left-right EEG asymmetry in left (F3, C3 P3 - top) vs right (F4, C4, P4 - bottom) during 7 second

epoch evaluated by S-transform.

ACKNOWLEDGEMENTS

The study was partially supported by the National Science

Fund of Bulgaria, Grant TK02/60 (www.cleverstance.com)

and EU FP7 project - A European Network of Excellence in

Managing Threats and Vulnerabilities in the Future Internet:

Europe for the World (www.syssec-project.eu) under the

agreement No 257007. The authors also express their grati-

tude to the organizers and sponsors of XI Summer School of

Mathematics and Informatics, August 17-19, 2011.

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1999, 45-60.

6. Lopatovska, I . , I . Arapakis . Theories, Methods

and Current Research on Emotions in Library and In-

formation Science, Information Retrieval and Hu-

man-Computer Interaction, Information Processing

and Management, 2010.

7. Lazarus, R. S. Thoughts on the relations between

emotion and cognition. Approaches to emotion.

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247-259, 1984.

8. LeDoux, J .E. Emotion circuits in the brain, Annu.

Rev. Neurosci, 2000, 23, 155-184.

9. Minchev Z. , G. Dukov, S. Georgiev. EEG

Spectral Analysis in Serious Gaming: An Ad Hoc Ex-

perimental Application, Int. J. Bioautomation, 13 (4),

2009, 79-88.

10. Minchev, Z. , M. Petkova. Information Pro-

cesses and Threats in Social Networks. A Case Study,

Proceedings of Conjoint Scientific Seminar "Model-

ling and Control of Information Processes", Sofia,

November 20, 2010, 85-93, Available at:

http://www.math.bas.bg/telecom/old_site/seminar201

0/doc9.pdf

11. Plutchik, R. A general psychoevolutionary theory

of emotion. Emotion: Theory, research and experi-

ence. Theories of emotion, New York: Academic, 1,

1980, 3-33.

12. Russel l , J . , A. Weiss , G. Mendelsohn. Affect

grid: a single item scale of pleasure and arousal, Jour-

nal of Personality and Social Psychology, 57(3),

1989, 493-502.

13. Scherer , K. R. What are emotions? And how can

they be measured? Social Science Information, 44(4),

2005, 695-729.

14. Scherer , K.R. , and P. Ekman. (Eds.) Affect

theory. Approaches to emotion. Hillsdale, New Jer-

sey: Lawrence Erlbaum Associates, 1984.

15. The Cisco Connected World Technology Report,

September 21, 2011, Available at:

http://www.cisco.com/en/US/solu-

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pter1-Report.pdf

128

Psychophysiological evaluation of emotions due to ...

MELATONIN AND THE FIBRINOLYTIC SYSTEM IN THE RAT:

EFFECTS ON THROMBIN ACTIVATABLE FIBRILOLYSIS

INHIBITOR (TAFI)

Decheva L.1, I. Pashalieva

1, E. Stancheva

1, Y. Nyagolov

2, N. Negrev

1

1Department of Physiology and Pathophysiology, Medical University "Prof. Paraskev Stoyanov" -

Varna, 2Department of Physiology, Medical University - Sofia

SUMMARY

There is scanty and contradictory data about the effect of the hormone melatonin on fibrinolysis. The purpose

of the present investigation is to study the effects of melatonin and its antagonist luzindolå on TAFI

(Thrombin Activatable Fibrinolysis Inhibitor) activity and plasma antigen level of TAFIa/TAFIai (TAFI ac-

tivated/TAFIa inactivated) complex which are specific markers of plasma fibrinolytic activity. The study cov-

ered 52 male Wistar rats divided into four groups, i. e.: injected with melatonin (in a dose of 0.2 mg/kg b.m.),

with luzindole (in a dose of 0.4 mg/kg b.m.), with melatonin and luzindolå both as luzindole pretreatment was

performed one hour earlier. The control group was injected with the vehicle. All the substances were adminis-

tered two times daily for three consecutive days. It was established that melatonin induced significant alter-

ations in the examined parameters as TAFI activity increased (p<0.001) while the plasma level of

TAFIa/TAFIai complex was strongly reduced (p<0.001). Luzindolå being a non-selective antagonist of

melatonin receptors diminished TAFI activity (ð<0.001) and elevated the plasma concentration of

TAFIa/TAFIai complex (ð<0.001). Luzindolå pretreatment and subsequent melatonin application caused

changes of the examined parameters which, in their essence, repeated the effect of the independent luzindolå

administration at ð<0.001. The reptilase time was significantly decreased in all tested groups (ð<0.001). The

results obtained allowed us to accept that melatonin significantly reduced TAFI activity and elevated

TAFIa/TAFIai. The blockade of MT1 and MT2 melatonin receptors by luzindolå eliminated the effect of this

hormone on TAFI activity and TAFIa/TAFIai.

Key words: melatonin, luzindolå, reptilase time, TAFI, TAFIa/TAFIai

INTRODUCTION

Melatonin is a neurohormone secreted by the pineal gland.

It regulates other hormones and maintains the body's circa-

dian rhythms. In addition to the involvement of the hor-

mone in the transduction and processing of the

photoperiodical information and modulation of various

neuronal and endocrine functions in mammalians, a wide

spectrum of biological effects, including regulation of re-

production, liver protein synthesis control, adipogenesis

and osteogenesis, oncostatic, antiapoptotic, immune-mod-

ulating and antioxydant effects are attributed to melatonin

(1-5). However, data about involvement of the hormone in

the complex mechanisms of haemostasis regulation are

relatively limited and partially controversial (6,7).

Recently a special scientific interest is directed towards

TAFI (thrombin activatable fibrinolysis inhibitor), a novel

molecule which is proposed to play a key role in the inter-

action between procoagulant, anticoagulant and

fibrinolytic systems expressing various additional biologi-

cal effects (8,9). TAFI (Plasma pro-carboxypeptidase B,

carboxypeptidase U) is a single chain glycoprotein

zymogen (Mr=60,000) synthesized in the liver and circu-

lating at plasma concentration of 50 nM/l as a

plasminogen-bound zymogen. Thrombin (plasmin,

trypsin) cleavage of the zymogen releases a 92 amino acid

N-terminal activation peptide containing 4 N-linked

glycosylation sites (N22, N51, N63, N86) and the proposed

plasminogen recognition site. The rate of thrombin cata-

lyzed activation of TAFI is increased 1250 fold by forma-

tion of a ternary complex with thrombomodulin (10). The

309 amino acid C-terminal (Mr=35,783) catalytic domain

(TAFIa, pCPB) displays the properties of a basic

carboxypeptidase, hydrolyzing lysine and arginine from the

C-terminal position of polypeptides. This portion of the

molecule is homologous to tissue carboxypeptidase B and

contains 7 conserved cystine residues

(64,77,136,151,160,165,291), the active site Zn2+ coordi-

nation site (H67, E69, H196) and the basic C-terminal

amino acid substrate binding pocket (D257, G244, S207).

129



L. Decheva, Dept. of Physiology and Pathophysiology, Medical

University "Prof. Paraskev Stoyanov" - Varna, 55 Marin Drinov str.,

9002 Varna


After its activation by thrombin, the

thrombin-thrombomodulin complex, or plasmin, the acti-

vated form of TAFI (TAFIa) attenuates fibrinolysis by re-

moving the carboxyl-terminal lysine residues from partially

degraded fibrin that mediate positive feedback in the

fibrinolytic cascade. These residues are required for ad-

sorption of tissue-type plasminogen activator (t-PA) and

plasminogen to fibrin. Therefore, TAFIa decreases plasmin

formation and protects the fibrin clot against lysis. An in-

creased of pro-TAFI/TAFIa levels have been reported in

some clinical conditions associated with thrombotic ten-

dency, as type II diabetes mellitus, deep vein thrombosis

and symptomatic artery disease. The thrombin-activatable

fibrinolysis inhibitor pathway defines a novel molecular

connection between blood coagulation and both

fibrinolysis and inflammation. A role for TAFIa in modu-

lating inflammation is suggested by the ability of this en-

zyme to down-regulate pericellular plasminogen activation

and to inactivate the inflammatory peptides bradykinin and

the anaphylatoxins C3a and C5a. TAFIa undergoes a rapid

conformational change at 37°C to an inactive isoform

called TAFIai.

This study was designed to investigate the effects of

melatonin and its nonselective receptor inhibitor luzindole

on TAFI pathway in rats (11).


The experiments were performed on 52 male Wistar rats

weighing 200-220 g, in accordance with the European

Convention for protection of experimental animals (Protec-

tion of animals used for experimental purposes, Council

Directive 86/609/EEc of November 1986). The rats were

kept with free access to standard food and water at

dark/light regimen 12:12 hours and were divided into four

equal groups, 13 animals each. The daily doses for

melatonin (Ìårck, Germany), 0.2 mg/kg, and luzindole

(Sigma Chemicals/St. Louis, MO, USA), 0.4 mg/kg, were

applied twice daily s.c. for three consecutive days. The rats

from one of the groups were injected with melatonin; an-

other group of animals were treated with luzindole; and a

third group with luzindole, followed after 1 hour by

melatonin. The control group was treated with the vehicle

by the same dose and schema. Melatonin and luzindole

doses were determined by literature data and by experi-

mental study on dose/effect dependence. The necessary

blood volume from a rat was acquired by cardiac puncture

under urethane narcosis. Sodium citrate (0.11 mol/l) was

used as an anticoagulant (blood/citrate ratio 9:1). The citrat-

ed blood was centrifuged for 10 min at 3000 revolutions

per minute. The citrated plasma (the supernatant) was sepa-

rated and stored at 4 0C in haemostatic test-tubes. The pa-

rameters studied TAFI activity and plasma antigen level of

the complex TAFIa/TAFIai, were determined not later than

2 hours. TAFI activity was estimated by the commercial

enzyme kit Stachrom® Diagnostica Stago (Franñå) and

measured on STA® analyzer. TAFIa/TAFIai antigen con-

centration was determined by Asserachrom® en-

zyme-linked immunosorbent assay (ELISA). The reptilase

time was estimated by a routine coagulation method. The

data were analysed by variation analysis using

Student-Fisher's t-test and values of p<0.05 were

considered statistically significant.


The effects of melatonin and luzindole on TAFI activity are

presented in figure 1. Melatonin elevated this parameter

(presented in % of activity from the baseline) from

115.20±4.10 for the control group to 180.70±9.97

(p<0.001), while both luzindole applied separately, and

luzindole applied 1 hour before melatonin decreased TAFI

activity to 50.09±4.81 (p<0.001) and to 40.00±4.82

(p<0.001), respectively. The other parameter,

TAFIa/TAFIai antigen concentration (ng/ml) was de-

creased by melatonin from 13.00±0.82 for the control

group to 5.02±0.49 (p<0.001) and was increased signifi-

cantly to 24.09±0.84 (p<0.001) and to 23.72±0.82

(p<0.001) both by the separate luzindole and combined

luzindole/melatonin treatment (figure 2). The reptilase time

130

Melatonin and the fibrinolytic system in the rat: effects on ...

Fig. 1. Melatonin and luzindole effects on TAFI activity.

M – melatonin; L – luzindole; C – control * - ð<0,001

Fig. 2. Melatonin and luzindole effects on TAFIa/

TAFIai complex.

M – melatonin; L – luzindole; C – control; * - ð<0,001

(figure 3) was shortened significantly from 11.77±0.57 sec

for the control group to, respectively 5.11±0.45 (p<0.001),

8.09±0.33 (p<0.001) and 7.00±0.45 sec (p<0.001) after

treatment with melatonin, luzindole alone and combined

luzindole/melatonin.

Striking is the observation, the three day application of

melatonin in this study in rats (24 hour dose 0.2 mg/kg

body mass) significantly increased the activity of and de-

creased TAFIa/TAFIai antigen concentration. Similar

findings indicating opposite effects on plasma activity and

concentration are abundant in literature and are not new in

relation to various clotting factors. The most tempting hy-

pothesis for explanation of this phenomenon is the suppres-

sion of the biosynthesis of TAFI by melatonin expressed by

TAFIa/TAFIai decrease, as well as suppression of its inac-

tivation presented by the stimulated TAFI activity. Having

in mind TAFI is synthesized and released in the liver (12),

and the various melatonin liver effects related to protein

synthesis reported, this hypothesis might be acceptable

(13,14). We should hardly use the upper facts to specify the

mechanism of the effect of melatonin, but it is quite proba-

ble the stimulating effect of the hormone to be accom-

plished at the level of the liver. Having in mind the abun-

dant expression of melatonin receptors in various areas of

the central nervous system (15), we might consider other

possible central effects of the hormone via other

mechanisms of haemocoagulation regulation.

Luzindole is the first competitive nonselective melatonin

receptor antagonist (16). Separate luzindole treatment (24

hour dose - 0.4 mg/kg) in the course of three days decreased

significantly the levels of activity of TAFI and increased

TAFIa/TAFIai concentration. These findings are exactly

opposite to melatonin effects discussed above and may be

attributed to one of the basic luzindole effects resulting

from its capability to block nonselectively melatonin MT1

and MT2 receptors. This is may be an evidence of a direct

participation of MT1and MT2receptors in the implementa-

tion of the melatonin effects in haemocoagulation via

TAFI.

Melatonin treatment of the rats one hour after luzindole

pre-treatment by the same doses and schema of application,

significantly decreased TAFI activity and increased

TAFIa/TAFIai concentration. The data indicate luzindole

pre-treatment effectively inverses the effect of melatonin on

the parameters studied. In addition, melatonin applied after

luzindole pre-treatment almost completely reproduces the

shifts of the parameters observed after separate luzindole

application. These results are in support of the presumtion

that luzindole eliminates the exogenous melatonin effect. In

addition the non-selective MT1 and MT2 receptor antago-

nist luzindole to a certain extent may inhibit the endoge-

nous melatonin too.

This study followed up the melatonin effect on reptilase

time (RT). RT is a basic integral haemocoagulation test to

estimate the functions of the factors involved in the trans-

formation of fibrinogen to fibrin. This parameter was sup-

pressed by melatonin, luzindole and melatonin applied af-

ter luzindole pretreatment. The registerred RT shifts are not

in a complete coherence with the effects on TAFI observed

and represent another pattern of the effects of melatonin

and luzindole compared to the effects on TAFI. This find-

ing may indicate a different mechanism of melatonin and

luzindole involved in transformation of fibrinogen to fibrin.

CONCLUSIONS

In conclision, the results of the study indicate: 1). Three day

treatment of male Wistar breed rats with melatonin in-

creases the activity of TAFI and may lead to activation of

fibrinolysis via TAFI pathway. 2). Separate luzindole ap-

plication for three consecutive days leads to a suppression

of TAFI activity. 3). TAFIa/TAFIai antigen concentration

is influenced in the opposite direction compared to TAFI

activity; decreased by melatonin and increased by

luzindole. 4). The treatment of the rats with melatonin after

luzindole pre-treatment completely reproduces the effect of

separate luzindole application. The data evidence the

nonselective melatonin receptor antagonist luzindole

blocks the effects both of endogenous and exogenous

melatonin on TAFI. 5). RT is shortened in all tested groups

versus the controls via mechanism(s) different from the

effect on TAFI.

REFERENCES

1. Boutin J . A. , Audinot V. , Ferry G. ,

Delagrange P. (2005). "Molecular tools to study

melatonin pathways and actions". Trends in Pharma-

cological Sciences 26 (8): 412-9.

2. Borj igin J. , Li X. , Snyder S.H. (1999) The pi-

neal gland and melatonin: Molecular and pharmaco-

logic regulation. Annu Rev Pharmacol

Toxicol.;39:53-65.

3. Chal let E. (2007). "Minireview: Entrainment of the

Suprachiasmatic Clockwork in Diurnal and Nocturnal

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4. A l t u n A . , U g u r - A l t u n B . (2007).

"Melatonin: therapeutic and clinical utilization". In-

ternational Journal of Clinical Practice 61 (5):

835-45.

131

Decheva L., I. Pashalieva, E. Stancheva ...

Fig. 3. Melatonin and luzindole effects on reptilase time (RT).

M - melatonin; L - luzindole; C - control; * - ð<0,001

5. Wolden-Hanson T; Mitton, D. R., McCants

R. L., Yellon S. M., Wilkinson C. W.,

Matsumoto A. M., Rasmussen D. D. (2000).

"Daily melatonin administration to middle-aged male

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ity, and plasma leptin and insulin independent of food

intake and total body fat". Endocrinology 141 (2):

487-97.

6. Claustrat B. , J . Brun, Chazot G. (2005) The

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U., von K�nel R. (2008) Oral melatonin reduces

blood coagulation activity: a placebo-controlled study

in healthy young men. J Pineal Res. Mar;

44(2):127-33.

8. Wang W., Hendriks D. F. , Scharpe S. S.

(1994) Carboxypeptidase U, a plasma

carboxypeptidase with high affinity for plasminogen.

J Biol Chem. 269:15937-15944.

9. Bajzar L. , Manuel R. , Nesheim M. E. (1995)

Purification and characterization of TAFI, a

thrombin-activatable fibrinolysis inhibitor. J Biol

Chem. 270:14477-14484.

10. Mosnier L. O. , Meijers J . C. M., Bouma B.

N. (2001) Regulation of fibrinolysis in plasma by

TAFI and protein C is dependent on the concentration

of thrombomodulin. Thromb Haemost.85:5-11.

11. Bouma B. N. , Mosnier L. O. (2005).

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the interface between coagulation and fibrinolysis."

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12. Kerr R . (2003) New insights into haemostasis in

liver failure. Blood Coagul Fibrinolysis 14(Suppl 1):

43- 45.

13. Brodsky V. Y. , Zvezdina N. D. (2010)

Melatonin as the most effective organizer of the

rhythm of protein synthesis in hepatocytes in vitro

and in vivo. Cell Biol Int. Dec; 34 (12) :1199-204.

14. Brodsky V. Y. , Dubovaya N. D. , Zvezdina

N. D. , Fateeva V. I . , Malchenko L. A.

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15. Wit t -Enderby P. A. , Bennet t J . , Jarzynka

M. J. , Firest ine S. , Melan M. A. (2003)

Melatonin receptors and their regulation: biochemical

and structural mechanisms. Life Sciences 72 :

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novel melatonin receptor antagonist. J Pharmacol

Exp Ther Sep; 246 (3) :902-10.

132

Melatonin and the fibrinolytic system in the rat: effects on ...

PREHYPERTENSION IN WOMEN WITH METABOLIC SYNDROME

Nikolova J.1, M. Orbezova

2, P. Atanasova

3, P. Nikolov

4, F. Nikolov

4, P. Hrischev

3

1Department of Physiology, 2Clinic of Embriology, 3Department of Anatomy, Histology and

Embriology, 4Clinic of Cardiology, Medical university - Plovdiv

INTRODUCTION

There is a continuous relationship between the level of

blood pressure (BP) and cardiovascular (CV) risk. The

2003 European Society of Hypertension - European Soci-

ety of Cardiology (ESH-ESC) guidelines for the manage-

ment of arterial hypertension referred the BP range

130-139/85-89 mmHg to as "borderline" hypertension ac-

cording JNC6 (Joint National Committee on Prevention,

Detection, Evaluation and Treatment of High Blood Pres-

sure - USA) and "high- normal" according ESH-ESC

guidelines. JNC 7 established new classification of BP,

coining the term "prehypertension" for those with blood

pressure ranging from 120 to 139 mmHg systolic and/or

from 80 to 89 mmHg diastolic. Prehypertension is not a dis-

ease. But increasing the BP with 20/10 mmHg for systole

and diastole respectively doubles the CV risk. Thus higher

CV risk in prehypertensive patients is an argument the cate-

gory "prehypertension" to be introduced. In one third of

181 followed up hypertensive people, jepaneese authors

register metabolic syndrome (MS) and morning

hypertention/MS ratio is significant. Prehypertension and

hypertension are components of MS, characterized by a

number of metabolic abnormalities including central obe-

sity, elevated triglycerides (TG) with decreased levels of

high-density lipoprotein - cholesterol(HDL-C), raised

blood pressure³135/80mmHg and impaired glucose toler-

ance. The MS predicts cardiovascular mortality in both

men and women. In the DECODE study, non-diabetic men

and women with MS had an increased risk of death from all

causes as well as CV disease. Leptin, one of the basic mast

tissue products, plays a major role as in carbohydrates' and

lipids' metabolism, as in appetite control and energy bal-

ance. Leptine/ insuline resistance correlation is an object of

discussion and intense examination.

The goal of our study was to follow up the frequency of

prehypertension in women with MS and the leptine's mast

tissue expression.


46 women of 36.68±6.48 years age with proven MS and 32

clinically healthy women of 35.54±5.39 years age were ex-

amined. The parameters: body mass index (BMI), arterial

blood pressure (ABP), based on multiple measurements

taken on separate occasions, plasma blood sugar (RA 1000

Technicon, USA), insulin and HOMA - index (MEIA,

ÀÂÂÎÒÒ, USA, AxSYM) and plasma lipid profile (Op-

tima KONE) were followed up. Imune histochemic investi-

gation of biopsy material for leptine was done. The material

was taken from gluteal under skin mast tissue region. The

immune histochemical reaction was followed on 5 m par-

affin slides by avidin biotidin peroxydase method (ABC)

using primary antibodies for leptin(Santa Cruz, USA), dis-

solved in PBS in 1:200 ratio.

RESULTS

Measuring ABP, it was established that in 37% of the

women with proven MS, the values are ³135/80 mmHg.

These values were registered in 20% of clinically healthy

women (p<0.05).

To value the degree of obesity, BMI was used, according to

the world health organization (WHO) criteria. In women

with proven MS, BMI was 35.43±3.18 kg/m2 and in clini-

cally healthy women - 23.43±5.11 kg/m2 (p<0.05) respec-

tively.

HOMA index in women with proven MS was 4.15±1.10

v/s 1.15±0.80 (p<0.05) in clinically healthy women.

Plasma lipid profile data were: total cholesterol (TC) - 4,1

0,9 mmol.L-1 in MS women v/s 6,3±0,28 mmol.L-1

(p<0,001) in clinically healthy women women; TG: 1,5 0,3

mmol.L-1 in clinically healthy women v/s 2,1 0,73

mmol.L-1 (p<0,05) in MS women.

Plasma glucose level (GLU) in MS women was 6,51 1,04

mmol.L-1 (p<0,05) v/s 4,46±1,53 mmol. L-1 in clinically

healthy women.

133


Èìóíîõèñòîõèìè÷íà åêñïðåñèÿ íà ëåïòèí ó çäðàâ.

Óâ. X400.

Imun histochemic reactions were positive in all MS

women. Under skin mast tissue adipocytes demonstrated

strong leptin expression - comparatively stronger expressed

than those of the controls.

DISCUSSION AND CONCLUSIONS

Hypertension affects 26% of the general population of the

world. It is an independent predictor of CV and brain ves-

sels incidents. Data from observational studies involving

more than one million individuals have indicated that death

from both coronary heart disease (CHD) and stroke in-

creases progressively and linearly from BP levels as low as

115 mmHg systolic and 75 mmHg diastolic upward. The

increased risks are present in all age groups ranging from

40 to 89 years old. The apparently simple direct relation-

ship between increasing systolic BP and diastolic BP and

CV risk is confounded by the fact that systolic BP rises the

throughout the adult age in the vast majority of populations

whereas diastolic BP peaks at about age 60 in men and 70

in women , and falls thereafter. For every 20 mmHg sys-

tolic or 10 mmHg diastolic increase in BP, there is doubling

of mortality from both CVD and stroke. In addition, longi-

tudinal data obtained from the Framingham Heart study in-

dicated that BP values in the 130-139/85-89 mmHg range

are associated with a more than two fold increase in relative

risk from CVD compared with those with BP levels below

120/80 mmHg. The analysis of different population inves-

tigations reveals that the rate of prehypertension among

adults is over 30%. In our study we established that the

range of prehypertension in women with proven MS is

twice higher.

Major risk factor for the appearance of MS is body over-

weight. The rate of MS increases with age. According to

wide Scandinavian study (Botnia Study), MS can be seen

in 10% of people with normal glucose tolerance, in about of

50% of prediabetic people and in about 80% of diabetes 2.

According to the National program for training of choles-

terol - third panel for treating adults MS exists in 10% of

men and in 7% in women. A Brazilian study reveals very

high frequency of MS in hypertensives - 71.6% è 82.4%

(after the criteria of IDF - International Diabetic Federa-

tion). Central obesity with mast gathering around the vis-

cera is a major factor for the appearance of MS. Central

obesity is connected with risk factors as impaired glucose

tolerance, dyslipidaemia, prehypertension and hyperten-

sion. Decreased insulin sensibility is usually connected

with impaired plasma lipid profile. High TG and low

HDL-C being part of the constellation of MS is associated

with 4 time increased risk of CVD and presence of

hyperinsulinaemia, increased apo-B and small dense LDL

particles - makes it 5 times higher.

In our study increased insulin resistance is established in

45% of the women with MS.

Leptin, one of the most important adipose derived hor-

mones, plays a key role in regulating energy intake and en-

ergy expenditure. Only leptin and insulin are known to act

as an adiposity signal. Leptin acts on receptors in hypothal-

amus of the brain inhibiting appetite by counteracting the

effects of neuroprptide Y - a potent feeding stimulant. Al-

though leptin is a circulating signal redusing appetite, in

general, obese people have an unusually high circulating

levels of leptin. They are said to be resistant to the effects of

leptin in much the same way that people with type 2 diabe-

tes are resistant to the effects of insulin. Leptin control in

obese people might be flawed at some point so the body

doesn't adequately receive the satiety feeling subsequent to

eating.

Leptin in our study is comparatively stronger expressed in

MS women. In MS there is a correlation between the level

of leptin gathered in the mast tissue and the state of insulin

resistance.

REFERENCES

1. National High Blood Pressure Education Program.

The sixth report of the Joint National Committee on

Prevention, Detection Evaluation and Treatment of

High Blood Pressure: the JNC 6 Report. JAMA 2003;

289:2560-2572.

2. Chobanian AV, Bakris GL, Black HR et

al l . The seventh report of the Joint national Commit-

tee on Prevention, Detection, Evaluation and Treat-

ment of High Blood Pressure: the JNC 6 Report.

JAMA 2003; 289: 2560-2572.

3. O'Brien E, Asmar R, Bei l in l et al l ., on be-

half of the European Society of Hypertension Work-

ing Group of Blood Pressure Monitoring, European

Society of Hypertension Recommendations for Con-

ventional, ambulatory and Home Blood Pressure

Measurement. J Hypertens 2003; 21:821-848.

4. Lakka HM, Laaksonen DE, Lakka TA et

al l . The metabolic syndrome and total and cardiovas-

cular disease mortality in middle-aged men. JAMA

2002; 288: 2709-2716.

5. Hu G, Qiao Q, Tuomilehto J et al l . Preva-

lence of the metabolic syndrome and all relations to

all-cause and cardiovascular mortality in nondiabetic

134

Prehypertension in women with metabolic syndrome

Èìóíîõèñòîõèìè÷íà åêñïðåñèÿ íà ëåïòèí ó

ïàöèåíò ñ ÌÑ. Óâ. X400.

European men and women. Arch Inter Med 2004;

164: 1066-1076.

6. Hunt KJ, Resendez RG, Wil l iams K,

Haffner SM, Stern MP. National Cholesterol Ed-

ucation Program versus World Health Organization

metabolic syndrome in relation to all-cause and car-

diovascular mortality in the San Antonio Heart Study.

Circulation 2004, 110: 1251-1257.

7. Malik S, Wong ND, Frankl in SS et al l . Im-

pact of the metabolic syndrome on mortality from

coronary heart disease, cardiovascular disease, and all

causes in United States adults. Circulation 2004; 110:

1245-1250.

8. Dekker JM, Girman C, Rhodes T et al l . Met-

abolic syndrome and 10-year cardiovascular disease

risk in the Hoorn study. Circulation 2005; 112:

666-673.

9. Qiao Q, Jousi laht i P, Erikson J,

Tuomilehto J. Predicted properties of impaired

glucose tolerance for cardiovascular risk are not ex-

plained with by the development of overt diabetes

during follow-up. Diabetes care 2003; 26:

2910-2914.

10. Third report of the National Cholesterol Education

Program (NCEP) Expert Panel on Detection, Evalua-

tion and Treatment of High Blood Cholesterol in

Adults (Adult Treatment Panel III) final report. Cir-

culation 2002; 106: 3143-3421.

11. McKenney JM, Davidson MH, Jacobson

TA, Guyton JR. Final conclusions and recommen-

dations of the National Lipid Association Statin

Safety ssessment Task Force. Am J Cardiol 2006;

97:89C-97C.

135

Nikolova J., M. Orbezova, P. Atanasova ...

SOMATOTROPIN, SOMATOSTATIN AND PROLACTIN EFFECTS

ON VITAMIN K-DEPENDENT PLASMA CLOTTING FACTOR

(FII, FVII, FIX, FX) ANTIGEN CONCENTRATIONS IN RATS

Nyagolov Y.1, I. Pashaliewa

2, N. Doncheva

3, N. Negrev

2

1Medical University - Sofia, Department of Physiology; 2Medical University - Varna, Department

of Physiology; 3Medical Institute, Ministry of Interior, Department of Clinical Laboratory

ABSTRACT

Somatotropin, somatostatin and prolactin hormonal disorders in clinical practice are often associated with

modulated haemostasis. However, the precise role of those hormones in haemocoagulation is not yet clearly

defined. The present survey was designed to measure the effects of somatotropin, somatostatin and prolactin

on plasma concentrations of vitamin K-dependent clotting factors FII, FVII, FIX and FX in rats. Experi-

ments were performed on male Wistar rats. Hormones enrolled in the study were applied in three consecutive

days as follows: somatotropin (0.2 mg/kg body mass, s.c.); somatostatin (0.1 mg/kg, s.c.); somatostatin ap-

plied 3 h before somatotropin in the same doses and scheme; and prolactin (2 õ 0.1 mg/kg, i.p.). Blood samples

were gathered by cardiac puncture under ether narcosis. Plasma clotting factor concentrations were

estimated by ELISA methods. Somatotropin reduced significantly plasma antigen concentration (p<0.001) of

FII, FVII, and FX, but did not affect FIX. Somatostatin, as well as somatostatin applied 3 h before somatotro-

pin and prolactin increased (p<0.001) FII, FVII, and FX, and reduced FIX (p<0.001). Prolactin elevated all

parameters studied (p<0.001). These results evidence somatotropin, somatostatin and prolactin are substan-

tially involved in regulation of plasma levels of the factors studied, as somatotropin reduces FII, FVII, and FX,

while somatostatin and prolactin provide the opposite effects. The completely different pattern of the effects

of the hormones on F IX suggest different mechanism(s) of actions related to this clotting factor.

Key words: vitamin K-dependent clotting factors, FII, FV, FIX, FX, somatotropin, somatostain, prolactin

INTRODUCTION

Hemostatic system is a well balanced mechanism acting to

protect organism of hemorrhages and thrombotic events.

The complex hemostasis regulation is a precisely tuned

machinery maintaining the functions of coagulation,

anticoagulation and fibrinolytic systems in harmony and

synchrony with platelets, endothelium, hepatocytes and

leukocytes (1). Endocrine system is undisputedly substan-

tially involved in the regulation of those processes. Addi-

tionally to the well known effects of sex hormones, insulin,

thyroid hormones, oral contraceptives, hormone replace-

ment therapy in various hormonal disorders on multiple

hemostasis parameters, literature data for direct influence of

almost all hormones are on primary or secondary

hemostasis are available (2-4). A lot of endocrine diseases

are related to larger or smaller anomalies of laboratory co-

agulation tests or manifestations of clinical tendencies of

thromboses or hemorrhages (5).

As far as Somatotropin, somatostatin and prolactin are con-

cerned, literature data evidence the hormonal disorders in

clinical practice related to these hormones are often associ-

ated with modulated haemostasis (6-8). However, the pre-

cise role of those hormones in haemocoagulation is not yet

clearly defined. We have previously reported effects of

growth hormone (somatotropin) and somatostatin on

plasma activity of clotting factors FII, FVII, FIX and FX in

rats (9). A point of specific scientific and clinical interest is

whether those hormones exert similar effects on plasma an-

tigen concentrations of the same clotting factors.

The present survey was designed to estimate the effects of

somatotropin, somatostatin and prolactin on plasma antigen

concentrations of vitamin K-dependent clotting factors FII,

FVII, FIX and FX in rats.


Experiments were performed on 78 male Wistar rats

weighing 200-220 grams. The animals were bred under

standard laboratory conditions with free access to standard

food and water. The experiments were performed follow-

ing the requirements of the European Convention for the

Protection of Experimental Animals (Protection of animals

used for experimental purposes, Council Directive

86/609/EEC of November 1986). The rats were received

from the internationally accredited vivarium of the Medical

University, Varna, Bulgaria and the experiments were

approved by the ethical committee of the University.

137


In order to avoid possible diurnal effects the animals were

exposed to 12/12 hours dark/light cycle and hormone ap-

plication, as well as, blood acquisition were performed at

defined times of the day.

Six groups of rats, 13 animals in a group, including two

control groups were used for determination of plasma anti-

gen concentrations of clotting factors F II, F VII, F IX and

F X. The hormones enrolled in the study were applied in

three consecutive days as follows: somatotropin (Somato-

tropin, human, Boehringer Mannheim, activity 1.8

units/ml) - 0.2 mg/kg body mass, s.c.; somatostatin

(Tyr-somatostatin, Sigma, activity minimum 97%) - 0.1

mg/kg, s.c.; somatostatin applied 3 h before somatotropin

in the same doses and scheme; and prolactin (prolactin ace-

tate, Ferring) - 2 õ 0.1 mg/kg, i.p.), and the animals in the

control groups were treated by the solvent. The doses used

were determined, on the bases of literature data, and

through preliminary analysis of logarithmic dose-effect de-

pendence. Blood samples were gathered by a cardiac punc-

ture under ether narcosis. Sodium citrate (0.11 mol/l) was

used as an anticoagulant (blood/citrate ratio 9:1). The citrat-

ed blood was centrifuged for 10 minutes at 3000 revolu-

tions per minute. The citrated plasma (the supernatant) was

separated and stored at 40Ñ in haemostatic test-tubes. The

coagulation parameters studied were determined not later

than 2 hours.

Plasma clotting factor concentrations were estimated by

ELISA methods using tests of Diagnostica Stago, France.

ELISA assays were done in duplicate and each sample was

measured twice. Optical densities were recorded on

Organon Teknika ELISA reader and the corresponding pa-

rameters (concentrations) were calculated by 4 parameter

fit logarithmic curves by the means the software of the

ELISA reader. Histological screening and macroscopic in-

spection for microhemorrhages and intravascular

thromboses were performed. Slices from internal organs

(liver, kidney and intestine) were prepared and stained by

hematoxylin-eosin and by Weigert for fibrin for detection

of micro-hemorrhages and/or intravascular coagulation.

All data were analyzed by variation analysis by GraphPad

Prism 4 software package, using the t-test of Student-Fisher

and the normality hypothesis was checked. Values of p less

than 0.05 were considered significant.

138

Somatotropin, somatostatin and prolactin effects on ...

Fig.1. Somatotropin effects on plasma clotting factors

II, VII, IX and X.

Data are presented as MEAN ± SEM; * - p<0.001, N.S.

Fig. 2 Somatostatin effects on plasma clotting factors II,

VII, IX and X.

Data are presented as MEAN ± SEM; * - p<0.001

Fig. 3. Somatotropin applied after somatostatin

pretreatment. Effects on plasma clotting factors II, VII,

IX and X.


Fig. 4. Prolactin effects on plasma clotting factors II,

VII, IX and X.


RESULTS AND CONCLUSIONS

Somatotropin reduced significantly plasma antigen con-

centration (p<0.001) of FII, FVII, and FX, respectively

from 130.40±6.00 to 50.08±5.22, 59.32±5.14 and

38.97±5.59 versus control group, but did not affect FIX.

(Fig. 1) Somatostatin, applied separately increased

(p<0.001) FII, FVII, and FX from 100.10±6.32 in the con-

trol group to, respectively 190.0±8.81, 169.30±6.99 and

189.30±13.65, while F IX was decreased to 47.85±5.80

(Fig. 2). Somatostatin applied 3 h before somatotropin in-

creased (p<0.001) FII, FVII, and FX from 100.00±3.62 in

the control group to respectively, 158.5±6.66, 162.40±9.49

and 175.00±10.85, and decreased F IX (p<0.001) to

50.83±8.02. (Fig. 3) Prolactin elevated all parameters stud-

ied (p<0.001) from 69.26±5.44 to 120.10±9.15,

155.50±8.31, 115.10±5.55, 125.10±9.67, respectively for F

II, F VII, F IX and F X versus controls (Fig. 4).

These results evidence:

� Somatotropin, somatostatin and prolactin are

substantially involved in regulation of plasma levels of

the factors studied, as somatotropin reduces FII, FVII,

and FX , while somatostatin and prolactin provide the

opposite effects.

� The completely different pattern of the effects of the

hormones on F IX suggest different mechanism(s) of

the hormones applied related to this clotting factor.

� Somatostatin application 3 h before somatotropin

completely abolishes the effect of the exogenous

somatotropin and this combination of the hormones

reproduces the effect of somatostatin applied separately.

� The results of the effects of somatotropin, somatostatin

and prolactin on vitamin K-dependent plasma clotting

factor (F II, F VII, F X and F IX) antigen concentrations

reproduce the effects of the same hormones on F II, F

VII and F X plasma activities, but not F IX reported

previously (9).

� Having in mind the basic site of biosynthesis of the

vitamin K-dependant clotting factors enrolled in the

study, as well as the site of their gamma-carboxylation,

the liver may be considered the place where the

hormonal effects are exerted. However, other

mechanisms of the actions of the hormones applied

should not be excluded.

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1. Hoffman M., Monroe D. M. III . (2001) A

cell-based model of hemostasis. Thromb Haemost

85:958-65.

2. Rosendaal F. R. , van Hylckama V. A.,

Tanis B. C. , Helmerhorst F. M. (2003)

Estrogens, progestogens and thrombosis. J Thromb

Haemost 1:1371-1380.

3. Orwoll E. S. , Orwoll R. L. (1987) Hematologic

abnormalities in patients with endocrine and meta-

bolic disorders. Hematol Oncol Clin North Am

1:261-279.

4. Banga J. D. (2002) Coagulation and fibrinolysis in

diabetes. Semin Vasc Med. Feb;2(1):75-86.

5. Targher G. , Pichir i I . , Zoppini G. , Bonora

E. , Chonchol M. (2009) Hemostatic and

fibrinolytic abnormalities in endocrine diseases: a

narrative review. Semin Thromb Hemost.

Oct;35(7):605-12.

6. Scarpignato C. , Pelosini I . (1999) Somatostatin

for Upper Gastrointestinal Hemorrhage and Pancre-

atic Surgery A Review of Its Pharmacology and

Safety. Digestion 60:1-16.

7. S�vendahl L.S., Grankvist K., Engstr�m K. G.

(1995) Growth hormone deficiency impairs blood

clotting and reduces factor VII coagulant activity in

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8. Kerst in Landin-Wilhelmsen. (1999) Effects of

growth hormone on blood coagulation and

fibrinolysis. In Growth hormone. Ed. Bengt-Àke

Bengtsson. Klewer Academic Publishers. Dordrecht.

The Netherlands.

9. Negrev N., Nyagolov Y., Stanchewa E. .

(1995) Somatotropin and somatostatin effects on vita-

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2-3, Pages: 145-149.

139

Nyagolov Y., I. Pashaliewa, N. Doncheva ...

RESEARCH ON VARIATIONS IN SOME

ELECTROPHYSIOLOGICAL CHARECTERISTIC DATA OF

ACUPUNCTURE POINTS IN EXPERIMENTAL ANIMALS PUT

UNDER DIFFERENT ATMOSPHERE PRESSURE

Hristov K.

Department of Physiology and Pathophysiology, Medical University - Varna

There is scarce literature information on the issues of electrophysiological parameters of acupuncture points,

and changes these parameters undergo as a result of the impact of various factors. According to the literature

the acupuncture points are characterized by reactance, capacity, inductance, conductivity, thermal radiation

and semiconductor effect. Changes in some of these parameters, reactance and thermal radiation, have a def-

inite diagnostic importance for the practice of acupuncture. The purpose of this research was to study the

changes in some parameters of acupuncture points (reactance and semiconductor effect), and general

electrophysiological parameters (antenna effect and electromagnetic radiation in the radio range) as well in

animals placed at a stressful situation with respect - hyperbaric hyperoxia and hypobaric hypoxia. The exper-

imental study was conducted with rats and rabbits. Animals were subjected to different regimes of hyperbaric

and hypobaric air. Relevant parameters were examined before and after loading. The results received during

the examination point to closely relative quality changes in both extreme loads. This gives grounds to assume

for the presence of a similar nature in the body energy exchange with the environment in both extreme loads

too. On the other hand the results are good basis for further studies of used electrophysiological parameters.

In addition these results confirm the change in some parameters of the acupuncture points, which in turn has

serious grounds to seek a direct link between the Eastern (Chinese) theosophical teachings and modern

medical science.

Key words: acupuncture points, electrophysiology, semi-conductor effect, antenna effect, hyperbaric

hyperoxia, hypobaric hypoxia

Biologically active points (BAP) according to the literature

are characterized by a number of electrophysiological and

physical parameters, such as reactance, inductance, capaci-

tance, conductivity, semicunductor effect, temperature ra-

diation etc. It is not known enough about the dynamic

changes of these parameters under different conditions still.

However, there is sufficient evidence and reason to assume

that changes in these parameters have a direct connection

with the processes in organs and body parts whose repre-

sentatives are the relevant BAP. For example, lowering the

resistance and temperature rise in the points in comparison

with the surrounding tissue has important diagnostic signif-

icance in electroacupuncture therapy. Accumulated in re-

cent years data provide serious grounds to adopt a working

assumption that the organisms communicate with the en-

vironment through electromagnetic phenomena in certain

frequency ranges, and the acupuncture points are the bio-

logically active agents in such type of communication. This

phenomena suggests the possibility of using electromag-

netic radiation in certain frequency bands from the body

and antenna effect of the body in order to obtain more

complete information about the general state of

experimental animals.

Biologically active corporal point Ying-Tang in traditional

Chinese medicine is considered to be the major energiser of

the body (the gateway to the temple - from Chinese). It is

used mainly for treatment to restore energy relationships in

the body.

Biologically active auricular point Sheng Men (Door to

heaven from Chinese) is one of the most common biologi-

141


Figure 1. Localisation of corporal point Ying-Tang in

rats

cally active points. It is believed that this point has a serious

impact in the treatment of various pathological abnormali-

ties, including respiratory and cardiac diseases, pain, in-

flammation, dysfunctional states, general stimulating effect

and others.

Biologically active auricular point Sheng Men (Door to

heaven from Chinese) is one of the most common biologi-

cally active points. It is believed that this point has a serious

impact in the treatment of various pathological abnormali-

ties, including respiratory and cardiac diseases, pain, in-

flammation, dysfunctional states, general stimulating effect

and others.

In the second experimental setup research was conducted

with 30 white nonbreed rats of both sexes with an average

body weight 170 g. Animals were divided into 5 groups

that undermine the response to hypobaric hypoxia: 830 hPa

equal to 1500 m above sea level, 710 hPa - 3000 m above

sea level, 480 hPa - 6000 m, 320 hPa - 8850 m and 220 hPa

- 12,000 m. Speed of decompression and recompression in

the chamber for all groups - 50 hPa / min, exposure time -

15 minutes. Before and after loading in all animals the

electrophysiological parameters of antenna effect were

traced.

The obtained results were processed by variance analysis.


The results of studies of animals from the first experimental

setup with hyperbaric load are set as follows:

The changes of resistance the in straight and reverse direc-

tion and the resulting changes in the semiconductor effect

under hyperbaric pressure in our opinion are evidence for

increased getting into contact of the BAP with the energy

sources of the environment. Increasing of the antenna effect

142

Research on variations in some electrophysiological charecteristic data of ...

Figure 2. Location of auricular point Sheng-Men in rats

Figure 3. Biosensoral detector for electromagnetic

radiation

Figure 4. Hyperbaric chamber MKBK – 42

Figure 5. Resistance in a straight and reverse direction

under hyperbaric pressure.

and the electromagnetic radiation suggest an increased

communication with the environment in the electromag-

netic spectrum range of 100MHz. In animals subjected to

different modes of response to hypobaric hypoxia is seen

that the greatest changes are in the antenna effect at 6,000

meters above sea level - 480 hPa. Proceeding from the no-

tion that antenna effect could be considered as a criteria for

communicative capabilities of the organism with environ-

mental energy at a given radio range, we can say that com-

municative opportunities are higher at a pressure of 830 to

480 hPa (1500 to 6000 m above sea level).

CONCLUSIONS

1. There were credible changes in the values of the

antenna effect of different modes of response to

hypobaric hypoxia, the biggest of these changes are

from 1500 to 6000 m asl.

2. There were significant changes in the values of

electrophysiological parameters measured in animals

exposed to hyperbaric stress where despite the lack of

statistical reliability there is a serious trend in all animals

to change the parameters.

3. In two disparate impact on different states between

unpaid animals - response to hypobaric and hyperbaric

pressure - in practice the deviations in the studied

parameters are at common mode.

4. The results raises serious questions for further

investigating about the communicative possibilities of

experimental animals in the electromagnetic spectrum

range.

BIBLIOGRAPHY

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143

Hristov K.

Figure 6. Antenna effect under hyperbaric pressure.

Figure 7. Electromagnetic radiation under hyperbaric

pressure.

Figure 8. Antenna effect in response to hypobaric

hypoxia

100 Hz electroacupuncture analgesia in the rat.

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ÃÁÎ, 1989, Ìîñêâà.

13. K.Par tr idge. Associating Users and Devices using

Attention-Correlated Communication. PhD thesis,

University of Washington, 2005.

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- 10 mb/s adaptive frequency hopping transciever for

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2009.

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T.Asahi . Human area networking technology:

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perimental evaluation of auricular diagnosis: the

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acupuncture points. 1980.

144

Research on variations in some electrophysiological charecteristic data of ...

LONG-TERM SURVIVAL OF A SERIES OF CANCER PATIENTS

WITH PURULENT SEPTIC POSTOPERATIVE COMPLICATIONS

Daskalov Ì.

Medical University of Sofia

ABSTRACT

The author analyzed the results from the postoperative management of the septic complications of eight pa-

tients, six males and two females, with advanced colorectal cancer. These patients have undergone radical and

enlarged operations. All of them survived longer than 10 years. A paradrainage transwound economic

laparostomy after Daskalov was also performed. All the patients were postoperatively treated with chemo-

therapy, immune stimulation with decaris and large amounts of vitamin C. The probable reason for the long

survival was the stimulation of cell immunity. T-lymphocytes activated during inflammation could destroy

the etiological agents of inflammation and single cells having undergone carcinomatous modifications. The fa-

vourable survival was promoted by hyperthermia and stimulation of the cellular immune functions during

the septic process.

Key words: advanced colorectal cancer, septic postoperative complications, hyperthermia, survival, cell

immunity

INTRODUCTION

Term of survival represents the main criterion for success

of cancer treatment. Thus it is a parameter for a well-per-

formed surgical intervention favourably combined with

chemo-, x-ray, immunotherapy and other treatment modal-

ities as well (3-9,11,13,14). During the advanced stage of

colorectal cancer presenting with lymph node metastases

and local progress up to T3-T4, the survival rate over 5

years is very low and there are almost no cases survived

longer than 10, 12, 15 and more years. In such cases of

serously proliferating cancer and operative metastases

some authors induce hyperthermia with a liquid that con-

tains cytostatic drugs such as mitomycin and cistplatine

(1,2,6-11,13,14). Hyperthermia combined with x-ray ther-

apy is administered, too. When the temperature of 410C is

reached, cell proliferation remains unaffected, at 42-430C

and during perfusion hyperthermic hyperpyrexia there oc-

curs apoptosis while at 440C cell necrosis and fibrous

histiocytosis can be seen. Hyperthermia influence varies in

different cytostatic combinations, however, as a whole, it is

considered positive.

The patients with serously proliferating gastric cancer pre-

senting with metastases are favourably influenced by

hyperthermia combined with cytostatic drugs (6,9,14). This

occurs also in cases with peritoneal metastases of ovary

cancer (10,14) and colorectal one as well (14).

In such cases, some authors apply, similarly to S. Hadzhiev

(cited after 1), a variety of vaccines, e.g. BCG, herpes virus

and other vaccines as well as other forms of immunologic

management (1).


Our study covers six male and two female patients with

colorectal cancer. The local progression consists in the af-

fection of the three layers of the organ, or in proliferation

into adjacent organs along with first- and second-range

lymph node metastases. It deals with carcinoma at stage Ò3

and Ò4, with or without lymph node metastases (N1 or N2).

These patients have undergone radical and enlarged opera-

tion. Due to current reasons, purulent septic complications

develop which are successfully treated. All the patients sur-

vive longer than 10 years as two of them survive even 16

and 21 years after the operation.

Case reports

The first patient was 60 years old. He underwent

hemicolectomy with lymph node dissection and resection

of 5th and 6th liver segments. A postoperative subhepatic ab-

scess in the right abdominal part occurred. It healed follow-

ing drainage aspiration and opening of the dermal and

fascial sutures along the bundle of tube drainages

(paradrainage transwound economic laparostomy after

Daskalov, 1). Septic fever lasted more than two weeks.

Healing resulted in the occurrence of a postoperative repo-

sition hernia that did not hamper his physical activity. He

survived longer than 20 years without any relapses or

metastases at all.

The second patient presented with rectal cancer of T3N1/2

as well as with empyema vesicae felleae, diabetes mellitus

and obesity of second degree. We performed

cholecystectomy, lymphadenectomy, abdomino-perineal

rectum amputation, and definite iliac anus praeter. Postop-

145


erative sepsis lasted longer than two weeks and consisted in

a purulent secretion originating from the bundle of tube

drainages located in the pelvis and from those draining the

operative wound itself. A paradrainage laparostomy after

Daskalov was carried out as both adjacent skin and fascial

sutures were removed (1). Healing was achieved through

active reanimation and antibiotic therapy. At the site of

laparotomy, subsequently, a postoperative hernia devel-

oped which relapsed after the plastic correction done sev-

eral years later on. His work capacity was restored and he

survived longer than 17 years without any relapses. He died

of adhesion ileus following surgical intervention in another

health institution. He suffered from diabetes mellitus

controlled by maninil.

The third patient was 59 years old and underwent an

abdominoperineal rectum amputation on the occasion of a

carcinoma at stage Ò4 N1/2 which removal caused

parenchymatous bleeding and necessitated tamponade with

local Mickulicz compress. A suppuration and sepsis devel-

oped at the background of diabetes mellitus. The patient

survived 21 years long.

The fourth patient presented with rectum amputation as a

consequence of carcinoma at T3N1/2 and accompanying

pelvic peritonitis. Sepsis occurred and lasted more than one

week. It was managed with drainage aspiration and

paradrainage transwound economic laparostomy (1) as

well as by antibiotic therapy and plasty with an

antimicrobial cusp for the emerged postoperative hernia

was performed resulting in a durable healing lasting more

than 20 years.

The fifth patient was 60 years old and presented with rec-

tum amputation for a carcinoma of Ò4N1/2. Postopera-

tively, septic fever occurred and lasted more than one week

favourably managed by the aspiration of the purulent con-

tent from the pelvic space through drainages as well as by

the accompanying antibiotic treatment. Two years later on,

the patient was operated for a localized carcinoma of the

urinary bladder and healed. The patient survived longer

than 20 years under good health conditions and with a pre-

served work capacity. He died of another disease.

The sixth patient was 60 years old and presented with a rec-

tal cancer at T3 stage. Postoperatively, he developed a pu-

rulent sepsis. He restored and reached up the age of 82

years.

The seventh patient was a 60-year-old female. She was op-

erated on the occasion of a genital neoplasm and developed

a purulent collection. After the successful operation she re-

mained under a good condition and reached up the age of

90 years without any relapses.

The eighth patient was a 45-year old female and presented

with gastric lymphosarcoma. She developed a postopera-

tive peritonitis and a purulent fistula. She was re-operated

after Daskalov's method (1) and enjoyed a good health sta-

tus longer than 21 years.

All the patients were postoperatively treated with chemo-

therapy according to a scheme approved by the Clinic of

Chemotherapy, Tsaritsa Giovanna University Hospital of

Sofia. Besides, an immune stimulation with decaris was

performed. Additionally, large amounts of vitamin C were

administered.

The long survival of these patients presenting with carcino-

mas of the size of children's hand and fist and affecting the

entire wall of the colon and rectum can't be explained nei-

ther by the radical intervention, nor by the cytostatic ther-

apy only. Probably, hyperthermia caused by the purulent

septic condition experienced by the patients is of impor-

tance, too. This coincides with the opinions of numerous

authors about the significance of the hyperthermia (11,13).

The involvement of the autoimmunization, similarly to the

influence of the fixation abscesses during the rheumatic

conditions can't be definitely considered. Suppuration and

fever in four of our cases occurred along with diabetes

mellitus which was routinely treated.

The probable reason for the long survival of these patients

who experienced sepsis and suppuration is the stimulation

of cell immunity. T-lymphocytes activated during inflam-

mation are capable of destroying not only the etiological

agents of inflammation but also single cells having under-

gone carcinomatous modifications. This reduces the proba-

bility of metastases during the postoperative period.

CONCLUSION

Herewith we report the unusually long-lasting survival of a

group of patients with advanced colorectal cancer who

have undergone radical interventions, with postoperative

hyperthermia based on the emerged suppuration and mi-

crobial septic process. The principle of radical operation

was observed in an optimal volume. However, the favour-

able survival was, probably, promoted by hyperthermia and

stimulation of the cellular immune functions during the

septic process.

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146

Long-term survival of a series of cancer patients with ...

5. Furukawa, H. , M. Hiratsuka, T. Iwanaga. A

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7. Panteix, G. , M. Guil laumont , L. Cherpin, J .

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8. Sayag, A. C. , F. N. Gil ly , P. Y. Carry , J . P.

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9. Sel l ins , K. S. , J . J . Cohen. Hyperthermia in-

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10. Takano, Y. S. , B. V. Harmon, J . F. Kerr .

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11. Yonemura, Y. , T. Fuj imura, S. Fushida, S.

Takegawa, T. Kamata, K. Katayama, et al .

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12. Yonemura, Y. , K. Katayama, T. Kamata, S.

Fushida, M. Segawa, S. Ooyama, et al . Sur-

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1991, No 4, 222-225.

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Sugiyama, K. Fuj imura, T. Sawa, et al . Pro-

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450-454; discussion 455.

14. Yonezawa, M., T. Otsuka, N. Matsui , H.

Tsuj i , K. H. Kato, A. Moriyama, et al .

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147

Daskalov Ì.

During the recent year, the eminent Bulgarian surgeon, professorMarko Daskalov, MD, celebrated his 80th anniversary. He was bornon June 19, 1931, in the village of Smilets, Pazardzhik district. In1955, he graduated with excellent scores from the Higher Institute ofMedicine of Plovdiv. Then he was occupied as surgeon and gynae-cologist in the towns of Ivailovgrad and Rakovski. In 1959-1960, hemade the specialities of 'surgery' and 'orthopaedics and traumatology'and was occupied in the corresponding clinics in the city of Plovdiv.In 1961, he became an assistant in the Clinic of Surgery and Ortho-paedics in Alexandrovska Hospital of Sofia. Later on he made this spe-ciality in Paris, France. He consecutively became associate professorand then full professor in the Second Department of Surgery of theMedical Academy of Sofia where he delivered the courses of lecturesto the medical students and the postgraduate students as well.Professor Marko Daskalov has authored more than 280 scientific pub-lications, including 4 monographs. He was the author of numerousown unique operative techniques as well as of new methods for simul-

taneous operation of several organs, the so-called 'en-bloc' operations. Besides we elaborated surgi-cal techniques for the 'creation' of new organs such as neogaster, ampula recti, etc. Additionally, heintroduced a series of modifications of already existing operative methods into the clinical practice.Professor Marko Daskalov performed the first successful experiments in dogs in order to transplantthe spleen and pancreas which were filmed and broadcasted by the national TV.He is an active member of the Bulgarian Society of Surgery. He is our first board member in the Coun-cil of Eurosurgery where he represented our country during a lot of years. Professor Marko Daskalovwas awarded the Medal of Cyril and Methodius of first degree for his active creative, scientific, teach-ing and surgical work.

Happy Birthday to You, our Teacher! And Happy Anniversary!

AUTOR'S INDEX

Achkakanov I. ........................... 55

Alexandrova A. ......................... 55

Alexandrova M. ........................ 63

Alexiev K. ................................. 109

Angelova P. ............................... 81

Atanasov P. ............................... 55

Atanasova P. ............................. 133

Bekyarova G. ............................ 31, 35

Belcheva I. ................................ 59

Belcheva S. ............................... 59

Bocheva N. ............................... 11, 73, 109

Boyadjiev N. ............................ 81

Danovska M. ............................ 63, 145

Decheva L. ............................... 129

Doncheva N. ............................. 137

Dushanova J. ............................ 15

Encheva E. ................................ 19

Gatev P. .................................... 125

Gendzhev Z. ............................. 101

Genova B. ................................. 11, 109

Georgiev K. .............................. 51

Georgiev Ts. ............................. 93

Georgieva O. ............................ 73

Hadzhibozheva P. ..................... 93

Halacheva L. ............................. 77

Hrischev P. ............................... 133

Hristov I. .................................. 67

Hristov K. ................................. 141

Hristova M. .............................. 31

Hristova M. ............................... 35

Ivanov I. .................................... 23

Ivanova A. ................................. 81

Ivanova M. ................................ 59

Klisurov R. ................................ 19

Kolarova R.tragvam .................. 97, 101, 105

Kolev N. .................................... 77

Kossev A. .................................. 5

Kupenova P. .............................. 23

M. Krustev S. ............................ 117

Mihaylova M. ............................ 67

Miladinova E. ............................ 51

Minchev Zl. ............................... 125

Mitov D. .................................... 15, 67

Muletarov S. .............................. 81

Negrev N. .................................. 59, 117, 129,137

Nikolov F. ................................. 133

Nikolov P. ................................. 133

Nikolova J. ................................ 133

Novoselski M. ........................... 19

Nyagolov Y. .............................. 129, 137

Orbezova M............................... 133

Pajpanova T............................... 51

Pashalieva I. .............................. 129

Pashaliewa I............................... 137

Pavlova E. ................................. 27, 89

Pechlivanova D. ........................ 47

Petkov V.................................... 19

Petkova Zl. ................................ 47

Petrov L. .................................... 55

Popova E. .................................. 23

Racheva K. ................................ 67

Sarov G...................................... 39, 43

Somlev P. .................................. 27, 85,89

Stancheva E. .............................. 129

Stefanov S. ................................ 11, 109

Stefanova M. ............................. 11, 73

Stephanova D. I. ........................ 117

Stoynev Al................................. 47

Tancheva L................................ 19

Tashev R.................................... 59

Tchekalarova J........................... 47

Tolekova A................................ 93

Totev Ts. ................................... 67

Tsaneva M. ................................ 35

Tzvetkov S. ............................... 113

Uzunova G. ............................... 27, 89

Zaekov N. .................................. 55

Zsheliaskova-Koynova Z. ......... 55

149


PERMUTERM SUBJECT INDEX

acupuncture points, electrophysiology,

semi-conductor effect, antenna effect,

hyperbaric hyperoxia, hypobaric hypoxia_______141

advanced colorectal cancer, septic

postoperative complications, hyperthermia,

survival, cell immunity ____________________145

Ageing, Motion discrimination, Fuzzy

clustering, Mixed ANOVA___________________73

bioelectricity, human emotions, social

networks, spectral analysis, rhythm

dynamics ________________________________125

classification images, motion direction

discrimination, internal noise, directional

tuning, temporal integration _________________109

Contrast, VEP, Spatial frequency,

Receptive fields____________________________67

dark adaptation, dopamine,

electroretinogram, frog, retina _______________23

diabetes, hypertension, stroke,

inflammation, CRP _________________________63

dopamine, dark adaptation,

electroretinogram, frog, retina _______________23

electrophysiology, acupuncture points,

semi-conductor effect, antenna effect,

hyperbaric hyperoxia, hypobaric hypoxia_______141

endomorphins, morphiceptin, opioid

receptors, halogenated phenylalanines,

peptide mimetics ___________________________51

exercise, physical working capacity, fat

metabolism _______________________________81

experimental hypertension, myocardial

hypertrophy, cardiac histomorphological

changes, Vit. E ___________________________101


hypertrophy, myocardial enzyme activity,

Vit. E____________________________________97

FII, vitamin K-dependent clotting factors,

FV, FIX, FX, somatotropin, somatostain,

prolactin ________________________________137

Handgrip, muscle force, bilateral deficit,

sex differences ____________________________77

heart rate variability, heart rate, saliva,

salivary alpha-amylase activity, salivary

potassium ________________________________55

heart rate variability, paced breathing,

athletes __________________________________85

heart rate variability, soccer players,

training __________________________________89

heart rate, heart rate variability, saliva,

salivary alpha-amylase activity, salivary

potassium ________________________________55

holistic, structural, functional,

pathophysiology ___________________________43

HR recovery, PWC170, VO2 max,

coefficient of recovery, soccer players __________27

human emotions, bioelectricity, social

networks, spectral analysis, rhythm

dynamics ________________________________125

hypertension, diabetes, stroke,

inflammation, CRP _________________________63

hypokinesia, physical exercise, Vit. E,


metabolism ______________________________105

light immunohistochemistry, superoxide

dismutase, malondialdehyde, liver, burns,

melatonin ________________________________35

luzindolå, melatonin, reptilase time, TAFI,

TAFIa/TAFIai____________________________129

maximal oxygen consumption, ventilatory

response, maximal incremental exercise

test, taekwondo athletes ____________________113

melatonin, luzindolå, reptilase time, TAFI,

TAFIa/TAFIai____________________________129

morphiceptin, endomorphins, opioid

receptors, halogenated phenylalanines,

peptide mimetics ___________________________51

motion direction discrimination,

classification images, internal noise,

directional tuning, temporal integration ________109

Motion discrimination, Ageing, Fuzzy

clustering, Mixed ANOVA___________________73

muscle force, Handgrip, bilateral deficit,

sex differences ____________________________77

muscle vibration (MV), transcranial

magnetic stimulation (TMS), motor evoked

potential (MEP), intracortical inhibition

(ICI), intracortical facilitation (ICF), silent

period (SP) ________________________________5

myelin sheath aqueous layers,

strength-duration time constant,

demyelinating neuropathies,

computational neuroscience _________________117

myocardial hypertrophy, experimental

hypertension, cardiac histomorphological

changes, Vit. E ___________________________101

myocardial hypertrophy, experimental

hypertension, myocardial enzyme activity,

Vit. E____________________________________97

Olfactory bulbectomy, Vasoactive

intestinal polypeptide, Hippocampus,

Locomotor activity, Lateralization, Rat _________59

paced breathing, heart rate variability,

athletes __________________________________85

Perception, Vision, Motion, Speed,

Adaptation, Sensitivity ______________________11

physical exercise, hypokinesia, Vit. E,


metabolism ______________________________105

physical working capacity, exercise, fat

metabolism _______________________________81

151


PWC170, HR recovery, VO2 max,

coefficient of recovery, soccer players __________27

septic postoperative complications,

advanced colorectal cancer, hyperthermia,

survival, cell immunity ____________________145

sinusoidal gratings, visual event-related

potentials, orientation discrimination ___________15

smooth muscle contraction, uterus,

peptides, hyperactivity, in vitro

experiments_______________________________93

soccer players, heart rate variability,

training __________________________________89

spontaneous hypertensive rats (SHRs),

Wistar rats, kainate model of temporal

lobeepilepsy; melatonin; diurnal rhythms________47

strength-duration time constant, myelin

sheath aqueous layers, demyelinating

neuropathies, computational neuroscience ______117

structural, holistic, functional,

pathophysiology ___________________________43

superoxide dismutase, light

immunohistochemistry, malondialdehyde,

liver, burns, melatonin ______________________35

thiol groups, uric acid, malondialdehyde,

gastric mucosa, burns ______________________31

transcranial magnetic stimulation (TMS),

muscle vibration (MV), motor evoked

potential (MEP), intracortical inhibition

(ICI), intracortical facilitation (ICF), silent

period (SP) ________________________________5

uric acid, thiol groups, malondialdehyde,

gastric mucosa, burns ______________________31

uterus, smooth muscle contraction,

peptides, hyperactivity, in vitro

experiments_______________________________93

Vasoactive intestinal polypeptide,

Olfactory bulbectomy, Hippocampus,

Locomotor activity, Lateralization, Rat _________59

ventilatory response, maximal oxygen

consumption, maximal incremental

exercise test, taekwondo athletes _____________113

VEP, Contrast, Spatial frequency,

Receptive fields____________________________67

Vision, Perception, Motion, Speed,

Adaptation, Sensitivity ______________________11

visual event-related potentials, sinusoidal

gratings, orientation discrimination ____________15

vitamin K-dependent clotting factors, FII,

FV, FIX, FX, somatotropin, somatostain,

prolactin ________________________________137

Wistar rats, spontaneous hypertensive rats

(SHRs), kainate model of temporal lobe

epilepsy; melatonin; diurnal rhythms ___________47

152

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Scripta Scientifica Medica is the official publication of Medical University Prof. Dr. Paraskev Stoyanov, Varna, Bulgaria. It is

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153


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