international journal of biological & medical researchthamaraiselvi k*, mathangi dc a,subhashini...
TRANSCRIPT
Contents lists available at BioMedSciDirect Publications
Journal homepage: www.biomedscidirect.com
International Journal of Biological & Medical Research
Int J Biol Med Res. 2012; 3(2):1754-1759
Effect of increase in duration of REM sleep deprivation on lipid peroxidation* a bThamaraiselvi K , Mathangi DC ,Subhashini AS .
A R T I C L E I N F O A B S T R A C T
Keywords:
REM sleep deprivationFree radicalsLipid peroxidationMDA
Original article
1. Introduction
Sleep deprivation is becoming common in this fast moving world. Sleep deprivation has been
shown to have deleterious effect on the human body, giving rise to many diseases and disorders
and REM sleep deprivation is shown to increase free radicals and cause more harm as the
duration increases. The aim of this study is to assess the effect of increase in duration of REM
sleep deprivation on lipid peroxidation. REM sleep deprivation cause an increase of free radical
induced damage in blood. This can be shown by estimation of plasma malondialdehyde (MDA)
level. Wistar strain male albino rats were used to study the effect of REM sleep deprivation.
They were divided into two groups, control and REM sleep deprived. REM sleep deprivation
was obtained by inverted flowerpot technique. Heparinised blood sample was taken and
assessed for plasma MDA level. Body weight and food intake was monitored. The values
obtained were statistically analysed using one way analysis of variance (ANOVA). On
significant f test ratio, Tukey's multiple comparison tests were also performed. The level of
significance was kept at P<0.05. All the results obtained from the study are expressed as mean +
standard deviation (SD). The result showed an increase in plasma MDA level as the duration of
sleep deprivation increased, 48hrs (8.9* ± 2.87) and 72hrs (10.8* ± 2.4). There was only
minimal change in 24hrs (7.1 ± 0.76) and 96hrs (6.4 ± 1.6) when compared to the control
animals (5.4 ± 0.79) which were not significant. This increase in MDA level can be attributed to
increase in free radical formation and damage, as the duration of REM sleep deprivation
increases. So sleep is important in life, especially REM sleep.
In this fast moving world, with cost of living soaring sky high
people are finding it difficult to make both ends meet. To meet the
requirements money is needed and money is earned by sacrificing
the basic need of the body i.e. sleep. Sleep is one of the important
needs like oxygen, nutrition for survival. Sleep has been classified
into two stages, non rapid eye movement (NREM) and rapid eye
movement (REM) sleep. It has been shown that rapid eye
movement (REM) sleep is important as REM deprivation has shown
many deleterious and unwanted harmful effects. REM sleep might
participate in the genesis or regulation of at least the following
functions, dreaming experience, basic rest activity cycle,
motivational and adaptive behaviours, internal stimulation of the
immature brain, programming of the behavioural code, memory
consolidation, unlearning of trivial memory traces, amplifying of
Number of theories has been proposed on sleep like cerebral
anaemia, fatigue theory, theory of diminished sensory input and
chemical theories. Free radical flux theory of Reimund,[5] is a
recent addition. Free radical flux theory of sleep by Reimund, [5],
states that free radicals accumulate during wakefulness and are
removed during sleep. Removal of excess free radicals during sleep
is accomplished by decreased rate of free radicals and increased
efficiency of endogenous antioxidant mechanism. Thus sleep
essentially has antioxidative role.
BioMedSciDirectPublications
Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.ISSN: 0976:6685.c
International Journal ofBIOLOGICAL AND MEDICAL RESEARCH
www.biomedscidirect.comInt J Biol Med ResVolume 2, Issue 4, Jan 2012
*Department of Physiology, SRM Medical College Hospital & Research Center, Kattangulathur, a Department of Physiology, Chettinadu Medical College Hospital, Chennai, b Department of Physiology, Sri Ramachandra Medical College Hospital & Research Institute, Chennai.
* Corresponding Author : Dr. K.ThamaraiSelvi
Associate Professor, Department of Physiology, SRM Medical College Hospital & Research Center, Kattankulathur, 603209, Kancheepuram District, Tamilnadu. E-mail: [email protected]
Copyright 2010 BioMedSciDirect Publications. All rights reserved.c
1755Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759
Paradoxical sleep deprivation was found to be followed by
deep destructive changes in mitochondrial membranes of the
brain and erythrocytes. At the same time the activity of the
antiradical defence system was abnormal changes in the ratio of
particular lipid components and impairing the functional activity
of biological membranes [6].
Experimental studies on sleep deprivation and the harmful
effects have been recognised for some time [7]. If the assumption
of Reimund, [5], ie., freeradicals produced during waking, are
removed during sleep were correct, one would predict that sleep
deprivation would be associated with an accumulation of free
radicals. It was suggested that free radicals are involved in the
pathogenesis of various human disease, psychological stress and
the normal ageing process [8]. In rats it was shown that prolonged
sleep deprivation leads to a syndrome characterized by increased
food intake, weight loss, increased energy expenditure,
progressive decline in body temperature which is invariably fatal
[9]. It was also proposed that the neural activity associated with
sleep deprivation for longer durations may damage brain cells and
eventually to cell death [10, 11]. Reimund, [5], showed that
increase in free radicals is associated with sleep deprivation.
According to Zepelin and Rechtschaffen, [12], sleep seems to limit
metabolic requirements. Therefore sleep deprivation could
enhance the metabolic rate and in turn increase oxidative stress.
But Almeida et al., [13], showed that there is no oxidative stress
following paradoxical sleep deprivation in rats. Cirelli,[14], has
shown that there is no evidence of brain cell degeneration after a
long –term sleep deprivation. No change was detected in TBAR
substances [15] .Gopalakrishnan et al showed that there was no
evidence of oxidative damage at the lipid or the peripheral tissues
[16].Neurons are more prone to oxidative stress relative to other
cell types [17].
As the oxidative products of free radicals in the body may be
present in the blood, so an estimation of these products in the
blood will show the level of free radical induced damage in the
body and the importance of REM sleep.
Reports of free radical level and free radical induced cell
damage following sleep deprivation are scanty and also
contradictory. Aim of the study is to study the effects of REM sleep
deprivation on free radical induced damage in the blood in Wistar
strain rats and to elucidate the effect of duration of REM sleep
deprivation on these damages.
Wistar strain male albino rats weighing between 150- 180
gms, were used as the experimental animal for this study. Ethical
clearance was obtained from the Ethical Committee through the
institution before the study.
The animals were divided into three groups of Control, REM
sleep deprived and REM sleep control.
Group I – Control animals (n=6): These animals were used for
evaluating the baseline values for the various parameters to be
assessed.
2. Materials and method
2.1 Food intake and body weight
2.2 Estimation of plasma MDA level for lipid peroxidation [19]
2.3 Statistical Analysis
Group II – REM sleep deprived animals: These animals were used
to study the effect of REM sleep deprivation on lipid peroxidation
in the blood.
Group III – REM sleep control animals : These animals were used to
assess whether the REM sleep deprivation or any other effect like
isolation, immobilisation involved in the REM sleep deprivation
procedure.
The groups II and III were further subdivided into four groups
based on the duration of study as (a) 24 hrs. (b) 48 hrs. (c) 72 hrs.
(d) 96 hrs. With six animals in each group.
The animals were provided with food and water ad libitium, with
12 hrs light and 12 hrs dark cycle at ambient room temperature (24
- 28°C).
A ] REM sleep deprivation
An ingenous method of Jovet et al.,[18], was used for REM sleep
deprivation. Here the animals were placed on small platform
(diameter 6.5 cms) surrounded by a pool of water, where the
platform is small enough with reference to the body size of the
animal. This technique is based on the loss of skeletal muscle tone
typical of REM sleep is accompanied by the animal falling into
water.
B] REM sleep control
The REM sleep control animals were also placed in the similar
condition but with the platform size increased (diameter15 cms)
which allowed the relaxed position of REM sleep. This was done to
rule out the possibilities of non- specific effects like isolation.
The body weight of the animals was estimated before and
during the REM sleep deprivation and amount of food intake was
also measured during the period of REM sleep deprivation. This
was done to see whether there was any change in the body weight
and amount of food due to REM sleep deprivation.
The animals were sacrificed with mild ether anaesthesia at the
stipulated time as per the subgrouping (ie.24, 48, 72 and 96 hours).
Heparinised blood samples were collected from jugular vein.
The heparinised blood was centrifuged at 3000 rpm for half an
hour and plasma was obtained. Malondialdehyde reacts with
thiobarbituric acid to generate a coloured complex which was read
in spectrophotometer at an absorbance of 532nm.
The values obtained were statistically analysed using one way
analysis of variance (ANOVA). On significant f test ratio, Tukey's
multiple comparison tests was also performed. The level of
significance was kept at P<0.05.
All the results obtained from the study are expressed as mean +
standard deviation (SD).
1756
The animals subjected to REM sleep deprivation showed an increase in food intake in all the subgroups, 24hrs (57 + 19), 48hrs (57 +26) ,
72 hrs (73 + 25) and 96hrs (70 ±27), as compared to control (17 ±2.4)
b) Body weight (gms) (Table -1, Figure-II)
Body weight monitored showed a steady decline in all subgrouping of REM sleep deprivation inspite of being hyperphagic. i.e.,24hrs (166
+ 19), 48hrs (160±+18) , 72 hrs (157 ± 19) and 96hrs (147 ± 17), as compared to control (180 ±18)
c) Lipid peroxidation ( moles MDA /ml) Table -2, Figure-III)
MDA level estimated in the animals showed an increase following REM sleep deprivation for 48 hrs (8.9 ± 2.87) and 72 hrs (10.8 ± 2.4).
But there was only minimum change in 24hrs (7.1 ± 0.760 and 96 hrs (6.4 ± 1.6) when compared to the control animals (5.4 ± 0.790 which
were not significant.
To rule out the possible non-specific effects attributed to the technique of REM sleep deprivation like isolation, immobility, all the results
were compared with sleep control animals.
3. Results
I.REM sleep Deprivation
a) Food Intake (gms) (Table -1, Figure-I)
Table -1. Effect of REM sleep deprivation on food intake & body weight changes
Food intake (gms)
Body weight (gms)
REM deprived
REM control
REM deprived
REM control
n=6 in each group, values given as mean ± SD, *P<0.05
17 2.4
17 3
180 18
168 8
±
±
±
±
57* 19
37* 9
166 19
159 12
±
±
±
±
57* 26
56* 11
160 18
153 13
±
±
±
±
73* 25
71* 71
57 19
153 12
±
±
±
±
70* 25
72* 61
47* 17
150 12
±
±
±
±
6.531
54.252
2.25
2.02
Control 24hrs 48hrs 72hrs 96hrs 'F' test ratio
Table -2. Effect of REM sleep deprivation on lipid peroxidation
Figure I Effect of REM sleep deprivation on food intake. Figure II Effect of REM sleep deprivation on body weight
LPO (n moles MDA / ml plasma
n=6 in each group, values given as mean ± SD, *P<0.05
5.4+ 0.79 7.1+ 0.76 8.9*+ 2.87 10.8*+ 2.4 6.4+ 1.6 7.745
Control 24hrs 48hrs 72hrs 96hrs 'F' test ratio
Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759
1757
II- REM sleep control
a) Food intake (gms) table-1, Figure-I)
The food intake estimated showed an increase in all the
subgroups of 24hrs (37 ± 9), 48hrs (56 ± 11), 72hrs (71 ± 7) and 96
hrs (72 ± 6) compared to control animals.
b) Body weight (gms) (table-2. Figure-II)
Body weight monitored showed only minimal changes in all the
subgroups of 24hrs (159 ± 12), 48hrs (153 ±13) , 72hrs (153 ± 12),
and 96 hrs (150 ± 12) compared to control animals (168 +8)
c) Lipid peroxidation (n moles MDA/ml) (Table -3 Figure-III )
REM sleep control showed minimum changes in MDA level in all
subgroups of 24hrs (4.75 ± 1), 48hrs (6.319 ± 2.24), 72hrs (5.29
± 1.5) and 96hrs (4.888 ± 1.1) when compared to the control
animals (5.4 ± 0.79 ) which were also not significant.
The above table shows comparison between control, REM sleep
deprived and REM sleep control on lipid peroxidation values of 24
hrs duration.
The above table shows comparison between control, REM sleep
deprived and REM sleep control, on lipid peroxidation values of 48
hrs duration.
The above table shows comparison between control, REM sleep
deprived and REM sleep control, on lipid peroxidation values of 72
hrs duration.
n=6 in each group, values given as mean ± SD, *P<0.05
n=6 in each group, values given as mean ± SD, *P<0.05
n=6 in each group, values given as mean ± SD, *P<0.05
Table 3
Table 4
Table 5
LPO (n moles MDA / ml plasma)
LPO(n moles MDA / ml plasma)
LPO(n moles MDA / ml plasma)
5.4 0.79±
5.4 0.79±
5.4 0.79±
7.1 0.76±
8.9* 2.87±
10.8* 2.4±
4.75 1.0±
6.319 2.24±
5.29 1.5±
12.077
4.398
20.428
24 hrs
48 hrs
72 hrs
Control
Control
Control
REM deprived
REM deprived
REM deprived
REM control
REM control
REM control
'F' test ratio
'F' test ratio
'F' test ratio
The above table shows comparison between control, REM sleep
deprived and REM sleep control, on lipid peroxidation values of
96hrs duration.
Free radicals are very reactive molecules or fraction of
molecules, which contain one or more unpaired electrons; they
exist independently and are very unstable. The free radical often
reacts non-specifically with any other molecules, which are usually
paired and forms another free radical. Oxidative stress refers to a
disturbance in the pro and antioxidant balance in favour of the pro
oxidant [20]. Oxidative stress ensues when free radicals evade or
overwhelm the antioxidant protective mechanisms of cells and
tissues. It induces cytotoxic effects that are mediated by
perturbation of intracellular free calcium and thiol homeostasis. An
early response to oxidative stress is depletion of cellular soluble
and protein bound thiols. Therefore it causes decreased plasma
membrane Ca2+ ATPase activity, plasma membrane blebbing,
decreased mitochondrial ability to retain Ca2+, decreased Ca
sequestration capacity into endoplasmic reticulum and alters
microsomal Ca homeostasis. Free radical damage includes lipid
peroxidation of membrane, mitochondrial damage, lesions in DNA
and cross linking of proteins [21]
n=6 in each group, values given as mean ± SD, *P<0.05
Table 6
Figure III Effect of REM sleep deprivation on lipid peroxidation
LPO(n moles MDA / ml plasma)
5.4 0.79± 6.4 1.6± 4.888 1.1± 2.551
96 hrs Control REM deprived REM control 'F' test ratio
4. Discussion
Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759
In todays world free radicals are held culprit to various
diseases that affect human from infection to ageing and cancer. Co
morbidities linked to disturbed or impaired sleep such as
cardiovascular disease, diabetes, arthritis are increased.[ 22,23]
increased lipid peroxidation was seen in diabetes.[ 24 ]
This effect is enhanced by increase in amount of sources that
produce free radicals like pollution, smoking etc. the effect of free
radicals on our body are due to the damage caused to cellular
elements, and lipid peroxidation is one of them.
Reimund [5], hypothesised that free radicals which
accumulate during wakefulness are removed during sleep.
Removal of excess free radicals during sleep is accomplished by
decreased rate of formation of free radicals and increased
efficiency of endogenous antioxidant mechanisms. Thus sleep has
an antioxidative role.
Studies have been done on the effect of REM sleep deprivation
on lipid peroxidation, but these studies are scanty and also
contradictory, so the aim of our study is to know the effect of REM
sleep deprivation on the lipid peroxidation in the blood of Wistar
strain rats.
To deprive REM sleep in rats the technique developed by
Jouvet et al., [15], was followed where the animals are placed on a
small platform surrounded by water. The advantages of this
technique are that several animals can be deprived
s i m u l t a n e o u s ly, w i t h o u t l a b o r i o u s m o n i t o r i n g o f
electrophysiological characteristics of sleep.
Coenen and Van Luijtelaar [25], have shown that there was not
much difference in the various REM sleep deprivation protocols
like classical platform, pendulum, multiple platform. Both stress
index, (Selyes classical indices, adrenal hypertrophy, thymus
atrophy, body weight loss and stomach ulceration) as well as EEG
recording did not show significant differences between the
various REM sleep deprivation procedures [26].
But this technique was heavily criticised for its several stress
factors like restriction of movement, isolation, recurrent wetting,
emotional distress which are inherent to the procedure [27]. This
technique is at present accepted as a valid method if comparisons
were made with animals on large platform allowing the relaxed
position of REM sleep and without danger of falling into water
[2,29]. Hence a REM sleep control was also included in this study.
The body weight and food intake were monitored in this study
and it was found that there was a steady decline in the body weight
as the duration of REM sleep deprivation increases, though there
was a steady increase in food intake. This loss of weight despite
h y p e r p h a g i a w a s a l s o s h o w n b y o t h e r w o r k s
[25,29,30,31,32].Study by Kushida et al.,[31], also showed that
REM sleep deprivation caused weight loss in spite of increased
food intake, which they attributed to increased energy
expenditure with mean levels reaching more than twice base line
values. Though increased food intake was also observed in REM
sleep control animals it was not accompanied by decreased body
weight is peculiar to REM sleep deprivation as also shown [32].
4.Conclusion
5.References
The 24hrs and 96hrs REM sleep deprived animals of the
present study showed no change in the MDA level. This shows that
both short term sleep deprivation and long term sleep deprivation
do not produce symptoms of oxidative stress. This in agreement
with the reports of Cerelli et al.[14] ( REM sleep deprivation of 8hrs
) and Almedia et al.[13] ( REM sleep deprivation of 96hrs
respectively. 48hrs and 72hrs of REM sleep deprived animals
showed an increase in the level of MDA and REM sleep control
animals showed minimal changes. This is in correlation with
studies which showed that 96 hrs REM sleep deprivation caused
severe motor weakness, lesion in various organs and systems and
eventually death in the animals [9]. This was attributed to
accumulation of free radicals. Halliwell and Gutteridge [8], also
showed an increase in the MDA level in REM sleep deprived
animals. According to Zeppellin, and Rechtschaffen [12] sleep
seems to limit metabolic requirements. Therefore sleep
deprivation could enhance the metabolic rate and in turn increase
oxidative stress.
From the results obtained by this study and in comparison
with other studies we can infer that REM sleep has an antioxidative
effect and deprivation leads increase in free radical generation and
free radical induced cell damage. This study supports Reimunds
hypothesis of antioxidative function for sleep. In spite of the
number of studies conducted and various contradictory results
that were put forward, the problem to arrive at a definite
conclusion is still an open question. So the future to such study lies
in devising an easy technique of REM sleep deprivation without the
non specific effects, study the behavioural changes due to REM
sleep deprivation, and study the antioxidant defense mechanism.
Study the effect of reabsorptive sleep, to see the return to baseline
value. Free radicals, the cause of many diseases, formed during
awake state are removed during sleep and the effect is more as the
duration of sleep deprivation increases. So sleep is very important
for a person to lead a normal healthy lifes.
1] Elomaa E. Effects of Rapid eye movement sleep deprivation on the feeding behaviour in the laboratory rat with a description of the cuff pedestal technique. Acta Physiol. Scand. Suppl. 1985; 545: 1 – 35.
[2] Vogel GV., A review of REM sleep deprivation., Arch, Gen Psychia., 1975; 32:749 – 761
[3] Coris MC, Poncedeleon M. Juarez J, Ramos J. Effects of Paradoxical sleep
deprivation and stress on the waking EEG in the rat. Physiol. Behav.1994;55 (6): 1021 – 1027.
[4] Sarada Subramanyam, Madhavankutty K., Sleep., Text book of Human Physiology., 2001; 6thed: 789 – 793.
[5] Reimund E. The free radical flux theory of sleep. Med.Hypo. 1994; 43: 231 – 233.
[6] Krosiun – VI, Rohzshoskii – LaV. Mechanisms of systemic labilization o cellular membranes during long term deprivation of paradoxical sleep. Patol – Fiziol – Eksp – Tor. 1995; 1:9 – 12.
[7] Bentivoglio M, Grassi Zucconi G., The pioneering experimental studies on sleep deprivation., Sleep., 1997; 2: 570 – 576
[8] Halliwel B., Gutteridge JMC, Free radicals in Biology and medicine 2nd ed, Clarendon press Oxford., 1989; 543.
[9] Rechtschaffen A, Gilliland MA, Bergman BM, Winter JB., Physiological correlates of prolonged sleep deprivation in rats., Science., 1983; 221: 182 – 184
Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-17591758
1759
[10] Inoue S. Honda K, Komoda Y. Sleep as neuronal detoxification and restitution. Behav.Brain Res. 1995; 69: 491 – 496.
[12] Zeppelin H, Rechtschaffen A, Mammalian sleep, longevity and energy metabolism. Brain Behav. Evol.1974; 10: 425 – 470.
[13] Almeida VD, Hipolide DC, Azzalis LA, Lobo L, Junqueira VBC, Tufik S., Absence of oxidative stress following paradoxical sleep deprivation in rats., Neuroscilett., 1997;235:25-28
[14] Cirelli C, Shaw P J, Rechtschaffen A, Tononi G., No evidence of brain cell
degeneration after long term sleep deprivation in rats., 1999; 840: 184 – 193
[15] Gopalakrishnan A, Ji L, and Cirelli C. Sleep deprivation and cellular responses to oxidative stress. Sleep 27: 27–35, 2004.
[16] Gopalakrishnan A; Ji LL; Cirelli C. Sleep deprivation and cellular responses to
oxidative stress. Sleep; 2004;27(1):27-35.
[17] Gutteridge JM, Halliwell B. Free radicals and antioxidants in the year 2000. A Historical look to the future. Ann N Y Acad Sci 2000;899:136-47.
[18] Jouvet D.P. Vimont F, Delorme, Jouvet M. Etude de la privation selective de la phase paradoxale de summeil chezle chat. C.R.Seanc Soc Biol. 1964; 158: 756 – 759.
[19] Devasagayam TPA, Tarachand.V, Decreased LPO in the rat kidney during gestation Biochem.. Biophys.Res.comm.., 1987;145: 134 – 138
[20] Sies H., strategies of antioxidant defense Eur.J Biochem., 1993; 215: 213 – 221
[21] Vinay kumar, Ramzi S Cotran, Stanley L Robbins. Basic pathology (5ed). Prism Indian Ed.1992; 8 – 9
[22] Melchiorri D, Reiter RJ, Sewerynek E, Hara M, Chen L, and Nistico G. Paraquat toxicity and oxidative damage. Reduction by melatonin. Biochem Pharmacol.,1996; 51: 1095–1099.
[23] Institutes of Health. National Sleep Disorders Research Plan. Bethesda, MD: National Institutes of Health, US Department of Health and Human Services., 2003; 138.
[24] Palanisamy Pasupathi, Jwahar Farook, Palanisamy Chinnaswamy.Oxidant – antioxidant status, high sensitive C reactive protein and homocysteine levels in type 2 diabetic patients with and without microabuminuria. Int.J Biol Med Res.,2010;193);04-08
[25] Coenen AML, Van Luijtelaar ELJM., Stress induced by three procedures of deprivation of paradoxical sleep., Physiol and Behav., 1985; 55) 501 – 504
[26] Van Luijtelaar ELJM, Coenen AML, Electrophysiological evaluation of three
paradoxical sleep deprivation techniques in rats., Physiol, and Behav. 1986; 36: 603 – 609
'[27] Albert IB., REM sleep deprivation.,Biol,phsychia., 1975;33:341-351
[28] Ellman S J, Spielman A J, Luck D, Steinerss, Halperin R., REM deprivation. A review., In the mind in sleep psychology and psychophysiology Eds: An Arkin J S Aritrobus, S J Ellman, Lawrence Erbaum Associates, New Jersey., 1978; 419 – 457
[29] Adam K., Body weight correlates with REM sleep., Br.Med.J., 1977a;1: 813-814
[30] Bregmann BM, Everson CA, Kushida CA., Sleep deprivation in rat: V Energy use and mediation., Sleep., 1989; 12 (1): 31 – 41
[31] Kushida CA, Bergmann BM, Rechtschaffen A. Sleep deprivation in rats – IV, paradoxical sleep deprivation. Sleep. 1989; 12 (1): 22 – 31.
[32] Brock JW, Farooque SM, Ross KD, Payne S, Chandan Prasad. Stress related
behaviour and central norepinephrine concentrations in the REM sleep deprived rat. Physiol. And Behav. 1994; 55 (6): 997 – 1003.
Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.
ISSN: 0976:6685.c
Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759