for peer revie · exodontia. adult wistar rats were submitted to unpredictable chronic mild stress...

For Peer Review

Positive effects of benzodiazepine therapy on masticatory

muscle dysfunctions induced by chronic stress in a rodent

model of occlusal instability

Journal: Journal of Cellular Physiology

Manuscript ID JCP-18-0787

Wiley - Manuscript type: Original Research Article

Date Submitted by the Author: 21-May-2018

Complete List of Authors: Nascimento, Glauce; University of Sao Paulo, Morphology, Physiology and

Basic Pathology Malzone, Bruno; USP Yomasa, Daniela ; University of Sao Paulo Leal-Luiz, Gabrielli; USP Pereira, Yamba; University of Sao Paulo Issa, Joao; USP Watanabe, Ii ; University of Sao Paulo Leite-Panissi, Christie; USP Iyomasa, Mamie; University of Sao Paulo Dias, Fernando; Dental School, Universidad de La Frontera, Temuco, Chile. Del-Bel, Elaine; USP,

Key Words: Muscle, Stress, Anxiety, Temporomandibular Disorders

John Wiley & Sons, Inc.

Journal of Cellular Physiology

For Peer Review

Positive effects of benzodiazepine therapy on masticatory muscle dysfunctions

induced by chronic stress in a rodent model of occlusal instability

*Glauce Crivelaro Nascimento1, *Bruno Lima Malzone

1, Daniela Mizusaki Yomasa

2,

Gabrielli Leal-Luiz1, Yamba Carla Lara Pereira

1, João Paulo Mardegan Issy

1, Ii Sei

Watanabe3, Christie R. A. Leite-Panissi

1, Mamie Mizusaki Iyomasa

1, Fernando José

Dias4**

a, Elaine Ap. Del Bel Belluz Guimarães

1 **

b

1 Department of Morphology, Physiology and Basic Pathology of Dentistry School,

Ribeirão Preto Campus, Universidade de São Paulo, 14040-904, SP, Brazil.

2Psychobiology Graduate Program, School of Philosophy, Science and Literature,

Ribeirão Preto Campus, Universidade de São Paulo, Ribeirão Preto, 14040-901, SP,

Brazil.

3Department of Anatomy, Institute of Biomedical Sciences, Universidade de São Paulo,

São Paulo, SP, Brazil

4 Department of Integral Dentistry, Research Centre for Dental Sciences (CICO), Dental

School, Universidad de La Frontera, Temuco, Chile

*Authors contributed equally to the work.

** Corresponding authors:

a Dental School, Universidad de La Frontera, Temuco, Chile. Phone: 56 9 8474-6702.

E-mail: [email protected]

b Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, 14040-904

Ribeirão Preto, SP, Brazil. Phone: 55 16 3315-4094 Fax: 55 16 3633-0999. E-mail:

[email protected]

Running head: Stress and diazepam on muscle metabolism

Keywords: stress, masticatory dysfunction, benzodiazepine, medial pterygoid

muscle, transmission electronic microscopy, metabolism.

5 figures

Contract grant sponsor: FAPESP; Contract grant number: 2012/22128-6 and

2015/03053-3).

Page 1 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Abstract

Psychological stress and occlusal alteration are important etiologic factors for

temporomandibular/masticatory muscular disorders, but the exact physiologic

mechanism underlying this relation remains unclear. Our purpose was to test the

hypothesis that benzodiazepine therapy is able to prevent metabolic and vascular

changes in the medial pterygoid muscle of rats under chronic stress and unilateral

exodontia. Adult Wistar rats were submitted to unpredictable chronic mild stress and/or

unilateral exodontia and their plasma and medial pterygoid muscles were removed for

analysis. A pre-treatment with diazepam was used to verify its effect on stress. The

parameters evaluated included anxiety behavior, plasma levels of corticosterone,

metabolic activity by succinate dehydrogenase, capillary density by laminin staining

and ultrastructural findings by transmission electron microscopy. Occlusal instability

induced anxiety-like behavior on elevated plus-maze test and diazepam administration

blocked the appearance of this behavior. Unilateral exodontia promoted in the

contralateral muscle an increase of oxidative fibers and capillaries and modification of

sarcoplasmatic reticulum. Chronic stress caused increased glycolytic metabolism,

reduced capillary density and morphological changes in mitochondria on both sides.

Association of both factors induced a glycolytic pattern in muscle and hemodynamic

changes. Pharmacological manipulation with diazepam inhibited the changes in the

medial pterygoid muscle after stress. Our findings provide insights into the mechanisms

by which chronic stress and exodontia might be involved in the pathophysiology of

masticatory muscular dysfunctions, possibly inducing metabolic and capillary

alterations, and shows, for the first time, an effective preventive benzodiazepine

treatment for stress and occlusal instability conditions affecting masticatory muscle

disorders.

Page 2 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Introduction

Temporomandibular dysfunction (TMD) can present itself as muscular, articular,

or of mixed character, when it affects both systems (Kostrzewa-Janicka et al., 2013).

Particularly, many findings suggested these dysfunctions are associated with abnormal

physiology of the masticatory muscle and consider this an important risk indicator for

TMD development (Furquim et al., 2015). In addition, their risk factors englobe

structural, physiological, behavioral, and environmental characteristics. Emotional

stress and instability in the maxilomandibular relationship, in case of orthodontic

treatment, occlusal, or masticatory dysfunction and the extraction of third molars, have

been involved with the development and maintenance of pain during TMD (Fricton,

2014).

Regarding occlusal interferences, these are associated with clinical signs and

symptoms, such as fatigue and masticatory muscle pain (Carvalho et al., 2010). In this

context, the adaptation of these muscles has been analyzed after unilateral exodontia

(Bazan et al., 2008; Iyomasa et al., 2008) and other occlusal interferences in

experimental models. Bazan et al. (2008) found that exodontia in guinea pigs decreases

metabolic activity of the medial pterygoid muscle ipsilateral to the exodontia. In

addition, the influence of occlusal alteration in the medial pterygoid muscle of gerbils,

previously described by Iyomasa et al. (Iyomasa et al., 2009), shows muscle fibers with

a central nucleus and decreased diameter six days after unilateral exodontia. It has been

admitted, therefore, that modified occlusion can affect not only morphology, but also

the functionality of masticatory muscles (Takeda et al., 2009).

TMD is related with anxiety, stress and depression (Schmitter et al., 2018,

Nascimento and Leite-Panissi, 2014). For this reason, the involvement of this disorder

with emotional disturbances arouses the interest of researchers and stimulates basic and

clinical research in the area. In accordance, emotional stress alters oral functioning

(Antonova, 2008) and sensory perception (Schweinhardt et al., 2008). Also, stress-

induced muscle disorders cause pain (Vedolin et al., 2009) through a mechanism that

involves modifications in muscle metabolism or pH and the release of inflammatory

mediators (Louca Jounger et al., 2017). However, although mandibular elevator muscles

are the most affected by stress (Bakke et al., 2004), the mechanism by which stress can

alter orofacial muscular physiology has not yet been elucidated. To better study the

consequences of stress and its relationship with these so-called stress-related disorders,

animal models have been developed. The unpredictable chronic mild stress (UCMS)

Page 3 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

protocol is an animal model that presents good face validity and that has been broadly

used in the past years to investigate both the physiological and the behavioral

consequences of chronic stress (Cameron and Schoenfeld, 2018). Briefly, in this test,

rodents are exposed to a variety of relatively mild stressors intermittently, generally for

two to four weeks (Loyola et al., 2016).

This study hypothesizes that unpredictable chronic mild stress influences on

morphophysiological changes in the right and left medial pterygoid muscles induced by

occlusal instability and that classic pharmacological manipulation with benzodiazepine

reverses the alterations caused, since diazepam shows anti-stressor and anxiolytic-like

activities. The aim of this work is to contribute to proposing a new therapeutic target

alternative for orofacial muscular dysfunctions and the emotional risk factors

associated. In addition, this study aims to propose mechanisms by which stress acts to

modify metabolism and capillary patterns of the medial pterygoid muscle.

Methods

Animals

All experiments were conducted in accordance with the National Institute of Health

Guidelines for Welfare of Experimental Animals. The experimental protocol was

approved by the Animal Use and Ethics Committee of Ribeirão Preto (certificate

number: 12.1.418.53.0) and all efforts were made to decrease the number of rats used

and to minimize animal suffering. A total of 128 male Wistar rats, weighing 275 ± 25 g,

were obtained from Ribeirão Preto Campus, at Universidade de São Paulo, Brazil. The

animals were kept in a temperature-controlled room (24 ± 1 °C) with 12 h light/dark

cycles and water and food ad libitum.

The animals were distributed in the following groups (n=9 for each group):

- Control: rats were submitted to simulation of exodontia and were not exposed to

stress protocol;

- AO: Altered occlusion, rats were submitted to unilateral exodontia;

- S: Stress group, rats were submitted to unpredictable chronic mild stress

protocol;

- AO+S: rats were submitted to unilateral exodontia and unpredictable chronic

mild stress protocol.

All rats of these groups received the anxiolytic diazepam (DZP) or its vehicle (VEH).

Page 4 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Anxiolytic Drug

Anxiolytic diazepam (2 mg/kg) 30 min before the stress protocols, during 10 days of

stress (Jindal et al., 2013; Shen et al., 2012) via gavage. The vehicle was prepared with

polyethylene sorbitan monooleate (tween 80) in 2% saline solution and administered

200 µl in each animal under the same conditions as diazepam.

Induction of altered occlusion

The dental occlusal alteration was induced in this study by upper molar teeth extraction

on the left side. The control group was subjected to the same trauma during jaw opening

for teeth extraction. Under aseptic conditions, all animals, control and experimental

groups, received intraperitoneal anesthesia—tribromoethanol (0.25 g/kg of body

weight). As a prophylactic measure, we administered a single dose of antibiotic

(Pentabiotic) – penicillin 24,000 IU/kg of body weight, as well as anti-inflammatory

and analgesic Bananine (Schering-Plorigh, flumexine meglumine, 25 mg/kg, 10 mg/ml).

On the 14th day, visually, it was possible to verify the complete healing of the

extraction region. Also, the socket was almost totally occupied by reticular bone and the

trabecular surfaces showed intense bone activity on this day.

Chronic unpredictable stress protocol

After surgical recuperation from exodontia procedure (14 days), unpredictable chronic

mild stress (UCMS) protocol was applied in animals from the stress groups (S and

AO+S groups). This protocol was performed according to a previous study by our

research group (Loyola et al., 2016). Five different methodologies were performed in a

ten-day period (14th

to 23rd

day) according to the following table 1:

Table 1. 10-day representative schedule of stressor agents used during the experiment

Page 5 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

The laboratory has a technical team, composed of veterinary and technical staff,

which helped maintain animal health and welfare. Furthermore, the animals were

monitored daily, which in checking animal activity in the housing, position, self-

cleaning, appearance, vocalization, and weight loss (control every 3 days). There were

no deaths correlated to the stress protocol.

Evaluation of anxiety levels with the Elevated Plus-maze (EPM) test

The elevated plus-maze test (EPM) has been proven to be bi-directionally sensitive to

manipulations of anxiety in rats. This test includes approach-avoidance based

behavioral paradigms with the conflict between rodents’ innate exploratory drive and

the drive to avoid brightly lit, open or elevated environments.

EPM test was used to assess the rats’ anxiety levels. The EPM was constructed of wood,

according to the specifications described in Morato and Brandão (1997). Behavior in the

EPM was recorded by a video camera linked to a monitor. This device, located outside

the experimental room, allowed the recordings to be analyzed later. Rats were placed

individually in the center of the maze facing a closed arm and allowed to explore the

maze. We analyzed the number of entries into the open arms of the maze and time spent

in the open arms during a 5-min test period. Open-arm activity was evaluated as the

time spent in the open arms relative to total time spent in the maze (300 s) and

expressed as a percentage. The behavioral categories were scored using Geo Vision

software. At the end of the session, animals were returned to their home cages, and the

area was wiped clean with a 70% alcohol solution.

Plasma Assay

This experiment was carried out to define the efficacy of unpredictable stress protocols

in inducing stress-like hormonal modifications. The plasma concentrations of

corticosterone hormones, a stress index, were measured by immunoassay using the

Luminex method (Milliplex) according to the protocols provided by the manufacturer.

This method allows a variety of bioassays on the surface of fluorescence-encoded

plastic beads (microspheres) that are subsequently read in a flow analyzer. By means of

two lasers and digital signal processors, a high-speed flow analyzer detects the

fluorescence of each individual microsphere. We used 9 samples from each group

analyzed (control; AO; S and AO+S, with or not diazepam treatment).

Page 6 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Histochemical Technique for Demonstration of Succinate Dehydrogenase (SDH)

The demonstration of Succinate Dehydrogenase was used to evaluate metabolic activity

of medial pterygoid muscle fibers in different experimental conditions. Both medial

pterygoid muscles (right and left) were dissected and each specimen was mounted in an

aluminum board with Tissue-tekR, O.C.T. (optimal cutting temperature) compound,

snap frozen in isopentane cooled by liquid nitrogen (−150 °C) and kept at −80 °C until

use. Serial cross-sections, 10 µm thick, cut at −20 °C using a Leica cryostat microtome

were stained for histochemical and histological studies. Serial cross-sections stained

with succinate dehydrogenase following the procedure described by Didier and

Westphal (1957) demonstrated the muscle metabolic pattern. On each image we

quantified the points that fell on each fiber type: high, low, or intermediate metabolic

activity. The dark fibers were considered as the most oxidative metabolic activity. Each

area was estimated according to Weibel et al., 1966, point-counting method, using a

test-system composed of 90 points. The area of each fiber type was determined on each

microscopy field, photographed using a photomicroscope (Leica MZ125 connected to a

digital camera, Germany). The Image J software (Free software,

http://rsb.info.nih.gov/ij) was used to count the points on the test-system.

Assessment of capillary density

We identified capillaries by immuno-histochemical analysis for αCD31 and Laminin

proteins. αCD31 is a membrane glycoprotein present in the vascular endothelium.

Laminin is a component in the basement membrane of capillaries. Reaction was

performed by the fluorescence method. Sections of 5µm thickness were immersed in

3% solution of BSA (Pentex, USA) and after three PBS washes, the slides were

incubated overnight with the primary antibodies: Laminin and αCD31. Then, sections

were washed in PBS and subjected to amplification reaction using the biotinylated

secondary antibody (Kit LSAB – HRP, DAKO Cytomation, Denmark). The reactions

were revealed and counterstained with Harris hematoxylin (Dynamic, Brazil). For each

animal, the highest quality cross-section was used to systematically capture five images,

randomly, with 40x magnification, with the aid of a camera attached to a microscope

(ZEISS AxioImager. Z2, Germany). The laminin antibody stains the intermediate

filament and basal lamina in brown, when positive. We made a photomicrographin

high-power field (400X) with a blade positive control, thus allowing laminin

quantification by the method of colorimetry.

Page 7 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Transmission electron microscopy (TEM)

For TEM analysis, we excised the medial pterygoid muscles, cutting them to small

fragments (1mm). These fragments were immersed in modified Karnovsky solution

containing 2.5 % glutaraldehyde and 2 % paraformaldehyde in sodium phosphate buffer

0.1 M. Then, they were post-fixed in a solution of osmium tetroxide at 4 °C, and they

were immersed in 5 % uranyl acetate aqueous. After 24 hours, we dehydrated the

muscles in an ascending series of alcohol, immersed in propylene oxide. So, they were

infiltrated by mixture of pure resin and propylene oxide (1:1) and embedded in Spurr

resin until complete polymerization occurred. Ultrathin sections (55 nm) of the tissues

in resin were obtained, collected on 200 mesh networks, contrasted with uranyl acetate.

Using a transmission electron microscope Jeol 1010, the sections were examined, and

several micrographs were taken from each fragment at different magnifications (4,800

K, 20,000 K, 40,000 K and 60,000 K).

Statistical Analysis

Data are expressed as mean ± SD. Two-way ANOVA was employed to evaluate the

effects of chronic unpredictable stress and exodontia. Post hoc analysis was conducted

using Student-Newman-Keuls. The level of significance was set at P < 0.05.

Results

Anxiety-like behavior

Considering the percentage of time spent in open arms, the application of a two-way

ANOVA showed a significant decrease (Newman Keuls test) in time spent by rats that

underwent molar exodontia and/or stress (Figure 1A) (F3.63 = 6.21, P < 0.05). Also, the

benzodiazepine treatment proved effective in reducing anxiolytic effect for both

conditions (F1.35 = 18.98, P < 0.001). Regarding mean entries into open arms (Figure

1B), we observed significant decreases in terms of time and treatment (F1.63 = 1.54, P <

0.05) in the groups under stress protocol and/or occlusal instability when compared with

the control and diazepam groups (F2.55 = 0.68, P < 0.05).

Plasma corticosterone

The plasma corticosterone concentrations in experimental groups are shown in Figure 2.

The chronic unpredictable stress group revealed a significant increase in plasma

concentrations of corticosterone (F3.33 = 10.42, P < 0.001) compared to the other

Page 8 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

experimental groups. Diazepam administration reverted this state in stressed rats.

Metabolic activity of muscle fibers

The representative photomicrographs from experimental groups are presented in Figure

3A. Quantitative analysis of this reaction may be seen in Figure 3B. The contralateral

muscle to the exodontia of rats under dental occlusion alteration (OA) administrated

with diazepam vehicle, expressed increased oxidative fibers, represented by dark

staining compared to the control group (F2.95 = 6.03, P < 0.05), while in the ipsilateral

side, there were no alterations. In the stress group (S), the muscles with vehicle

treatment presented an increase in glycolytic fibers in both right and left muscles

compared to the exodontia and control groups (F4.44 = 3.49, P < 0.001). The same

condition was observed in rats under dental occlusion alteration and chronic

unpredictable stress protocol (OA+S) for both sides, contralateral and ipsilateral to

exodontia (F4.39 = 6.03, P < 0.001). There were no significant differences in the control

group. The increased glycolytic fibers in both sides observed in stress isolated or

exodontia groups did not occur for rats treated with diazepam.

Capillary assessment

For analysis of local vascularization, we quantified laminin in the pterygoid muscles

(Figure 4). Representative photomicrographs from the experimental groups are

presented in Figure 4A. Graphs present quantitative analysis from this reaction (Figure

4B). In groups injected with diazepam vehicle, dental occlusion alteration promoted

increased laminin densities in the right medial pterygoid muscle (F3.33 = 2.83, P < 0.001)

and there was no difference in the left side compared to the control group. The chronic

stress protocol decreased laminin in both sides, contralateral and ipsilateral (F4.48 = 4.03,

P < 0.01) and did not alter alpha CD. Exodontia and stress associated groups also

reduced laminin in the muscle contralateral to exodontia (F1.11 = 6.03, P < 0.001).

Diazepam administration was able to reverse laminin levels in the stress and stress with

exodontia groups (F2.33 = 4.22, P < 0.05), but did not modify vascularization in the

occlusion alteration isolated group.

Transmission electron microscopy

Under transmission electron microscopy (Figure 5), medial pyterygoid muscle of

control rats showed muscle fibers with several well-preserved structures. There were

Page 9 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

well-preserved mitochondria with dilated double membrane interspace and space

between mitochondrial cristae. Blood capillaries between the muscular fibers evidenced

thick endothelial cells with intraluminal protrusions, large cytoplasmic vacuoles and

increased cytoplasmic organelle content (endoplasmic reticulum, ribosomes and

mitochondria). The intercellular substance showed fibroblast extensions rich in granular

endoplasmic reticulum with dilated cisternae containing secretion products and large

quantities of collagen fibrils.

The rats submitted to unilateral exodontia (Altered Occlusion) showed dilated

sarcoplasmatic reticulum and T-tubules. There were numerous smoothly contoured

mitochondria, with ample inter-membrane spaces and dilated space between

mitochondrial cristae. The main characteristics of the stressed group (Stress) included

muscular fiber cytoplasm – made up of myofibrils surrounded by several vesicles of

sarcoplasmic reticulum – and sarcolemma folds that were poorly delineated with

irregular contours, some filled with amorphous substance. Additionally, there were

disorganized mitochondrial cristae and subsarcolemmal mitochondria with varied sizes.

The diazepam pre-treatment (Stress + Dzp) was able to maintain muscle characteristics

similar to the control.

Discussion

The present study follows a model of dental occlusion alteration used in

previous studies with effective results (Iyomasa et al., 2009, Loyola et al., 2016,

Fernandez et al., 2018). Again, the unpredictable chronic stress protocol used herein

constituted a valorous tool, providing a realistic model for the stresses of daily life.

Compared with repeated chronic stress, this protocol is closer to reality, since stress

varies in an individual's life (Cameron and Schoenfeld, 2018). On the other hand, acute

stress is not adequate since there is hardly a single and punctual stressful factor, rather,

there is a set of stress factors occurring in different periods (Cameron and Schoenfeld,

2018).

The present work investigated corticosterone levels in the different experimental

groups in order to measure the effectiveness of this protocol, evidencing that the group

of animals treated with diazepam vehicle and submitted to the stress protocol alone or

with exodontia presented significant increases of corticosterone when compared to the

other groups under analysis. Again, diazepam was effective in preventing this increase,

suggesting its efficacy on emotional alterations. Additionally, the anxiety-like behavior

Page 10 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

analyzed here via EPM presents an important result regarding the ability of the altered

occlusion experimental model to induce anxiety behaviors in the same way as stress,

represented by the reduction of rats entering open arms of the apparatus. This result

suggests a strong relationship between occlusal instabilities and anxiolytic behavior,

mainly due to the fact that benzodiazepine treatment prevents onset. These data are

innovative as the same anxiolytic effect is acquired when comparing a stress model and

a model of dysfunction in the orofacial region, and additionally the suggestion that

diazepam therapy may be used for physio-pathological conditions concentrated in the

orofacial region is valuable.

The medial pterygoid muscle is an even muscle and one of the four main

muscles responsible for the chewing function. It has an important relationship with the

temporomandibular joint, as a potent masticatory muscle involved in mandibular

elevation and eccentric movements of protrusion and laterality (Gray et al., 1994). The

analysis of a muscle that is involved in all these actions will provide more complete

information about possible changes associated with non-physiological muscular activity

that stress and altered occlusion can generate, such as clenching and griding (Okeson,

1995).

Regarding muscular modifications, the results of this study for the groups

administered with diazepam vehicle indicated that the unilateral exodontia promoted an

increase of oxidative fibers in the contralateral muscle, increase of capillaries evidenced

by laminin, and modification of the sarcoplasmatic reticulum in the medial pterygoid

muscle fibers. Stress alone, on the other hand, caused an increase in glycolytic fibers, a

decrease of laminin, and morphological alteration of the muscle fiber mitochondria in

the muscles studied on both sides. The association of exodontia and stress, in turn,

promoted an increase of glycolytic fibers, reduction of laminin and mitochondria in the

muscles contralateral to the exodontia. In the animals pharmacologically manipulated

with diazepam, we noted an inhibition of changes in the medial pterygoid muscle after

stress, which presented similar characteristics to the control group. These data indicate

that benzodiazepine action was specific to the stress used and did not act as a muscle

relaxant, since only the groups with the stress protocol showed a reversal of the altered

muscle states. Also, the preventive character of this drug on medial pterygoid muscles is

emphasized, considering that the treatment was performed 30 minutes before daily

stresses.

Page 11 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

With respect to the types of skeletal muscle fiber, these have been classified

based on their contraction rate and type of metabolism recruited. They include slow-

contracting oxidative fibers; fast-contracting glycolytic-oxidative fibers; and fast-

contracting glycolytic fibers (Blaauw et al., 2013). Exodontia in this study promoted an

increase of oxidative fibers in the right medial pterygoid muscle, which use oxidative

phosphorylation for metabolism. These fibers have a high amount of myoglobin as an

oxygen transport molecule, which presents pigmentation that leaves the fiber darker in

relation to glycolytic fibers, which are clear (Hoppeler and Vogt, 2010). This increase of

oxidative metabolism after exodontia, together with an increase in local vascularization,

allows greater diffusion of oxygen to the mitochondria. These results suggest, therefore,

that the muscle will take longer to enter into fatigue (Grassi et al., 2015). Therefore,

modified occlusion of the left side, from onset, promotes a protection mechanism in the

contralateral muscle, since it will have increased function to compensate the affected

side.

In addition, our results show that the induction of chronic stress alters the

protective character of the contralateral muscle promoted by exodontia, since it entails

an increase in glycolytic metabolism, which depends on anaerobic glycolysis for the

synthesis of ATP, and therefore, the muscle tends to enter into fatigue more quickly

(Grassi et al., 2015; Vissing and Haller, 2012). In addition, we found that there was a

reduction of local vascularization and altered morphology of mitochondria, suggesting a

decrease in energy for this muscle’s fibers. The stress stimulus alone, in turn, also

promotes increased glycolytic metabolism, reduced vascularity and mitochondrial

morphological alteration in both sides. Furthermore, glycolytic fibers have a larger

diameter than oxidative fibers. The combination of larger size, smaller quantity of

myoglobin and fewer blood vessels makes it more likely that glycolytic fibers will run

out of oxygen after repeated contractions. Diazepam, in this context, was able to prevent

the state produced by isolated stress or by the association of stress and exodontia, as it

maintains the oxidative capacity of muscular fibers at normal levels.

This work is pioneer in highlighting the preventive effectiveness of a

benzodiazepine drug on masticatory muscular dysfunction changes induced by an

unpredictable chronic stress experimental model. Furthermore, the data from this study

suggest the change of respiratory metabolism and capillary alterations as possible

mechanisms involved in the emergence of orofacial muscular dysfunctions in conditions

of emotional disorder. These emotional disorders deserve special mention in this study,

Page 12 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

since, for the first time, there is evidence for anxious behavior induced by an

experimental model of occlusal instability. The pathway by which stress promotes

muscular metabolic pattern modification remains an important topic for further

research.

Conflict of interest statement: The authors report no conflict of interest.

Acknowledgement

This work was supported by FAPESP (2012/22128-6 and 2015/03053-3). We

thank Sonia Almeida, for the technical support.

References

Antonova IN. 2008. Changes in the masticatory muscles, periodontal tissues, and the

pharyngeal ring in Wistar rats in chronic psychophysical stress. Neurosci Behav Physiol

38(9):891-6.

Bakke M, Tuxen A, Thomsen CE, Bardow A, Alkjaer T, Jensen BR. 2004. Salivary

cortisol level, salivary flow rate, and masticatory muscle activity in response to acute

mental stress: a comparison between aged and young women. Gerontology 50(6):383-

92.

Bazan E, Issa JP, Watanabe IS, Mandarim-de-Lacerda CA, Del Bel EA, Iyomasa MM.

2008. Ultrastructural and biochemical changes of the medial pterygoid muscle induced

by unilateral exodontia. Micron 39(5):536-43.

Blaauw B, Schiaffino S, Reggiani C. Mechanisms modulating skeletal muscle

phenotype. Compr Physiol 3(4):1645-87.

Cameron HA, Schoenfeld TJ. 2018. Behavioral and structural adaptations to stress.

Front Neuroendocrinol 2018 Feb 5.

Carvalho CM1, de Lacerda JA, dos Santos Neto FP, Cangussu MC, Marques AM,

Pinheiro AL. 2010. Wavelength effect in temporomandibular joint pain: a clinical

experience. Lasers Med Sci 25(2):229-32.

Didier G, Westphal A. 1992. Evaluation of a histochemical method specific for myosin

ATPase activity in the masticatory muscles of the rat. J Biol Buccale 20(2):103-9.

do Nascimento GC, Leite-Panissi CR. 2014. Time-dependent analysis of nociception

and anxiety-like behavior in rats submitted to persistent inflammation of the

temporomandibular joint. Physiol Behav 125:1-7.

Page 13 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Fernández RAR, Pereira YCL, Iyomasa DM, Calzzani RA, Leite-Panissi CRA, Iyomasa

MM, Nascimento GC. 2018. Metabolic and vascular pattern in medial pterygoid muscle

is altered by chronic stress in an animal model of hypodontia. Physiol Behav 185:70-78.

Furquim BD, Flamengui LM, Conti PC. 2015. TMD and chronic pain: a current view.

Dental Press J Orthod 20(1):127-33.

Grassi B, Rossiter HB, Zoladz JA. 2015. Skeletal muscle fatigue and decreased

efficiency: two sides of the same coin? Exerc Sport Sci Rev 43(2):75-83.

Gray RJ, Davies SJ, Quayle AA. 1994. A clinical approach to temporomandibular

disorders. 1. Classification and functional anatomy. Br Dent J 176(11):429-35.

Iyomasa MM, Issa JP, Siéssere S, Regalo SC, Watanabe IS. 2008. Effect of unilateral

extraction of molar teeth on suprahyoid muscles: macroscopic and ultrastructural

aspects. Micron 39(8):1111-8.

Iyomasa MM, Issa JP, Sosthines MC, Oliveira AM, Stuani MB, Motoyama AA,

Watanabe IS. Ultrastructural characteristics of the gerbil pterygoid medial muscle after

experimental occlusal alteration. Micron 40(2):206-11.

Jindal A, Mahesh R, Kumar B. 2013. Anxiolytic-like effect of linezolid in experimental

mouse models of anxiety. Prog Neuropsychopharmacol Biol Psychiatry 40:47-53.

Kostrzewa-Janicka J, Pietrzak B, Jurkowski P, Wielgos M, Binkowska M,

Mierzwinska-Nastalska E. 2013. Effects of oral contraceptives on the treatment for

internal derangements in temporomandibular joints in women. Neuro Endocrinol Lett

34(6):566-72.

Louca Jounger S, Christidis N, Svensson P, List T, Ernberg M. 2017. Increased levels

of intramuscular cytokines in patients with jaw muscle pain. J Headache Pain 18(1):30.

Loyola BM, Nascimento GC, Fernández RA, Iyomasa DM, Pereira YC, Leite-Panissi

CR, Issa JP, Iyomasa MM. 2016. Chronic stress effects in contralateral medial

pterygoid muscle of rats with occlusion alteration. Physiol Behav 164(Pt A):369-75.

Morato S, Brandão ML. 1997. Paradoxical increase of exploratory behavior in the

elevated plus-maze by rats exposed to two kinds of aversive stimuli. Braz J Med Biol

Res 30(9):1113-20.

Okeson JP. 1995. Occlusion and functional disorders of the masticatory system. Dent

Clin North Am 39(2):285-300.

Schmitter M, Kares-Vrincianu A, Kares H, Malsch C, Schindler HJ. 2018. Chronic

stress and temporalis muscle activity in TMD patients and controls during sleep: a pilot

study in females. Clin Oral Investig. 2018 May 10.

Page 14 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Schweinhardt P, Sauro KM, Bushnell MC. 2008. Fibromyalgia: a disorder of the brain?

Neuroscientist 14(5):415-21.

Shen Q, Fuchs T, Sahir N, Luscher B. 2012. GABAergic control of critical

developmental periods for anxiety- and depression-related behavior in mice. PLoS One

7(10):e47441.

Takeda M, Oshima K, Takahashi M, Matsumoto S. 2009. Systemic administration of

lidocaine suppresses the excitability of rat cervical dorsal horn neurons and tooth-pulp-

evoked jaw-opening reflex. Eur J Pain 13(9):929-34.

Vedolin GM, Lobato VV, Conti PC, Lauris JR. 2009. The impact of stress and anxiety

on the pressure pain threshold of myofascial pain patients. J Oral Rehabil 36(5):313-21.

Vissing J, Haller RG. 2012. Mechanisms of exertional fatigue in muscle glycogenoses.

Neuromuscul Disord 22 Suppl 3:S168-71.

Vogt M, Hoppeler H. 2010. Is hypoxia training good for muscles and exercise

performance? Prog Cardiovasc Dis 52(6):525-33.

Wieckiewicz M, Boening K, Wiland P, Shiau YY, Paradowska-Stolarz A. 2015.

Reported concepts for the treatment modalities and pain management of

temporomandibular disorders. J Headache Pain 16:106.

Figures legends

Page 15 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

Figure 1. Effects of stress and unilateral exodontia on time spent in open arms (A) and

on the percentage of entries into the open arms (B) in the EPM test. Two-way ANOVA

showed an effect of diazepam therapy in rats submitted to altered occlusion (AO),

chronic stress (S) or both (AO+S) (n=10 per group). Data are expressed as the means ±

SEM. *P < 0.05 Newman-Keuls when compared with diazepam and control groups.

SEM: standard error means.

Figure 2. Analysis of plasma corticosterone levels of control rats and rats submitted to

altered occlusion (AO), chronic stress (S) or both (AO+S) (n=8 per group). Two-way

ANOVA showed an effect of chronic unpredictable stress, in which S and AO+S groups

presented increased plasma corticosterone compared to the other groups (*P < 0.05,

Newman-Keuls test). Data were expressed as mean ± SEM. SEM: standard error means.

Figure 3. A: Panels show representative photomicrographs of succinate dehydrogenase

(SDH) staining of the right MPM of control rats and rats submitted to Altered

Occlusion, chronic stress (Stress) or stressed group treated with diazepam (Stress+Dzp).

40x magnification. B: Demonstration of SDH in different groups of rats. Graph

indicates number of glycolytic fibers (light), glycolytic-oxidative fibers (intermediate)

and oxidative fibers (dark) in control rats and rats submitted to altered occlusion (AO),

chronic stress (S) or both (AO+S) (n=8 per group) in contralateral (right) and ipsilateral

(left) muscles. Two-way ANOVA indicated an effect of exodontia and stress, isolated or

associated. *P <0.01, S and AO+S with vehicle groups vs all other experimental groups.

#P<0.05, AO groups vs all other experimental groups. Data were expressed as mean ±

SEM. SEM: standard error means.

Figure 4. A: Panels show representative photomicrographs of immunofluorescence for

capillary marked by laminin in the right MPM of control rats and rats submitted to

Altered Occlusion, chronic stress (Stress) or stressed group treated with diazepam

(Stress+Dzp), 40x magnification. B: Graph indicates number of capillaries marked by

laminin in control rats and rats submitted to altered occlusion (AO), chronic stress (S) or

both (AO+S) (n=8 per group) in contralateral (right) and ipsilateral (left) muscles. Two-

way ANOVA indicated an effect of stress and exodontia, isolated or associated.

#P<0.05, AO groups vs all other experimental groups. *P<0.01, S and AO+S with

Page 16 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

vehicle vs all other experimental groups. Data were expressed as mean ± SEM. SEM:

standard error means.

Figure 5. Images (TEM) of right MPM of control rats and rats submitted to Altered

Occlusion, chronic stress (Stress) or stressed group treated with diazepam (Stress+Dzp).

Control and Stress+Dzp showed muscle fibers with several well-preserved structures.

AO group showed dilated sarcoplasma reticulum cisternae with irregular contours

(arrow). The stress group indicated myofibrils containing amorphous material (*) and

altered morphology mitochondria (m).

Page 17 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

123x84mm (300 x 300 DPI)

Page 18 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

71x55mm (300 x 300 DPI)

Page 19 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

341x190mm (150 x 150 DPI)

Page 20 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

272x148mm (150 x 150 DPI)

Page 21 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

198x163mm (150 x 150 DPI)

Page 22 of 22



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

for peer revie · exodontia. adult wistar rats were submitted to unpredictable chronic mild stress...

Documents