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
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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:
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).
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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.
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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)
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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).
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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
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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).
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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.
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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
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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
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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
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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.
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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,
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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.
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Figures legends
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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
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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).
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123x84mm (300 x 300 DPI)
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71x55mm (300 x 300 DPI)
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341x190mm (150 x 150 DPI)
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272x148mm (150 x 150 DPI)
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198x163mm (150 x 150 DPI)
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