| With the rapid advance of China’s economy, the dramatic changes of society patternand alterations of lifestyle, stress from study, work and family constantly confoundspeople and results in many mental illnesses and psychosomatic diseases, the incidence ofwhich climb each year. Psychological stress caused by negative life events not only resultsin mental illnesses in human body, but also plays important roles in the pathogenesis anddevelopment of several stomatognathic system diseases, including temporomandibularjoint disorder syndrome (TMD) and nocturnal bruxism (NB). Prolonged stress causesmental changes, such as anxiety and depression, as well as pathological changes in oral muscles, the underlying mechanisms of which remains unclear. In addition, exposure topsychological stress also leads to a significant increase of masticatory muscle tone whichis not merely a body’s response, but also may be a pathological process of induction andaggravation of TMD. However, to date the underlying mechanism of this phenomenon isstill enigmatic. Understanding to the intrinsic mechanism and accordingly establishingeffective preventive measures and reasonable treatments have a great benefit on the peopleaffected by psychological stress.For these reasons, we have established a rat model of chronic restraint stress (CRS)and studied the impact of long-term restraint stress on rats’ masseter and brain nucleus,and the mechanisms of the corresponding changes by means of muscleelectrophysiological recording, high performance liquid chromatography, ELISA, westernblot, qRT-PCR and other molecular biology techniques. The effect of lamotrigine on thesepathological changes were also evaluated.The study is comprised of two parts as following:1. Part one: The establishment and evaluation of the psychological stress animal model.A psychological stress animal model was established with CRS. Rats were randomlydivided into five groups, including control group, psychological stress group, low-dose(20mg/kg) group, moderate-dose (30mg/kg) group and high-dose (40mg/kg) group. At theend of the3-week restraint stress, bodyweight of rats were measured, behavioralevaluations such as sucrose preference test and open field test, and measurement of serumhormone such as adrenocorticotropic hormone (ACTH) and corticosterone (CORT) wereemployed for the evaluation of the CRS model. Experimental results showed thatbodyweight of the rats of psychological stress group significantly decreased after3-weekstress (p <0.05).CRS also resulted in a decrease of sugar preference of sugar preference test (p<0.005), decrease of distance moved (p <0.005) and velocity (p <0.005) and an increase ofcentral residence time (p <0.005) in open field. Significantly upregulated concentrations of serum hormone (ACTH (p <0.005) and CORT (p <0.005)) were also observed on the ratspurely challenge by CRS. These results demonstrated that the CRS model established inthe present study characterized by significant stress-related behaviors and hormonecharacteristics, satisfied the demands of the subsequent experiments.Pat two: The studies of influence of psychological stress on masseter muscle tone (MMT)and brain neurotransmitters, and the effects of lamotrigine on the changes.1. Study of impact of psychological stress on masseter energetic metabolism andeffects of lamotrigineThe results showed CRS resulted in an increase of content of creatine kinase (p<0.005), lactate dehydrogenase (p <0.005) and lactate (p <0.005) in masseter muscle,whereas a decrease of creatine phosphate (p <0.005) was also observed. Administration oflamotrigine with a dosage of30(p <0.005) and40mg/kg (p <0.005) bodyweighteffectively reversed the above changes, while lamotrigine of20mg/kg dose had nosignificant effects (p>0.005).2. Study of the impact of psychological stress on MMT and effect of lamotrigineThe results showed that MMT of rats significantly increased at the end of the CRSprocess compared to the MMT before the CRS both at the quiet wake and active wakestates (p <0.05). Administration of lamotrigine with a dosage of30(p <0.005) and40mg/kg (p <0.005) bodyweight effectively reversed the upregulation of MMT, whilelamotrigine of20mg/kg dose had no significant effects (p>0.005).3. Study of the impact of psychological stress on free radical metabolism in brain andeffect of lamotrigine on free radical metabolism in brainThe results showed CRS resulted in decrease of activity of superoxide dismutase (p<0.005), catalase (p <0.005) and glutathione peroxidase (p <0.005) in central amygdaloidnucleus (CeA), parvicellular reticular formation (PCRt) and motor nucleus of trigeminalnerve (Vm), whereas an increase of malondialdehyde (p <0.005) was observed in the aforementioned areas. Administration of lamotrigine with a dosage of30(p <0.005) and40mg/kg (p <0.005) bodyweight effectively reversed the above changes, whilelamotrigine of20mg/kg dose had no significant anti-psychological-stress effects (p>0.005).4. Study of the impact of psychological stress on brain neurotransmitters and effect oflamotrigineThe results showed CRS resulted in increase of activity of glutaminase (GLS)(p<0.005), and content of glutamate (Glu), vesicular glutamate transporter1(VGluT1)(p<0.005) and vesicular glutamate transporter2(VGluT2)(p <0.005) in the aforementionedbrain areas, whereas a decrease of glutamine synthetase (GS)(p <0.005) was observed inthe above brain areas. Expression of N-methyl-D-aspartate receptor2（p<0.005）wasincreased in CeA after CRS, while the expression of N-methyl-D-aspartate receptor1(NMDAR1)（p<0.005）was downregulated in Vm. Administration of lamotrigine with allof the three dosage had no significant effects on the above changes (p>0.005).In the present study, we demonstrated that CRS induces an increase of MMT viaupregulation of Glu level in Vm, further causing the disorders of masseter muscleenergetic metabolism. The increase of Glu was attributed to the alteration of GLS and GSactivity, and upregulated expression of VGluT1and VGluT2(mainly influence theextracellular content of Glu) in Vm. In addition, increased expression of NMDAR1resulted in the upregulated sensitivity of neurons to the increased content of Glu. All thesefactors caused abnormal impulses on motor neurons of Vm, eventually leading to a rise ofMMT. Moreover, CRS also induced overactive oxidative stress response and brain injury.Increase of Glu in Vm likely partially due to the rise of excitatory neurotransmittertransmission from its superior neurons in PCRt and CeA. The efficacy of the lamotrigineat dose of30and40mg/kg bodyweight on the increased MMT does not rely on theregulation of the activity or content of GLS,GS, VGluT1, VGluT2and NMDAR, butlikely on the inhibition of the release of Glu into synaptic cleft. The current study first illustrated the underlying mechanism of the increase of MMTinduced by psychological stress and confirmed the potential therapeutic effects oflamotrigine on negative effect induced by psychological stress. The findings enriched theknowledge of the pathogenesis of diseases caused by psychological stress and providedexperimrntal envidence for the formulation of treatment methods of stress relatedstomatognathic diseases in the future. |
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