The Influence Of Chronic Unpredictable Stress On The Temporomandibular Joint In Rats

Temporomandibular disorders(TMD)is one kind of common and frequently-occurring diseases of stomatology,which often involves masseter muscle and temporomandibular joint,has complicated invasion reasons and pathopoiesis mechanism.Many studies have shown that TMD is a multiple factors disease that has a close relationship with psychological factors,such as anxiety,depression and so on.However,many studies nowadays about psychological factors in TMD just focus on evaluated subjects by clinical inquiry and SAS(self-rating anxiety scale),SDS(self-rating depression scale), MMPI(Minnesota Multiphasic Personality Inventory),the directly foundation of pathology is scarce.Thus,the purposes of our study were to establish the chronic unpredictable stress experimental animal model in rats and observe the changes of temporomandibular joint,masseter and correlated agent in this state. Meanwhile,we observed the changes of temporomandibular joint when rats recovered from this stress state,and tried to make a reasonable explanation of the relationship between stress and TMD.Part 1 The changes of temporomandibular joint induced by chronic unpredictable stress in ratsPurpose:To observe the changes of general body state,temporomandibular joint,masseter and correlated agent of rats in stress state induced by chronic unpredictable stress experiments.Methods:Thirty two 12-week-old male Wistar rats were randomly divided into 2 groups,the experimental group and the control group.Each group was divided into two subgroups according to execution time(3-week subgroup and 6-week subgroup),and each had eight rats.Each rat in experimental group was breeding in single cage,suffered chronic unpredictable stress(CUS) stimulations everyday.The control group drank and ate normally.1)Each rat was weighed every week,and the ethology performance scores of rats which evaluated by open-field box were recorded at the time of before experiment, after 3 weeks and 6 weeks respectively.2)The peripheral blood of rats' 3-week subgroup and 6-week subgroup were collected before sacrifice at the time of 3rd week and 6th week respectively.The blood-serum corticosteroid, Ca²⁺and Mg²⁺in peripheral blood were measured.3)Some of masseter and internal pterygoid muscle of rats were cut,the changes of ultrastructuralof masseter muscle under the transmission electron microscope were observed.4) The left condyles and articular discs of rats were taken out completely.The surface of tissue was left uninjured.Then the changes of ultrastructure of condyles and articular discs were observed under scanning electron microscope. 5)The fight condyles were taken out completely,trimed and reserved the apogee part of condyles,routine declalcification,dehydration,and embedment,make slice and dye with immunity.The images of slice were analyzed by image-pro plus 5.0 software,and the expressions of MMP-1,MMP-3 and TIMP-1 in condylar cartilage were compared between experimental group and control group.Results:1)The weight of rats in control group increased more rapidly than those of rats in the experimental group.There were significant difference between experimental group and control group in the scores of ethology performance of rats(P＜0.05).2)The concentrations of blood-serum corticosteroid of rats in experimental group were higher than those of rats in control group.The concentrations of blood-serum Ca²⁺of rats in experimental group were lower than those of rats in control group.Comparing with control group,the concentrations of blood-serum Mg²⁺also changed. 3)Under transmission electron microscope,ultrastructures of masseter muscle cells changed significantly in experimental group.It showed that the myofilament deranged,the "z-line" warped and the mitochondria with cristae loss and degrade were found in 3w experiment group.Severe vacuolus changes of the mitochondria were observed in 6w experimental group.There were no obvious changes in control groups.4)Under scanning electron microscope,the gelatum on the surface of condyles in experimental group was found to be cracked,and collagen fibrils were disorderly and broken,and deep layer collagen fibrils were exposed.The gelatum on the surface of articular discs was still integrated,but the arrangements of the gyrus were irregular.The 6w group was even more severe than 3w group.In control group,there were no such obviously changes on the surface of condyles and articular discs.5)The expression of MMP-1 in condylar cartilage was higher in experimental group than those of in control group.In 6w group,there was statistical significant difference between groups(p＜0.05).The expression of MMP-3 and TIMP-1 in experimental group was also higher than those of in control group,but it was not significant.Conclusions:Long-term chronic unpredictable stress stimulation could affect the general body state of rats,such as changing the behaviors,inhibiting the increase of weight,heightening the level of concentrations of blood-serum corticosteroid and vary concentrations of blood-serum Ca²⁺and Mg²⁺.It could also induce the ultrastructure injures of temporomandibular joints,including masseter muscle,condyles and articular discs.We found that there was a disbanlance expression of MMPs/TIMPs of condylar cartilage in experimental group rats,which showed that the ECM of condylar cartilage suffered a degradation state.So it is considered that long-term emotional stress can make rat's temporomandiblar anaplasia. Part2 The changes of temporomandibular joint after the emotional stress factors were removed in ratsPurpose:To observe and study the recovery of the general body state, temporomandibular joint,masseter and correlated agent of rats when the stress factors had been removed.Methods:Thirty two 12-week-old male Wistar rats were divided into 2 groups randomly,experimental group and control group.Every group was divided into two subgroups according to execution time respectively;named 9-week subgroup and 12-week subgroup,each had eight rats.Firstly,CUS animal model were established in experimental group in 6 weeks,then removed all the stimulation factors and breed normally.The control group drank and ate normally.1)Each rat was weighed every week,and the ethology performance scores of rats which evaluated by open-field box were recorded at the time of before experiment,after 9 weeks and 12 weeks respectively.2)We gained the peripheral blood of rats before 9-week subgroup and 12-week subgroup before execution at the time 9 weeks and 12 weeks respectively,and then measured blood-serum corticosteroid,Ca²⁺and Mg²⁺in peripheral blood.3)Some of masseter and intemal pterygoid muscle of rats were cut,the changes of ultrastructuralof masseter muscle were observed under the transmission electron microscope.4)The left condyles and articular discs of rats were taken out completely;and then the changes of ultrastructure of condyles and articular discs were observed under scanning electron microscope.5)The fight condyles were taken out completely,trimd and reserved the apogee part of condyles, routine declalcification,dehydration,and embedment,make slice and dye with immunity.The images of slice were analyzed by image-pro plus 5.0 software, and the expressions ofMMP-1,MMP-3,and TIMP-1 in condylar cartilage were compared between experimental group and control group.Results:1)The weight of rats in experimental group increased rapidly after 6 weeks.There were no significant difference between experimental group and control group in the scores of ethology performance of rats(P＞0.05).2)There were no significant difference between experimental and control groups in the concentrations of blood-serum corticosteroid and Ca²⁺of rats (P＞0.05).Comparing with control group,the concentrations of blood-serum Mg²⁺changed slightly.3)Under transmission electron microscope,there were some recoveries in experimental group,which expressed the myofilament,z line turned to trim and mitochondria with cristae loss and degrade found less.The 12w group was better than the 9w group.There were no obvious changes in control groups.4)There was some recovery in experimental group under scanning electron microscope.The gelatum on the surface of condyles increased,collagen fibrils became regular,and deep layer collagen fibrils less exposed.There were no such obviously changes on the surface of condyles and articular discs.5)The expression of TIMP-1 in condylar cartilage was higher in experimental group than control group.In 9w group,there was statistical significant difference between groups(p＜0.05).The expression of MMP-1 and MMP-3 in experimental group was also higher than control group,but it was not significant.Conclusion:When the stress factors were removed,there were no obviously difference between experimental groups and control group in general body state,and the ultrastructure of temporomandibular joint,masseter recovered.The expression of MMPs/TIMPs of condylar cartilage in experimental group rats showed that the degradation of ECM in condylar cartilage was inhibited.So we considered that the ultrastructure injures of temporomandibular joint in rats which induced by emotional stress could be reversed if the stress factors were removed.