Masseter Muscle Injury Induced By Experimental Created Disordered Occlusion On Rat

Masticatory muscles dysfunction, one of the symptoms of temporomandibular disorders characterized as the sour, fatigue and pain of the masticatory muscles, is generally associated with no organic injury and supposed to be related to the increasing of myocyte Ca²⁺ content. Our pilot experiments showed that the experimental created disordered occlusion (ECDO) leaded to the degeneration of temporomandibular joints on rats. In order to study the effects of the experimental created disordered occlusion on rats'masseter muscle and the underlying mechanism, this research was designed to observe the microstructure, ultrastructure and some other changes associated with the muscle injury and the physiological and pathological effects of the administrations of EDTA and Nifedipine.Female Sprague-Dawley rats were used in this research. First of all, the experimental created disordered occlusion was established by moving the left maxillary and right mandibular third molars distally about 0.8mm. Second, the microstructure and ultrastructure of masseter muscle, the mitochondria Ca²⁺ content, proteins sensitive to the muscle injury and substances in serum were observed. Third, the effects of EDTA and Nifedipine on changes of masseter muscle were observed.First part: 42 8-week-old female SD rats were adopted for the experiments. The rats were randomly assigned to 7 groups: blank control group, operation control group, ECDO group, ECDO + EDTA injection group, ECDO + saline injection group, ECDO + Nifedipine injection group and ECDO + DMSO injection group. EDTA is chelating agent, while Nifedipine is calcium-channel blocker (CCB). The microstructure and ultrastructure of masseter muscle were observed at the time points of 1w, 2w and 4w after the operation. At all check points, there was no difference of microstructure between the blank control group and ECDO group. The ultrastructure of operation control group, ECDO + EDTA injection group, and ECDO + Nifedipine injection group were similar with the appearance of blank control group. The swollen mitochondria and sarcoplasmic reticulum could be seen in the ultrastructure of ECDO group, ECDO + saline injection group and ECDO + DMSO injection group. Conclusion: ECDO result in slight ultrastructure changes of masseter muscle, and the changes could be prevented by EDTA and Nifedipine.Second part: 135 8-week-old female SD rats were randomly assigned to 7 groups: blank control group, operation control group, ECDO group, ECDO + EDTA injection group, ECDO + saline injection group, ECDO + Nifedipine injection group and ECDO + DMSO injection group. The mitochondria Ca²⁺ contents were tested at the time points of 1w, 2w, 4w after operation. First, the mitochondria were abstracted by using a commercial kit. Second, test the Ca²⁺ contents by atomic absorption spectrographic (AAS) method and the protein contents by BCA kit. Third, take the Ca²⁺ content/the protein content (μg/mg)as the mitochondria Ca²⁺ content. In ECDO, ECDO + saline injection and ECDO + DMSO injection groups, the mitochondria Ca²⁺ contents increased significantly (P < 0.001). In operation control, ECDO + EDTA injection and ECDO + Nifedipine injection groups, however, the mitochondria Ca²⁺ contents (P > 0.05) showed no significant difference from the blank control group. Conclusion: ECDO lead to the increasing of mitochondria Ca²⁺ level, and could be inhibited by EDTA and Nifedipine.Third part: 36 8-week-old female SD rats were randomly assigned to 4 groups: operation control group, ECDO group, ECDO + EDTA injection group and ECDO + Nifedipine injection group. Desmin and skeletal muscle troponin (sTnI) were observed by western-blot method at the time points of 1w, 2w and 4w after operation. The two proteins showed no significant difference in all groups(P>0.05). Conclusion: ECDO did not cause degradation of desmin and sTnI of SD rats'masseter muscle.Fourth part: 75 8-week-old female SD rats were randomly assigned to 4 groups: blank control group, ECDO group, ECDO + EDTA injection group and ECDO + saline injection group. At time point of 1w, 2w and 4w after operation, test the activity of creatine kinase (CK) and lactate dehydrogenase (LDH) in serum by means of automatic biochemical analyzer and test the serum myoglobin contents through the ELISA kit. The three substances showed no significant difference in all groups. Conclusion: ECDO could not cause the changes of serum CK, LDH and Mb. Conclusions:1. ECDO leads to slight ultrastructure injury of rat masseter muscle, such as swollen mitochondria and sarcoplasmic reticulum. The injury is related to the mitochondria Ca²⁺ overload, and the reason might be the Ca²⁺ inflow.2. ECDO causes no change in the contents of serum substance and proteins content sensitive to muscle damage.