| Objective: Our experiment is to establish experimental model of superior buccal branch of the facial nerve of rabbits with compressing injury,and using the treatment of electroacupuncture,studying on N-cad, E-cad and P-cad m RNA expression in facial nerve nucleus by in Situ Hybridization,and also studying on the electroacupuncture's effect on N-cad, E-cad and P-cad m RNA expression in facial nerve nucleus after.facial nerve damaged.Methods: 52 New Zealand rabbits were needed,they were randomly divided into three groups:normal control group ? operation group and electroacupuncture treatment group. There were 4 rabbits in the normal control group,while both operation group and electroacupuncture treatment group were 24 rabbits, and we setted six time points: 1 day?4 days?7 days?14 days?21 days and 28 days after operation,and chose 4 rabbits each time point. Normal control group was not given any treatment, took the middle and lower part of the pons in rabbit brain tissue after heart perfusion and fixation,and then made into paraffin sections, while operation group and electroacupuncture treatment group establishing experimental model of superior buccal branch of the right facial nerve of rabbits with compressing injury,and damage accorded with Sunderland IV.In electroacupuncture treatment group,electroacupuncture stimulation was performed at Jiache?Dicang?Yifeng?Quanmiao?Yangbai and Sibai for 30 minutes,two hours after operation,once a day, while Hegu was just needle-embedding therapy not electroacupuncture stimulation.Operation group was not given any treatment after operation. At six different time points, both in electroacupuncture treatment group and operation group, took the middle and lower part of the pons in rabbit brain tissue and made into paraffin sections.Three experimental groups were stained hematoxylineosin staining to confirm the position of facial nerve and observe the morphological changes of motor neurons in the nucleus of facial nerve under the microscope.Detected N-cad, E-cad and P-cad m RNA expression in facial nerve nucleus by in Situ Hybridization,and counted positive cells of the three calcium adhesion molecules under the light microscope.Using software of SPSS 17.0 for statistical analysis.Result: Facial nerve nucleus were located in the ventrolateral of pons, anterolateral of abducens nucleus in the three experimental groups under the light microscope. N-cad?E-cad and P-cad were no positive expression in normal control group, besides,both E-cad and P-cad in electroacupuncture treatment group and operation group were no positive expression in 1,4,7,14,21,28 days after operation.Shown as no brown granules in cytoplasm and axons after DAB staining,the cells were blue.After H-E staining, motor neurons in the nucleus of facial nerve showed irregular polygon in normal control group,the nucleus was round and the nucleolus is obvious, like "fish eye", scattered blue Nissl bodies were visible in the cytoplasm.N-cad both in electroacupuncture treatment group and operation group have positive expression at various time points except 28 days after operation in operation group.It Shown that there were brown yellow granules in cytoplasm and(or) axons after DAB staining.1 day after operation, operation group showed N-cad positive cells(N-cad 14.67±1.52), at the same time,the number of N-cad positive cells(N-cad 21.67±3.51) in electroacupuncture treatment group increased compared with the operation group, the difference was statistically significant(P < 0.05). Compared with the normal control group, the morphology of the neurons in the operation group and the electroacupuncture treatment group had no significant change,4 days after operation, the number of N-cad positive cells(N-cad 29.67±3.51)in operation group increased,and the number of N-cad positive cells(N-cad 37.00±1.00) in electroacupuncture treatment group increased compared with 1 day after operation too. the number of N-cad positive cells in electroacupuncture treatment group were more than operation group too, the difference was also statistically significant(P < 0.05). Cell degeneration such as :swelling of the body of neuronal cells, edged nucleus,the decrease of Nissl bodies were shown in operation group while electroacupuncture treatment group did not change significantly in cell degeneration. 7 days after the surgery, the number of N-cad positive cells(operation group N-cad 44.67±3.06, electroacupuncture treatment group N-cad 120.00±8.71) both in the operation group and the electroacupuncture treatment group further increased,in the treatment group, the number of N-cad positive cells reached the maximum value,and the number were much more than the operation group's, the difference was statistically significant(P < 0.01). Degeneration of neurons in operation group increased, the death of neurons showed nucleoli disappeared; electroacupuncture treatment group had no phenomenon of neuronal death. 14 days after the surgery, the number of N-cad positive cells in operation group reached peak(N-cad 58.00±7.21), while in the treatment group the number of N-cad positive cells compared with the previous time points decreased(N-cad 76.00±4.00), but the N-cad positive cell number in treatment group is higher than the operation group, the difference was statistically significant(P < 0.05). The operation group showed that neurons appeared nuclear membrane rupture, nuclear dissolution, and nucleolus disappeared,which were phenomena of neuronal death; in treatment group,only a few neurons showed neuronal death, we can saw neuron recovery phenomenon that nucleoli in neurons and Nissl body presented again. 21 days after surgery, the number of N-cad positive cells in operation group compared with the previous time points decreased(N-cad 31.67±4.73), the number of N-cad positive cells in electroacupuncture treatment group was not decreased obviously(N-cad 70.33±9.07), the number of positive cells of treatment group that N-cad marked were still much more than operation group, the difference was statistically significant(P < 0.01). At this time point,neurons lose their normal morphology in operation groups, but Nissl body in cytoplasmic increased, indicating that neurons began to recover;in treatment group, the number of restoring neurons increased,including nucleolus regression, "fish eye" construction.28 days after surgery, there was no N-cad positive expression in the operation group,while the electroacupuncture treatment group have N-cad expression(N-cad 52.33±2.52). Compared electroacupuncture treatment group with operation group, the difference is statistically significant(P < 0.01). At this time point, morphology of neurons in operation group and electroacupuncture treatment group basically recovered.Conclusion: 1. Positive expression of N-cad appeared 1 day after facial nerve injury, and its positive expression existed at every time point after operation(except 28 days after operation in operation group),while normal control group had no N-cad positive expression.We think that N-cad participate in the process of restoration of facial nerve.2.Electroacupuncture can promote the high expression of N-cad,letting the peak ahead of time and prolong the duration of high expression.And the high expression shows corresponding to the morphological changes of the motor neurons in H-E staining sections, the positive changes in the morphological indicates the functional status of neurons.It indicates that the promoting effect of electroaeupuncture on facial nerve regeneration may be achieved by maintaining the high expression of adhesion molecules, thus affecting the transmission of information.3.There was no positive expression of E-cad and P-cad in the study, indicating that these two adhesion molecules did not participate in the restoring process of facial nerve injury. |
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