The Experimental Study Of Repairing Facial Nerve Defect And Facial Motoneurons Death And Its Gene Control After Injury

Objective: To evaluate the feasibility and the superiority of repairing the longer facial nerve defects in rabbits by regenerating chambers and compound bridge grafts which made of acellular rat sciatic nerves and autogenous inside-out veins, and to find a simple and effective method for treatment of long nerve defects, which could be widely performed by surgeons. Meanwhile, to investigate the morphology of facial motoneurons and the expression of caspase 3,caspase 8,cyto-c,Bcl-2 and p53 following the facial nerve crush or transection at the level of the stylomastoid foramen in SD rats. At the same time, to assess the effect of Minocycline and GDNF in the function recovery of the facial nerve and the protection to its motoneurons after facial nerve injury.Methods: The study included two parts: The first part is an Experimental Study of Repairing Facial Nerve Defect by Regenerating Chambers and Tissue engineering in Rabbits and the second part is a Study on Facial Motoneurons Death and its Regulatory Gene Expression Following Facial Nerve Injury.In the first study, a 15mm nerve defect was created at the marginal mandibular branch of the facial nerve in rabbits, and then the defects were treated by different methods. Including, (1)chitolsan-collagen chambers which included double-bridging with little gap, (2)double-bridging without gap, and (3)combined bridge grafting which was made of acellular rat sciatic nerves by chemical extraction and autogenous inside-out vein, (4)nerve autografts and (5)normal control.In the second study, the facial nerve trunks were transected distally or crushed with different severities in the SD rats, the GDNF was applied locally for the transection injury group and the Minocycline was taken orally for the both groups. Gross observation, electrophysiologic evaluation, histologic morphometrical analysis and transmission electron microscope were performed. Moreover, the expression of Caspase 3,Caspase 8,Cyto-c,Bcl-2 and P53 protein in facial motoneurons was analyzed by immunohistochemical staining.Results: (1) chitosan-collagen chambers were obviously degraded in 12 weeks postoperatively and there was no foreign body reaction over all experimental periods. What's more, they also restrained the formation of neuroma and provided a good microcircumambience for nerve regeneration. (2)The nerve regeneration was good in double-bridging groups. There was no significant difference between the double-bridging and nerve autografts groups, but they all did not recover to the level before the surgery. (3)There was no significant difference between double-bridging groups and nerve autografts group according to the rate of regeneration of myelinated axon, the double-bridging groups were slightly lower than the nerve autografts group in the recovery rate and the maturity degree of myelinated axon. (4)The compound bridge did not show severe inflammatory reaction and provided a better microenvironment for nerve regeneration. (5)The result of facial nerve repairing was the same between the combined bridge and nerve autografts groups. (6)Different facial nerve crushes caused different results in the death of facial motoneurons; Severe nerve crushes resulted in death of facial motoneurons. (7) The loss of motoneurons was peaked in 21-28d after injuries and was mainly through by apoptotic pathway. The number of motoneurons loss in the transection group was larger than the crush group. (8) The Minocycline could promote the rapid recovery of crushed facial nerve. In addition, the protective effect of the Minocycline and GDNF for the facial motoneurons was detected in transection group. (9)The expression of Bcl-2 and P53 following facial nerve transaction was significantly higher than those of normal control group. (10)Caspase 3,8 protein and Cyto-c protein expressions were observed in wide spread areas of normal rat facial nucleus. Cells of the transection group stained more intense than that of crush group. Expressions of Cyto-c protein were correlated with expression of Caspase 3 protein.Conclusion: (1) The Double-bridging technique is proved as a simple and effective method for treatment of the long nerve defect. (2) The Chitosan-collage chamber shows the excellent biocompatibility in repairing of the nerve defects. There was no significant difference between the double-bridging with little gap group and double-bridging without gap group. (3)After acellularization by chemical extraction, the acellular heterogeneous nerve matrix has become a low antigenicity xenograft; the compound bridge provides a better microenvironment for nerveregeneration without severe inflammatory reaction. (4)There is a correlation between the facial motoneurons apoptosis and the patterns of facial nerve injuries. A severe nerve crush or a nerve transaction can cause the death of motoneurons. (5) The Minocycline can promote the rapid recovery of the crushed facial nerve; Both GDNF and Minocycline obviously prevent facial motoneurons from dying after transaction. (6)Bcl-2 and P53 genes might play a great role in the process of facial motoneurons apoptosis following facial nerve transection, the ratio of Bcl-2 / P53 may control facial motoneurons apoptosis. (7)The expressions of Caspase 3,8 and Cyto-c proteins were related with facial nerve injury severities. Cyto-c proteins' expressions were correlated with caspase 3 protein's expression.