Experimental Research Of The Role Of CGRP During Facial Nerve Injury And Regeneration

Calcitonin gene-related peptide (CGRP) is a neuropeptide generated by tissue-specific alternative splicing of the primary transcript of the calcitonin gene. CGRP is a member of a superfamily of neurotransmitters and hormones including calcitionin(CT), adrenomedullin(AM), amylin and calcitonin receptor-stimulating peptide(CRSP) that have about 25% sequence homology with calcitonin and adrenomedullin , about 50% with amylin and about 60% with CRSP. Since the discovery of CGRP, several groups have reported on the presence of CGRP mRNA as well as of CGRP-like immunoreactivity in both central and peripheral nervous systems. A wide variety of biological effects on various tissues have been reported for CGRP. Motoneurons themselves synthesize CGRP which is then anterograde transported to the nerve terminal and probably plays a role in the formation, maintenance and normal functioning of the neuromuscular junction..CGRP receptor have been reported in various central neurons, glial cells and Schwann cells. Several researchs suggest that CGRP might increase activity of glial cells with subsequence of promoting proteinsynthesis in vitro, and as a differentiation- promoting factor for embryonic midbrain dopaminergic neurons and cultured Schwann cells. It have been revealed that CGRP could reduce the death of hippocampus neurons induced by hypoxia in vitro. One report described a significant loss of CGRP in the cervical spinal cord and in motor and sensory cranial nuclei in Wobbler mouse. These results proposed that CGRP may act physiologically as a motor neuron-derived differentiation and survival promoting factor. But up to now , there have been no reports about the question whether CGRP have the effect in promoting motoneurons survival and axon regeneration.In present sdudies,we employed cultured Schwann cells and facial nerve injury models of adult rat,researched the influence of CGRP on synthesis and secretion of neurotrophic factors by Schwann cells,on astrocyte activity and the changes of growth associated peptide-43 after facial axon injuried. Our research is aimed to observe of the role of CGRP during facial nerve injury and regeneration.The major findings of the present work are as follows:1. Semiquantitative RT-PCR analysis and indirect ELISA indicated antisense CGRP primary gene oligonucleotides had the inhibitory effects on synthesis and secretion of CGRP in rat medullary thyroid carcinoma CA-77 cell line and facial motoneurons. Thereby, we established a new method to sdudy CGRPfunctions.2. CGRP promote cultured Schwann cells synthesize and secret glia cell line-derived neurotrophic factor with using Semiquantitative RT-PCR analysis,indirect ELISA and immunohistochemical method,the effect might be mediated by CGRP receptor 1.3. Pathomorphological changes were revealed by Histochemical,immunohistochemical method and TEM in the three facial axon injury model of adult rats-crush, cut off and suture, avulsion. Three adult rat models representing different facial nerve injury protocols were established for the further study of motor nerve regeneration.4. By the injection of exterior CGRP and antisense CGRP primary gene oligonucleotides into the facial nucleu of the avulsional animal model, the promotive effects of CGRP on the survival rates of motoneurons were confirmed.5. By the injection of exterior CGRP and antisense CGRP primary gene oligonucleotides into the facial nucleu of the cut off and suture animal model, the changes of the GAP-43, GFAP in facial nucleus and the GDNF in axons were studied. Results indicated that the mediating effects of CGRP between the motoneurons and the glia cells may play an crucial role in promoting the motoneuron survival and axon regeneration after injury.6. These results can lead to the following conclusion: 〤GRP synthesized by the motoneurons may be a self-serving neurotrophic factor after axotomy. (2) CGRP may serve as a "injury signal" activating both the central and peripherical gli...