Effects Of Physical Factors On Accelular Nerve Allografts Repairing Sciatic Nerve Gap In Rats

PrefacePeripheral nerve injury is very common and the reparation and regeneration mechanism of is very complicated and affected by many factors.The therapy of long gap peripheral nerve injuries is always a puzzle clinically.It usually require a graft to facilitate axonal regeneration into the distal nerve stump.Autograft neural transplantation therapy is thought as gold standard.The use of autografts is often limited because of graft availability and donor-site morbidity.While nerve allografts possess many good quality such as sufficient sources,not producing vice-injury and obtaining various kinds nerve segment easier.But the main shortage is immunological rejection.Liu Cheng Ji reported that handling sciatic nerves of rats with hypotonic-chemical detergent method can get rid of Schwann cell's extraneous coat, bundle theca cell,axon and myelin.By this way,we can obtain three diamensions tubiform stand materials of epineurium only including Schwann cell's vessel of basilar membrane.Zhang Cai Sun et al found that the allografts can facilitate axonal regeneration into the distal nerve stump and reach effector to form nerve-muscle synapse and make motor function restore partly by constituting an animal model of acellular nerve allografts(ANA)bridging sciatic nerve defect of rats.The study shows that acellular nerve allografts surpass other artificial precipitate grafts,not only their good biocompatibility,but also they are good materials to long gap nerve defect by preserving nerve out-matrix containing neurotrophic factor and nerve adherence factor to promote nerve regeneration.The linked study of physical factors and tissue engineering is other important topic. Many physical factors also play a part to promote nerve regeneration besides neurotrophic factors and some chemical drugs clinically.Former studied showed that low dose ultrashortwave(USW)can promote injured peripheral nerve regeneration by expanding blood vessel,ameliorating blood circle and nutrition of nerve and peripheral tissue and intensifying tissue metabolizability and nerve system function.Low energy laser can speed up neuraxon regeneration by ameliorating physiological activity of injured nerve,promoting axoplasm transportation and metabolizability and inhibiting scarring.Brain derive neurotrophic factor(BDNF)is an important neurotrophic factor.It possesses nerve protecting function and plays an important part to synaptic form and sustain nervous process's shape.Snyder et al thought the continuing increasing of calcitonin gene-related peptide(CGRP)may be thought as one of indicators of nerve regeneration.Vascular endothelial growth factor (VEGF)once regarded as a specific angiogenic factor,now implicated in neroprotection.It has not been reported if the two physical factors can promote nerve regeneration on accelular nerve allografts repairing sciatic nerve gap in rats and their action mechanism hasn't been extremely clear.The study aims at discussing and exploring the action mechanism of low dose USW therapy and low energy Ga-al-sn laser therapy on injured peripheral nerve restore and regeneration after ANA repairing the sciatic nerve gap of rats and provides a theory basis clinically.Materials and Methods1.We prepared acellular rats sciatic nerve(ARSN)in accordance with hypotonic-chemical detergent method of LIU Cheng-ji's.Then use ARSN and autografts to bridg the 10mm long gap of rats sciatic nerves.We administed low dose USW therapy and low energy Ga-al-sn laser therapy to some rats who were chosed from ARSN bridging rats.At 2w,4w,8w,12w after surgical procedure,we observated axon diameter of regeneration nerve,myelinated nerve number and myelin sheath thickness under general and microscope observation,detected expression of CGRP, BDNF,VEGF protein and gene in the spinal cord and muscle by immunohistochemistry dyeing and reverse transcription polymerase chain reaction (RT-PCR),and write down nerve conduction velocity,latent period and wave amplitude by electrophysiology examination.All the data were statistically analyzed by the first author with SPSS 11.0 software.The results were expressed as Mean±SD.Analysis of variance was used for significant difference test.T-test was used for intergroup comparison.P＜0.05 level was considered significant. Results1.The expression of BDNF and CGRP in operated site spinal cord had begun increasing at 2w,were up to the top at 4w,sustained to 8w,then decreased gradually, and were still higher than normal control group at 12w after operation.Compared with autograft group,the deference was not obvious.While the expression of BDNF and CGRP in operated site tibialis anterior muscle decreased gradually to the lowest at initial four weeks,then increased gradually and was up to the normal level at 12w after operation.The deference was not obvious between ANA group and autograft group. The gene expression of BDNF and CGRP was consistent with that of their protein on the whole.2.Twelve weeks later,muscular atrophy and motor function was restore partly. The adherence was very slight between grafts and around tissue and was easy to segregate.Compared with acellular nerve allografts alone group,both USW and laser therapy can increase nerve conductive velocity,the restoring rate of tibialis anterior muscle wet weight,myelinated nerve number,axon diameter.In addition,USW therapy can make VEGF mRNA expression of spinal cord and muscle at injury site increase, and laser therapy can make CGRP protein and mRNA expression increase obviously. The difference is significant(P＜0.05).Conclusion1.BDNF and CGRP deriving from central nerve played an important role on promoting nerve regeneration after ANA repairing sciatic nerve gap in rats.2.ANA had good biocompatibility,so it may substitute autonerve grafts to repair long gap of the sciatic nerve in rats.Its action mechanism may relate to upregulate the expression of BDNF and CGRP protein and mRNA of spinal cord.3.Low dose USW and low energy Ga-al-sn laser therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats.Low dose USW may play an important role on promoting nerve regeneration by upregulating the expression of VEGF mRNA in the spinal cord and muscle,while the action of low energy Ga-al-sn laser may be related to the upregulation of the expression of CGRP protein and mRNA in the spinal cord.