An Axon Guidance Pathway Mediated By Netrin-1 In Peripheral Nerve Defect Repair

Peripheral nerve defect repair is a complex process that is controlled by axon guidance molecules during the reconstruction of the neural circuit.At present,there are few reports on axon guidance pathways,and the types of molecular guides involved are very different.Based on the axon guidance pathway provided by our team's recent transcriptome analysis report,we examined axon attraction and axonal rejection in the axon guidance pathway mediated by Netrin-1.Signaling molecules involved in the pathway: The expression of Netrin-1,DCC,Nck1,Rac,and UNC-5 in the repair of peripheral nerve defects provides a reference for the further elucidation of the repair mechanism of peripheral nerve defects.This article used cellulose and polylactic acid as a material for the preparation of nerve catheters,named Nerve conduits prepared with cellulose and polylactic acid,CCP.In order to prevent the Schwann cells suspension from escaping from the catheter,preserving the Schwann cells in the catheter,first use the calcium alginate hydrogel to carry Schwann cells,then the calcium alginate hydrogel and Schwann A mixture of cells is filled in a nerve conduit to prepare a Nerve conduits filled with calcium alginate hydrogel and Schwann cells,CCHS.For this purpose,a calcium alginate hydrogel containing no Schwann cells was filled in a nerve conduit to prepare a Nerve conduit with calcium alginate hydrogel(CCH).The three kinds of artificial nerves were used to bridge 10 mm sciatic nerve defects on the rat.At a series of time points,morphological methods were used to assess the progress and efficacy of the nerve repair.Relative neo-fluorescence quantitative PCR was used to analyze the expression of signaling molecules in the axon guidance pathway mediated by Netrin-1 during repair of peripheral nerve defects,and to investigate the expression changes of related molecules in the axonal guidance pathway mediated by Netrin-1.The relationship with the repair process of peripheral nerve defects provides an experimental basis for the study of the repair mechanism of peripheral nerve defects.(1)In the early stage of repair of peripheral nerve defects,blood cells participate in this process,but the final repair of nerve defects depends on fibroblasts,Schwann cells,and axons.?-actin is an important component of the cytoskeleton,and the expression of ?-actin is positively correlated with the number of cells.Smear staining observation cannot be used as the basis for determining the number of cells,but it can be used as the basis for determining the relative proportion of different cells.Observation of smears of cells stained with HE showed that in each group,the proportion of non-blood cells increased significantly with time.Among them,the proportion of non-blood cells in the artificial nerves(CCHS)with Schwann cells added was significantly higher than that in the other two groups,suggesting that exogenous addition of Schwann cells is beneficial to the repair of peripheral nerve defects.The molecular expression of ?-actin can be shown that on the third day after nerve transplantation,the molecular expression of ?-actin was significantly lower in the CCHS group than in the other two groups.In addition,the expression levels of ?-actin in these two groups were similar.Although a large number of SCs were preset in the CCHS group,the number of cells was still significantly lower than the other two groups.Most likely,there are a large number of inflammatory cells in the CCP and CCH groups.This suggests that the exogenous addition of Schwann cells has an inhibitory effect on the inflammatory response at the initial stage of neural transplantation.At 7d and 14 d,the molecular expression of ?-actin in the CCHS group was significantly higher than that in the other two groups,showing that the exogenously added Schwann cells may be actively dividing in the cells.At 21 d and 30 d,the expression of ?-actin in the CCHS group was significantly higher than that in the other two groups,indicating that the number of cells in the other two groups was higher than that of the CCHS group.The smears at 21 days showed that there were inflammatory cells in each group.The proportion of inflammatory cells in the CCP and CCH groups was significantly higher than that in the CCHS group.Comprehensive observation of cell smears and expression of ?-actin,presumably,the inflammatory response is still the main event in the CCP and CCH groups in 21 to 30 days.In other words,the exogenous addition of Schwann cells inhibits the inflammatory response in nerve repair.In 3~30 days,the fluctuation of ?-actin expression also suggested that peripheral nerve defect repair is not a uniform process.(2)In order to facilitate a more accurate examination of the morphological effects of peripheral nerve defect repair,this article was obtained from the proximal,distal and graft sites of recipient nerves for immunofluorescence staining.The results showed that the fluorescence intensity of various markers in each group was different.At the same time point in the same group,the intensity of fluorescence at the proximal,distal,and graft sites of the recipient nerve is also different.Among them,at the 14 th day after transplantation,the fluorescence intensity of NGFR P75 in the CCHS group and the CCP group was higher,suggesting that the number of Schwann cells in the proximal,distal and vascular wall of the graft was significantly increased.Compared with the CCH group,it was suggested that the proximal Schwann cells in the CCHS group were less than the CCH group,whereas in the graft wall and the distal end,the CCHS group had more cells than the CCH group.Comparing the fluorescence signal intensity of NF200 and Fibronectin in the three groups,the number of fibroblasts and axons in the CCHS group was the highest,followed by the CCH group,and the CCP group was the lowest.Comparing the intensity of the fluorescence signal of Netrin-1 in the three groups indicated that the content in the CCHS group was the highest,followed by the CCH group,and the content in the CCP group was the lowest.The above results suggest that peripheral nerve defect repair is not a uniform process,and there is no difference in cell behavior at the proximal,distal and graft sites of the recipient nerve.(3)Real-time fluorescence quantitative PCR showed that: during 0 to 30 days after transplantation(1)In the attraction pathway mediated by Netrin-1 with DCC as a transmembrane receptor,The relative expression of Netrin-1 in the CCHS group showed a trend of decline,then rise again and then rise again.In the CCH group,there is a trend of falling first,then rising and then falling.In the CCP,it is the trend of falling and then falling.In the CCP group,the relative expression of DCC showed a trend of first increase and then decrease.In the CCH group and CCHS group,the expression of DCC first decreased,then increased,then decreased.The relative expression of Rac in CCP group increased first,then decreased,then increased,and then decreased.In CCH group and CCHS group,Rac showed a trend of first increase,then decrease and then rise.The relative expression levels of Nck-1 in CCP and CCH groups increased first,then decreased,then increased,and then decreased.In CCHS group,the expression of Nck-1 first increased and then decreased.(2)In the repulsive pathway mediated by Netrin-1 and DCC and UNC-5,the relative expression of UNC-5 in the CCP group was consistent with the DCC expression trend,showing a trend of increasing first and then decreasing.In the CCH group,the UNC-5 and DCC expression trends were consistent,showing a trend of decreasing first then rising and then falling;in CCHS The overall group showed a downward trend.In summary,peripheral nerve defect repair is not a uniform process.There are also differences in cellular behavior between the proximal,distal,and graft sites of the recipient nerve.Correspondingly,the amount of signal molecules expressed in Netrin-1 mediated axon guidance pathway fluctuates with time.Exogenous addition of Schwann cells inhibits the inflammatory response in nerve repair and is beneficial for promoting repair.To elucidate the role of signaling molecules in the repair of peripheral nerve defects in Netrin-1-mediated axon guidance pathways,more research to be done in the future.