The Effects Of TRPV1 Functional Inhibition On Nerve Regeneration After Sciatic Nerve Transverse Injury

Objective:1.To detect the changes in expression and function of transient receptor potential cation channel subfamily V member 1?TRPV1?based on rat sciatic nerve transection injury model.2.To investigate the effects of TRPV1 functional inhibition on the axonal and Schwann cell regeneration at proximal stem of sciatic transverse injury.3.To explore the effects of TRPV1 functional inhibition on the expression of growth associated protein-43?GAP-43?at proximal sciatic nerve.4.To provide a new strategy on searching more efficient clinical therapeutic agents for accelerating the rehabilitation process after peripheral nerve injury.Methods:1.Rats were divided into seven groups:1)the control group,2)only injury-1 week group,3)only injury-2 weeks group,4)injury+AMG-517 150?g/kg-1 week group?AMG-517,one of TRPV1 blockers?,5)injury+AMG-517 150?g/kg-2 weeks group,6)injury+AMG-517 300?g/kg-1 week group,7)injury+AMG-517 150?g/kg-2 weeks group.Left sciatic nerve of rats in experimental groups were transversely chopped off by microscissors.Two doses?150?g/kg,300?g/kg?of AMG-517 were injected into the surrounding area of L₃?L₄ and L₅ DRGs of rats after 30 min of sciatic nerve was cut.2.At 1 week and 2 weeks of model established,dorsal spinal cord,injury ipsilateral L₃-L₅ DRGs and sciatic stem were collected,the release of CGRP,the number of axons,the expression of TRPV1?GAP-43 and GFAP,the distribution of GAP-43 and GFAP in sciatic nerve were assayed and observed using the methods of ELISA?semi-thin section?western blot and immunofluorescence,respectively.Results:1.The result from ELISA showed that there was no significant difference for lumbar dorsal CGRP release from 1 week after sciatic transverse injury to the controls(the concentration of CGRP-C_CGRPGRP in these two groups were about 0.15±0.002 ng/g,P>0.05).But the release of CGRP was obviously increased after 2 weeks of sciatic injury(C_CGRPGRP was about 0.17±0.004 ng/g,P<0.05).The groups which plus AMG-517?both 1week and2 weeks groups?after injury displayed a dramatic arrest of CGRP release(1 week:C_{CGRP-150?g/kg}GRP-150?g/kg was about 0.11±0.0006 ng/g,C_{CGRP-300?g/kg}GRP-300?g/kg was about 0.11±0.003 ng/g,2weeks:C_{CGRP-150?g/kg}GRP-150?g/kg was about 0.095±0.005 ng/g,C_{CGRP-300?g/kg}GRP-300?g/kg was about 0.11±0.002ng/g,P<0.001).2.SDS-PAGE and western blot for TRPV1 in DRG displayed that the expression of TRPV1 increased continuously over time compared to the control group?P<0.001?.However,dramatic decrease was noted among tissue samples treated with AMG-517?both 1week and 2 weeks,P?0.001?.3.The results of semi-thin section showed that after 1 week of sciatic injury,the normal structures disappeared and much debris was observed from the terminal of proximal stump,axon was hardly founded.With the lasting of injury?2 weeks?,some myelinated axons were dotted on the background.In contrast to the case,there was no obvious difference about the number of axons from the 1 week injury only group to those treated with AMG-517 intervention.But the proximal stump with 2 weeks of AMG-517treatment manifested a dramatically regeneration of axons compared to its counter group?P<0.001?.At the same time,the sciatic axons in 2 weeks of AMG-517 treatment were well organized.4.There were a few karyocytes in the normal sciatic transverse section(the number of karyocyte-N_karyocytearyocyte was about 16±2).The quantization of karyocytes on sciatic transverse section showed the interstitial cellular response was notable obvious in each group of 1 week and that in the AMG-517 intervention groups was more remarkable(injury only group:N_karyocyte=61±3,AMG-517 150?g/kg:N_karyocyte=85±3,AMG-517300?g/kg:N_karyocyte=112±5,P<0.001),most of the cells seemed phagocytes.After 2weeks of sciatic injury,there was a remarkable decrease of cellular response from the nerve with AMG-517 treatment groups(injury only group:N_karyocyte=101±4,AMG-517150?g/kg:N_karyocyte=33±2,AMG-517 300?g/kg:N_karyocyte=24±2,P<0.001).Meanwhile,the number of phagocytes dropped,instead of Schwann cells.5.Results of immunofluorescence and western blot of GFAP showed that the expression of GFAP was obviously lower after 1 week of injury?P<0.001?,but was almost recovered after 2 weeks of injury.The expression of GFAP was obviously up-regulated by TRPV1 blockage using AMG-517?both 1 week and 2 weeks?.Ordinarily the expression of GAP-43 in normal sciatic nerve was very low and it was significantly increased aftere injury?P<0.01?.The intervention of AMG-517 could improve it's expression further?P<0.01?.Apart from this,the result of immunofluorescence showed that both GFAP and GAP-43 positive fibers from AMG-517 treatment groups formed buddle-like pattern,which seemed similar to the normal controls.Conclusion:1.The over-expression or over-activation of TRPV1,which caused by peripheral nerve injury,prevented nerve regeneration.2.Attenuating the over-expression or over-activation of TRPV1 can promote axon and Schwan cell regeneration,which were important facts in nerve repair after peripheral nerve injury.3.Attenuation of TRPV1 function facilitated the expression of GAP-43 in proximal stumps of sciatic nerves.