| Background:After the body's tissue injury, there are only two kinds of prothetic forms: regeneration and scar repair. These two forms both existed when peripheral nerves were injuried: several proximal ranvier's nodes and distal nerve fibre occured Wallerian degeneration at the injuried location and axons passed the cell bridges composed of Schwann cell through budding and formed complete regeneration. While perilemma,perineurium and endoneurium and so on occurred scar repair, that is, focal necrotic tissue and other corpus alienum were dissolved and absorbed through the hyperplasia and filling of granulation tissue. And later granulation tissue translated into scar tissue mainly composed of collagen fibres and scar repairing was finished. Many studies confirmed that regeneration capacity of nerve fiber were very stubborn, but formation of scar in the course of repair of nerve supporting structure obviously hinder the recovery of nerve function. Exterior and interior scholars investigated the repair following injury of peripheral nerves. But they redundantly pay close attention to promotional factor for nerve fiber and sheath and so on and ignored the rejection capability study of scar repair——repair form of nerve supporting structure which account the 50～70% of the cross section area. This may be the bottleneck resulting in our now research dilemma. In recent years, researchers found that the calcium channel blocking agent may affect the synthesis and metabolismof extracellular matrix through regulating Ca2+ concentration of the fibroblast and even affect the synthesis and secretion of collagen. In clinical practice, prevention and therapy on metabolism abnormity of xtracellular matrix gained good results, for example fibration,scleroderma,hyperplastic scar and past-operation adherence. Then whether calcium channel blocking agent can inhibit scarring after peripheral nerve injury? This experiment used common model of scar and peripheral nerve injury and designed correlated experiment to try explaining the question.Objective: To observe the effect of verapamil on secreting the collagen protein of fibroblast in vivo and vitro experiment and initially investigate the mechanism of action on inhibiting nerve scar.Methods and Results:1. Culture in vitro and observation on nerve scar fibroblast in ratsTo establish the model of sciatic nerve injury and take nerve scar at injuried location after two weeks. According to the tissue block method and repeated differential adherent method nerve scar fibroblasts were taken and further dissociated and purified. We choosed the 4～8 generation fibroblasts with good liveness and passage and prepared the verapamil condition medium with different density to interfere in fibroblasts. We observed cell generation by MTT and calculated half inhibition ratio. And we drew cell growth curve by cytometry, measured the cell cycle by flow cytometer, observed the cell cytotoxicity by lactate dehydrogenase, observed cell morphocytology and microstructure by Gimesa,SEM and TEM, measured collagen content in cell supernatant and endochylema by hydroxyproline chromatometry, and statistically analyzed the datum.Results showed:Verapamil may inhibit fibroblast multiplication and the effect was dose dependent. And it can prolong detention period of fibroblasts, shorten logarithmic growth phase and obviously slowed down cell multiplication. From lactate dehydrogenase we observed that Vap was not harmful to fibroblasts at certain density, but Vap may be harmful to fibroblasts at high density. The results of flow cytometry showed that 100 and 200μmol/L groups may make G0/G1 of fibroblasts standstill and S decreased. Giemsa and SEM manifested that Vap may make fibroblast rounder and make intracytoplasm ves more and prominency smaller. TEM disclosed verapamil can make rough endoplasmic reticulum decrease and ves. increase in fibroblast. And hydroxyproline chromatometry showed Vap may make decrease tropocollagen into outside of cell and make it into the endochylema.We observed the form,hyperplasy and function of fibroblasts in vitro and scarring,disposition of blood vessel,deposit ofâ… ,â…¢collagen,regeneration of medullated nerve fibers and nerve functional recovery after injury of sciatic nerve in vivo and carried out the image and statistic analyses.Vivo results showed Vap did not increase animal death rate in experimental phase. After 12 weeks rats, unfold claw reflex gradually recoveried and toe function partly recoveried in Vap group, while that was bad in control group. In addition, Vap may affect the skin healing. At the same time, there were less adhesion,better dialyneury,more blood vessel and branches,coincident caliber and alignment in Vap group.â… ,â…¢collagen manifested there were more obvious collagen hyperplasia,nerve sparseness,twisting with hyperplasia fibres and many vacuolar degeneration in control group. And TEM and toluidine blue showed medullated nerve fibers was more and even and sheath degeneration was less in Vap group. Collagen assaying manifested there was higher protein level in control group. And SFI and NCV showed nerve function of Vap group was superior to that of control group.Conclusions:1.we successfully dissociated nerve scar and make homogeneous,active and stable cells; Verapamil may inhibit fibroblast multiplication,affect cell growth cycle,decrease excretion of collagen protein in ecto-cell and make fibroblasts globular which was dose dependent in certain range;2.Nerve scar model was successful. And after verapamil interference there were diminished nerve stoma, more blood vessel, diminished deposit ofâ… ,â…¢collagen, less sheath degeneration and more medullated nerve fibers which had better functional recovery in Vap group.
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