脱细胞异体神经支架预防创伤性痛性神经瘤的实验研究

Experimental study of acellular nerve allograft scaffold in the prevention of traumatic painful neuroma

目的探讨脱细胞异体神经支架(acellular nerve allograft scaffold, ANAS)对创伤性神经瘤的形成及疼痛的影响。方法以SD大鼠坐骨神经为来源, 采用化学萃取方法制备ANAS, 通过HE和髓鞘染色检测评估ANAS脱细胞情况。取健康SD雌性大鼠20只, 随机分为对照组和ANAS组, 每组10只。对照组大鼠坐骨神经切断后, 无特殊处理;ANAS组大鼠坐骨神经切断后, 近侧残端连接长度为2 cm的ANAS。术后定期观察并评估两组大鼠失神经后自噬、神经瘤形成、神经纤维的组织形态学变化、免疫组化观察到的神经生长因子表达情况等。结果处理后的ANAS通过HE染色, 光镜下见神经轴突和髓鞘消失, 未见雪旺细胞核;髓鞘LFB染色脱细胞神经髓鞘消失, 无细胞核结构。术后自噬评分ANAS组明显低于对照组, 差异有统计学意义(P<0.05)。HE染色光镜下观察, ANAS组神经纤维排列规则, 结缔组织增生不明显;对照组神经纤维排列紊乱, 结缔组织增生明显, 发生神经瘤例数多, 差异有统计学意义(P<0.05)。甲苯胺蓝染色有髓神经纤维数量, ANAS组与对照组相比, 显著减少(P<0.01)。透视电镜下对照组可见大量紊乱排列的成纤维及胶原纤维细胞、无髓神经纤维, 少量杂乱密度不一的髓鞘;ANAS组可见髓鞘进一步成熟, 成纤维及胶原纤维细胞排列相对规整。免疫组化结果显示, 术后两组均有神经生长因子表达, ANAS组明显低于对照组, 均随着时间递减(P<0.01)。结论 ANAS能很好地预防创伤性神经瘤形成及疼痛的发生。

Objective To investigate the effect of acellular nerve allograft scaffold(ANAS)on the formation and pain of traumatic neuroma.MethodsANAS were prepared by chemical extraction from the sciatic nerve of SD rats.The acellular status of ANAS was evaluated by HE and myelin staining.Twenty healthy female SD rats were randomly divided into control group and ANAS group,with 10 rats in each group.There was no special treatment after sciatic nerve transection in the control group.The proximal stump of rats in ANAS group was connected with ANAS with a length of 2 cm after sciatic nerve transection.The autophagy,neuroma formation,histomorphological changes of nerve fibers and the expression of nerve growth factor(NGF)observed by immunohistochemistry were observed and evaluated regularly after denervation.ResultsUnder light microscope,the treated ANAS showed the disappearance of axons and myelin sheath by HE staining and there was no Schwann nucleus.Myelin LFB staining showed that the myelin sheath of acellular nerve disappeared and there was no nuclear structure.The postoperative autophagy score of ANAS group was significantly lower than that of the control group,and the dference was statistically significant(P<0.05).Under light microscope,the arrangement of nerve fibers in ANAS group was regular,and the proliferation of connective tissue was not obvious by HE staining.In the control group,the arrangement of nerve fibers was disordered,the proliferation of connective tissue was obvious,the number of neuromas was more,and the difference was statistically significant(P<0.05).Compared with the control group,the number of myelinated nerve fibers stained with toluidine blue in ANAS group was significantly reduced(P<O.01).A large number of disorderly arranged fibroblasts and collagen fiber cells,unmyelinated nerve fibers and a small amount of disordered myelin sheath with different density were observed in the control group under fluoroscopy electron microscope.In ANAS group,myelin sheath was further mature,and fibroblasts and collagen fibroblasts were arranged relatively regularly.Immunohistochemical results showed that the expression of NGF was significantly lower in the ANAS group than in the control group,and the expression of NGF of both groups decreased with time.ConclusionANAS can prevent the formation of traumatic neuroma and the occurrence of pain.