神经节苷脂促进周围神经再生的实验

Ganglioside in promoting peripheral nerve regeneration

目的:观察神经节苷脂对冷冻处理后的异体周围神经移植再生的影响。方法:实验于2001-06/2003-12在广西医科大学中心实验室完成。选取健康成年新西兰大白兔36只,取4只作为供体,切取双侧坐骨神经,制成10mm神经段,深低温冷冻储存2周备用。其余32只作为受体,随机分为神经节苷脂组与正常对照组,16只/组,将兔左侧坐骨神经造成10mm长缺损,用复温后的同种异体神经进行移植桥接。神经节苷脂组每天每只局部注射质量浓度为1g/L的神经节苷脂溶液1mL,连续14d;正常对照组不做任何处理。两组分别于术后2,4,8,12周随机取4只进行电生理检查、组织学观察、超微结构观察、肌肉湿重测定以及形态学定量分析。结果:作为受体的32只兔全部进入结果分析,中途无脱落。①两组兔术后大体观察比较:正常对照组均有足底及足趾溃疡,神经节苷脂组有6只发生足跟溃疡。两组动物左后肢均无力,行走不稳。②两组兔术后不同时期电生理检测结果比较:与正常对照组传导速度、电位波幅比较,术后2,4,8周神经节苷脂组基本无变化(P>0.05),术后12周明显升高[(28.62±2.42),(21.78±2.43)m/s;(5.48±0.36),(4.67±0.53)mV;P均<0.05]。③两组兔术后不同时期组织学观察结果比较:术后2周,正常对照组有髓神经纤维髓鞘崩解,许旺细胞增生不明显;神经节苷脂组崩解反应较正常对照组慢,许旺细胞增生明显。术后4周,正常对照组许旺细胞增生活性低,有髓神经纤维密度低;神经节苷脂组许旺细胞增生活跃,有髓神经纤维密度较大;术后12周,正常对照组有髓神经纤维数量少,神经束膜间毛细血管增生明显,神经纤维瘢痕化明显;神经节苷脂组有髓神经纤维数量较多,有少许神经纤维瘢痕化,神经束膜间毛细血管清晰。④两组兔术后12周超微结构观察比较:正常对照组可见结缔组织无定形结构中夹杂少量变性神经纤维,轴突电子密度低且有空泡,许旺细胞细胞器及有髓轴突稀少,部分再生轴突及髓鞘发生wallerian变性脱髓;神经节苷脂组可见大量神经纤维髓鞘增厚和)旺氏细胞细胞器更丰富,轴突电子密度深且均匀,许旺细胞外有完整基膜包绕,变性神经纤维少。⑤两组兔术后不同时期肌肉湿重Guadros指数的测定:术后4,8,12周,神经节苷脂组均明显高于正常对照组(P均<0.05),表明正常对照组肌萎缩较重。⑥两组兔术后不同时期再生神经纤维各项指标测定结果比较:与正常对照组有髓神经数目、纤维直径、髓鞘厚度及轴突直径比较,术后2,4,8周神经节苷脂组基本无变化(P>0.05),术后12周均明显高于正常对照组[(2183.6±184.3),(1520.2±124.3)个/400倍视野;(16.45±1.86),(10.08±1.62)μm;(6.48±0.72),(4.72±0.56)μm;(4.40±0.42),(3.04±0.34)μm;P均<0.05]。结论:再生神经能够顺利地通过移植体进入远端,使再生神经传导速度、有髓神经纤维数目、肌湿重等指标明显升高,证明兔同种异体神经经冷冻处理后桥接神经缺损可引导神经纤维再生,且外源性神经节苷脂可促进异体移植周围神经的再生。

AIM：To observe the effects of ganglioside on the regeneration of peripheral nerve allografts after cryopreservation in rabbits. METHODS：The experiment was carried out in the central laboratory of Guangxi MeMcal University between June 2001 and December 2003. Thirty-six health adult New Zealand white rabbits were used in this study. Four rabbits were selected as donors. Bilateral sciatic nerves were taken out from the rabbits and cryopreserved for 2 weeks. The other 32 rabbits as recipients were randomly divided into ganghoside group （n=16） and normal control group （n=16）. For every rabbit, 10-mm defect was created in left sciatic nerve, the nerve allograft for bridging the defect of peripheral nerves was done after rapid thawing. In the ganglioside group, the rabbits were treated with local injection of 1 mL ganglioside （1 g/L） for 14 continuous days. Not any treatment was given to the rabbits in the normal control group. Four rabbits in each group were chosen randomly at 2, 4, 8 and 12 weeks after operation for electrophysiological test, histological and ultrastructural observations,muscular wet mass detection and morphological quantitative analysis. RESULTS： All the 32 rabbits as recipients were involved in the analysis of results without deletion midway. ① Comparison of postoperative gross observation between the two groups： There were ulcers of soles and toes in all the rabbits in the normal control group. Ulcer of heels occurred in 6 rabbits of the ganglioside group. All the rabbits had asthenia of left hindlimb and unstable walking. ② Comparison of electrophysiological test at different postoperative points between the two groups： Compared with the normal control group, the conduction velocity and wave ampJitude of potential in the ganglioside group at postoperative 2, 4 and 8 weeks had no changes （P〉0.05）, and those at postoperative 12 weeks were obviously increased [（28.62±2.42）, （21.78±2.43） m/s; （5.48±0.36）, （4.67±0.53） mV; P〈0.05]. ③Comparison of histological observation at different postoperative points between the two groups： At 2 weeks,the myeline of myelinated nerve fiber collapsed and the proliferation of Schwann＇s cells was not obvious in the normal control group; and the collapse reaction in the ganglioside group was slower and the proliferation of Schwann＇s cells was more obvious as compared with those in the normal control group. At 4 weeks, the proliferative activity of Schwann＇s cells was low and the density of myelinated nerve was low in the normal control group; the proliferation of Schwann＇s cells was active and the density of myelinated nerve was greater in the ganglioside group. At 12 weeks, there were a few myelinated nerves in the normal control group, the proliferation of blood capillary among nerve fascicles was obvious, and cicatrization of nerve fiber was obvious; there were more myelinated nerves in the ganglioside group, a little cicatrization of nerve fiber, and clear blood capillary among nerve fascieles.④Comparison of uhrastructural observation at 12 weeks between the two groups： In the normal control group, a small amount of degenerated nerve fibers could be observed in the unfixed form structure of connective tissue, electron density of axis-cylinder was low and there were vacuoles,the Schwann＇s cells organell and myelinated axiscylinder were rare,wallerian degenerated demyelination occurred in part of the regenerated axis-cylinder and myelin sheath. In the ganglioside group, thickening of plenty of nerve fiber myelin sheath and richer organell of Schwann＇s cells were observed,electron density of axis-cylinder was deep and even, Schwann＇s cells were wrapped by complete basal membrane, there were fewer degenerated nerve fiber.⑤ Detection of muscular wet mass Guadros index at different postoperative points in both groups： At 4、8 and 12 weeks, it was obviously higher in the ganglioside group than in the normal control group （P〈0.05）, indicating that myatrophy was severer in the normal control group. ⑥ Comparison of the indexes of regenerated nerve fiber at different postoperative points between the two groups： Compared with the normal control group, the number of myelinated nerve, fiber diameter, myelin thickness and axis-cylinder diameter in the ganglioside group had no changes at 2, 4 and 8 weeks （P〉0.05）, but obviously higher at 12 weeks[（2183.6±184.3）, （1520.2±124.3）/400 power visual sight; （16.45±1.86）, （10.08±1.62）μm; （6.48±0.72）, （4.72±0.56）μm; （4.40±0.42）, （3.04±0.34）μm; P all〈0.05]. CONCLUSION： The regenerated nerves can smoothly enter the distal end through allograft, and obviously increase the regenerated nerve conduction velocity, number of myelinated nerve and muscular wet mass, it is suggested that allografted nerve bridged neurologic deficit after cryopreservation can induce the regeneration of nerve fiber in rabbits, and the exogenous ganglioside can accelerate the regeneration of allograft peripheral nerves.