大鼠陈旧性坐骨神经缺损修复期限的实验研究

TIME LIMIT OF REPAIRING OLD SCIATIC NERVE DEFECT IN RATS

目的探讨大鼠陈旧性坐骨神经缺损的修复期限,观察自体神经移植对大鼠陈旧性坐骨神经缺损的修复作用。方法清洁级SD大鼠36只,随机分为6组,每组6只。切除大鼠左侧部分坐骨神经,制作神经缺损模型。神经损伤后1、3、6个月用对侧自体神经修复神经缺损作为A1、B1、C1组,不予修复作为对照A2、B2、C2组。术后每周观察步态、足趾皮肤及腿部肌肉的变化。第2次术后3个月,进行电生理检查、荧光金(fluorogold,FG)逆行示踪、腓肠肌Masson染色、再生神经亮绿-变色酸2R-磷钨酸法染色与透射电镜观察。结果神经损伤后各组大鼠均有足部溃疡、跛行等失神经表现,修复术后2个月,A1、B1组溃疡愈合,跛行改善,其余各组仍有溃疡、跛行。修复术后3个月,A1、B1、C1组的运动神经传导速度分别为(21.84±6.74)、(20.02±4.17)、(16.09±8.21)m/s,组间差异无统计学意义(P>0.05)。复合肌肉动作电位(compound muscle actionpotential,CAMP)波幅分别为(12.68±4.38)、(9.20±3.43)、(1.22±0.39)mV,A1、B1组与C1组比较差异有统计学意义(P<0.05);A2、B2、C2组未记录到CAMP。FG逆行示踪观察,A1组阳性细胞最多,胞体较大,B1组居中,C1组阳性细胞最少,胞体最小。腓肠肌Masson染色示A1、B1组腓肠肌纤维形态接近正常,C1、A2、B2、C2组肌纤维明显萎缩。A1、B1、C1组肌纤维横截面积为(340.73±118.46)、(299.88±119.75)和(54.33±53.43)μm2,A2、B2、C2组为(78.60±51.38)、(65.62±25.36)和(40.93±28.22)μm2。A1、B1组与C1、A2、B2组比较差异均有统计学意义(P<0.05)。神经形态观察,A1组再生有髓神经纤维数量多,直径粗,髓鞘厚;B1组再生神经纤维的数量和形态与A1组相似;C1组中再生的有髓神经纤维数量少,纤维细,髓鞘薄;A2、B2、C2组则仅见SC及增生的胶原纤维。结论自体神经移植能不同程度修复缺损1个月和3个月的大鼠坐骨神经,但对缺损6个月的大鼠坐骨神经修复作用不明显。

Objective To investigate the time limit of repairing old sciatic nerve defect in rats and observe the repair effect of autogenous nerve transplantation on old sciatic nerve defect in rats. Methods Thirty-six SD rats of clean grade were randomized into 6 groups （n=6 per group）. The animal model of nerve defect was made by transecting left sciatic nerve at the mid-thigh level. For groups AI, B1 and C1, defects were repaired by the contralateral autogenous nerve transplantation 1, 3 or 6 months after nerve damage and for the control groups of A2, B2 and C2, defects were not repaired. After operation, the gait, toe skin and leg muscle were examined weekly. Three months after autograft, a combination of electrophysiology examination, fluoro gold （FG） retrograde tracing and histological assessment including light microscopy, TEM was utilized to investigate the nerve functional recovery. Results Lameness and foot skin ulcers were observed in each group after nerve damage. At 2 months after autograft, such denervation symptoms were only improved in groups A1 and B1. At 3 months after autograft, the motor conduction velocity was （21.84 ± 6.74）, （20.02 ± 4.17） and （16.09 ± 8.21） m/s in groups A1, B1 and C1, respectively, showing no statistically significant difference between them （P 〉 0.05）. The amplitude of compound muscle action potential （CAMP） was （12.68 ± 4.38）, （9.20 ± 3.43） and （1.22 ± 0.39） mV in groups A1, B1 and C1, respectively, indicating significant differences between groups AI, B1 and group C1 （P 〈 0.05）. No CAMP was evident in groups A2, B2 and C2. FG retrograde tracing conducted 3 months after autograft showed that the positive cells were most common in group A1 with big soma, mild in group B1 and lest in group C1 with smallest soma. Gastrocnemius Masson staining showed that the fiber morphology of gastrocnemius in groups A1 and B1 was close to normal, while the rest 4 groups had an obvious atrophy of muscle fiber. The fiber cross-section area was （340.73 ± 118.46）, （299.88 ± 119.75）, （54.33 ± 53.43）, （78.60 ± 51.38）, （65.62 ± 25.36）, and （40.93 ±28.22） um^2 in groups A1, B 1, C1, A2, B2 and C2, respectively, indicating a significant difference between groups A1, B1 and groups C1, A2, B2 （P 〈 0.05）. Neurohistology observation showed that more regenerated nerve fibers were observed in group A1 and B1, but less in group C1. The myelin sheath was thick in groups A1 and B1, while it was thin in group C1. Only SCs and hyperplastic collagen fiber were found in groups A2, B2 and C2. Conclusion Autogenous nerve transplantation is capable of repairing 1- and 3- month sciatic nerve defect to some degree in rat, but repair effect is not obvious on 6-month sciatic nerve defect in rats.