川芎嗪浓度对大鼠异体坐骨神经玻璃化保存后神经再生影响的实验研究

EFFECT OF TETRAMETHYLPYRAZINE ADDED TO VITRIFICATION SOLUTION ON PERIPHERAL NERVE ALLOGRAFTS REGENERATION

目的探讨不同浓度川芎嗪(tetramethylpyrazine,TMP)对玻璃化保存的大鼠异体坐骨神经再生的影响。方法取3月龄雄性清洁级成年健康SD大鼠48只,体重200～250g,作为供体;3月龄雄性清洁级成年Wistar大鼠96只,体重200～250g,作为受体。取供体动物,切取双侧坐骨神经,制成15mm神经段,按玻璃化保存液中TMP浓度不同随机分为A、B、C、D组,A组不含TMP,作为对照,B、C、D组玻璃化液中分别含100、200、400mg/LTMP,作为实验组。—196℃液氮保存3周。取受体动物,制备右侧坐骨神经10mm缺损模型,随机分为4组(n=24)。取上述保存神经段复温后移植至相应组的受体。术后4、8、12周行大体观察,术后16周取移植神经行透射电镜、电生理、轴突图像分析等检查。结果术后大鼠均存活至实验完成。术后4周A、B组移植神经与周围组织粘连最严重;术后8周A组粘连最严重,其余各组逐渐减轻;术后12周A组仍有粘连,B组与周围组织部分粘连,C、D组无粘连。术后16周,A、B组再生神经远端肌肉复合动作电位潜伏期及波幅、运动神经传导速度、单位面积髓鞘数、平均灰度值、单位面积髓鞘密度值及运动终板检测,均显著低于C、D组(P<0.01),A、B组间及C、D组间差异无统计学意义(P>0.05)。透射电镜示C、D组有髓神经纤维粗,髓鞘较A、B组厚,板层结构清晰。结论坐骨神经玻璃化保存液中加入200mg/LTMP可改善大鼠异体神经再生效果。

Objective To investigate the effect of tetramethylpyrazine （TMP） with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration. Methods Forty-eight healthy clean SD male rats were selected as donors, and 96 healthy clean Wistar male rats as recipients, all rats being 3 months old and weighing 200-250 g. The sciatic nerves segments of 15 mm were removed from the donors, then randomly divided into 4 groups according to vitrification solution containing TMP. No TMP was used in group A as the control group; 100 mg/L, 200 mg/L and 400 mg/L TMP were used in group B, group C and group D, respectively. Then them were cryo-preserved at - 196℃ for 3 weeks. Nerve defect of 10 mm in length was made in the sciatic nerves of recipients. After rewarming, the allografts were transplanted to the corresponding rats. The gross appearance, the morphological and electrophysiological changes, the image analysis of axons and motor end-plate were detected at 4, 8, 12 and 16 weeks. Results All rates survived to the end of the experiment. The adhesion and edema of allografts in group A and group B were obvious 4 weeks after operation; then adhesion and edema was obvious in group A and were improved in the other groups 8 weeks after operation. Adhesion was observed in groups A and B; no adhesion was observed in groups C and D at 12 weeks. The number of regeneration nerve, the latent, the amplitude, the nerve conduction velocity, the medullary sheath/μm^2, the medullary sheath density/μm^2 and the image analysis of axons and motor end-plate in groups A and B were significantly lower than those in groups C and D （P 〈 0.01）; and there were no significant differences between groups C and D （P 〉 0.05）. The observation of transmission electron microscope showed that medullated nerve fibers and myelin sheath of groups C and D were thicker than groups A and B, layers of groups C and D were clear. Conclusion The vitrification solution with 200 mg/L tetramethylpyrazine has protective effect on regeneration of peri pheral nerve allografls.