生物膜处理神经吻合口促进神经再生的实验

Effect of biofilm-embedded nerve anastomosis site in improving nerve regeneration

目的:观察生物膜防止神经粘连的作用并与静脉、己丁糖、强的松龙以及单纯缝合进行对比,探索促进神经再生的方法。方法:实验于2002-04/07在解放军总医院骨科实验室完成。将新西兰大白兔随机分为5组:生物膜组、己丁糖组、强的松龙组、静脉组及单纯缝合组,每组12只。①模型制作:采用250g/L乌拉坦静脉麻醉,显露兔的双侧坐骨神经,并在胫骨外髁上方1cm处切断胫神经,双侧作同样处理。在手术显微镜下,将胫神经端端吻合,并将吻合口周围肌肉作适当损伤以形成瘢痕。②分组处理:生物膜组:生物膜片包裹神经吻合口,并将两端缝合成管状,生物膜管两端各与神经外膜固定1针。己丁糖组:在吻合口周围涂抹0.5mL己丁糖。强的松龙组:在吻合口周围注射0.5mL强的松龙。静脉组:取一段兔颈外静脉,长约2cm,分为两段,分别套在双侧胫神经上,两端各固定1针。单纯缝合组:直接吻合后放入肌间隙,吻合口不作特殊处理。③指标测试:于术后2,4,10和16周用肉眼观察足底溃疡及愈合情况,神经吻合口的粘连程度;取各时间段神经吻合口上下5mm神经,光镜观察再生神经通过吻合口情况及结缔组织增生情况;取吻合口近远端各2mm长神经,用伪彩色图像分析行轴突数目分析;对16周组,用诱发电位仪检测其神经传导速度、诱发电位波幅及潜伏期;在透射电镜下观察其超微结构特征。结果:每组12只新西兰白兔均纳入结果分析。①大体观察:单纯缝合组修复段神经2周时为瘢痕组织包裹压迫固定,4和10周时粘连加重,神经无活动度。静脉包裹组修复段神经2周时与周围界限清楚,4,10和16周时与周围粘连固定,无活动度。生物膜组、己丁糖组及强的松龙组各时间段神经活动不受限。②组织学检查:生物膜组、己丁糖组和强的松龙组再生纤维通过吻合口较直,神经内无明显结缔组织增生。单纯缝合组神经外膜较厚,吻合口处结缔组织增生明显,神经再生纤维通过障碍。静脉包裹组神经外膜与分界短期内基本清楚,但长期组有静脉壁坏死的表现,吻合口周围有较多结缔组织增生。③电生理检测结果:静脉组及单纯缝合组的神经传导速度和潜伏期的恢复程度低于生物膜组、己丁糖组和强的松龙组(P<0.01);诱发电位波幅恢复率各组间无显著性差异。④轴突图像分析结果:术后16周,单纯缝合组及静脉组与其他3组比较有髓纤维再生率明显较低(P<0.01)。⑤透射电镜观察结果:16周时吻合口远端单纯缝合组有髓纤维髓鞘较薄,轴突直径较细,但各组神经远端轴突及髓鞘均较成熟,可见大量再生无髓纤维。结论:术中使用生物膜、己丁糖、强的松龙处理神经吻合口,能有效地防止周围神经粘连,促进周围神经再生,最大限度地恢复周围神经的功能。

AIM： To investigate the effect of biofilm in preventing scar adhesion after nerve injury, and compare the above effects with effects of vein, chitosan, prednisolone and simple anastomosis so as to probe into a new method to improve nerve regeneration. METHODS： The experiment was fulfilled in Department of Orthopaedics, General Hospital of Chinese PLA from April to July 2002. Sixty New Zealand big white rabbits were randomized into biofilm, chitosan, prednisolone, vein and simple anastomosis groups with 12 rabbits in each group. ①Modeling： The rabbits were anesthetized by 250 g/L urethane to expose the bilateral sciatic nerves; The tibial nerve was cut 1cm above lateral tibial condyle. The operation was same in both sides. The tibial nerve was anastomosed end-to-end under a surgical microscope, and the muscle around the anastomosed site was injured properly to induce scar. ②Treatment according to grouping： For the biofilm group, the anastomosis site was embedded by the biofilm and was sutured to a conduit, and thenboth sides were fixed to the epineurium by a needle. For the chitisan group, chitosan 0.5 mL was daubed around the anastomosed site. For the prednisolone group, prednisolone 0.5 mL was injected into the anastomosed site. For the vein group, rabbit external jugular vein about 2 cm was divided into 2 pieces. Each vein was knotted to one side tibial nerve and fixed by a needle. For the simple anastomosis group, the anastomosed site was not treated. ③Test of indexes： Plantar ulcer, healing and nerve adhesion degree were observed by naked eyes at 2, 4, 10, 16 weeks after operation. Nerve 5 mm of the upper and lower anastomosed site was obtained. The regenerative nerve fiber through the anastomoscd site and connective tissue hyperplasia were observed by light microscopy. Nerve 2 mm in length of the distal and proximal anastomosed site was analyzed by counting axons using the pseudo-color image analyzing system. Nerve conduction velocity （NCV）, evoked potential amplitude and incubation period were detected at 16 weeks after operation, and uhrastrueture was observed by transmission electron microscopy. RESULTS： All the 60 rabbits were analyzed in the resull. ①Gross observation： In the simple anastomosis group, the anastomosed site was embedded and fixed by scar at 2 weeks, became more serious at 4 and 10 weeks, and the nerve had no range of motion（ROM）. In the vein group, the anastomosed nerve had clear margin at 2 weeks, however fixed by scar at 4, 10, 16 weeks and had no ROM. Biofilm, chitosan and prednisolone groups were not limited at any time. ② Histological observation： The regenerative nerve fibers went through the anastomosed site straightly in biofilm, chitosan and prednisolone groups and no obvious scar was found in nerve. The regenerative nerve fibers were hindered hy the scar, connective tissue hyperplasia was obvious and epineurium was thicker in simple anastomosis group. In the vein group, nerve bound was clear in short time, however vein wall was necrotic and more sear was observed around anastomosed site in long time. ③Electrophysiological examination： The NCV was slower and the recovery of incubation period was shorter in the vein and simple anastomosis groups than in the biofilm, chitosan and prednisolone groups （P 〈 0.01）, however, the evoked potential amplitude had no difference among the above groups. ④Image analysis of axons： Medullated nerve fiber regeneration rate of simple anastomosis and vein groups were lower than the other three groups （P 〈 0.01）. ⑤Results of transmission electron microscopy： The medullary sheath of medullated nerve and the diameter of axon were thinner in simple anastomosis groupat 16 weeks. However, the axons and medullar sheaths of distal nerve were mature, and a lot of regenerative non-medullated nerve fibers were observed in all the groups. CONCLUSION： The biofilm, chitosan and prednisolone used intraoperatively can prevent nerve adhesion effectively, improve peripheral nerve regeneration, and recover the nerve function to the most extent.