在缝合基础上局部应用纤维蛋白胶对周围神经再生的影响

Local application of fibrin glue in peripheral nerve regeneration

目的:在缝合的基础上局部应用纤维蛋白胶治疗损伤的周围神经,观察纤维蛋白胶对周围神经再生的影响。方法:实验于2005-03/07在大连医科大学中心实验室完成。实验材料:纤维蛋白胶(广州倍绣生物技术有限公司,主要成分:纤维蛋白原50～70mg/支和凝血酶400U/支,从哺乳动物血中提纯,经过灭菌消毒,冻干制成,不含致热源)。实验分组:选择健康SD大鼠48只,按随机数字表法分为两组,每组24只:单纯缝合+纤维蛋白胶组、单纯缝合组。实验方法:大鼠麻醉后,于左大腿后外侧做2cm纵切口,显露坐骨神经。距梨状肌下缘远侧约1.5cm处切断坐骨神经,切除远端1～2mm,采用10-0无创伤线缝合神经外膜,使远近端保留约1～2mm间隙。单纯缝合+纤维蛋白胶组:对称缝合2针,将纤维蛋白胶注入缝合周围在神经对合端生成凝胶环,混合物固化形成再生室。单纯缝合组:单纯外膜缝合。实验评估:①术后连续观察动物行为学:手术侧后肢及足趾的运动情况,有无溃疡形成,足趾、趾甲的溃疡愈合情况,观察展爪反射。②术后8周两组各取4只大鼠行神经电生理检查,检测神经传导速度、潜伏期。③术后2,4,6,8周两组各取2只大鼠行苏木精-伊红染色后光镜下观察神经再生情况。④术后8周两组各取4只大鼠采用LUZEX-F彩色图像分析仪对甲苯胺蓝染色神经组织切片中轴突数目及轴突直径进行分析。⑤术后8周两组各取4只大鼠行醋酸铀枸橼酸铅染色,Phlip-10型透射电镜下观察轴突再生情况。⑥术后8周两组各取4只大鼠行辣根过氧化物酶标记观察脊髓前角运动神经元情况。结果:纳入大鼠48只,均进入结果分析。①术后大鼠行为学观察:术后8周单纯缝合+纤维蛋白胶组大鼠除足趾略见下垂、屈曲现象外,步态基本正常,展爪反射基本正常,下肢活动已接进正常,单纯缝合组下肢活动略差。②神经电生理检查:术后8周单纯缝合+纤维蛋白胶组神经传导速度快于单纯缝合组[分别为(11.13±0.37),(9.26±0.44)m/s],潜伏期短于单纯缝合组[分别为(1.83±0.18),(2.17±0.19)ms],差异有显著性意义(F=27.78,5.53,P<0.05)。③光镜下神经再生情况:单纯缝合+纤维蛋白胶组再生的有髓神经纤维髓鞘较厚、直径较大、数量多、排列规则,再生良好。单纯缝合组再生的有髓神经纤维髓鞘较薄、直径较小、数量少、排列不规则,再生较差。④轴突数目及轴突直径:单纯缝合+纤维蛋白胶组在轴突数目、轴突直径大于单纯缝合组[分别为(2187±107),(1847±96)个/400倍视野;(2.79±0.15),(2.05±0.17)μm],差异有显著性意义(F=80.70,42.92,P<0.05)。⑤透射电镜下轴突再生情况:术后8周单纯缝合+纤维蛋白胶组大鼠再生轴突发育良好,排列有序,轴突直径大小相差小,髓鞘厚薄一致,轴突染色均匀,雪旺细胞核呈卵圆型。单纯缝合组大鼠轴突发育差,排列不规则,髓鞘薄,可见扩张血管,部分区域有出血水肿。⑥辣根过氧化物酶标记观察脊髓前角运动神经元情况:单纯缝合+纤维蛋白胶组在实验侧腰骶段前角可见辣根过氧化物酶标记的大型运动神经元,且数目较多。单纯缝合组标记的数量较少。结论:在修复神经过程中应用纤维蛋白胶,可明显促进损伤的周围神经修复与再生,优于单纯缝合的效果。

AIM： To observe the effect of fibrin glue at local use on the regeneration of peripheral nerve based on the suture. METHODS： The experiment was conducted in the Central Laboratory of Dalian Medical University between March and July in 2005. Experimental materials： fibrin glue （Guangzhou Bioseal Biotech Co., Ltd., main component includes 50-70 mg/injection fibrinogen and 400 U/injection thrombin, purified from mammal blood, sterilized and freeze dried, without pyrogen）. A total of 48 SD rats were randomly assigned into suture group and combination of suture and fibrin glue group, with 24 rats in each. All the rats were anesthetized to expose sciatic nerve by a 2-cm incision posterior to lateral left thigh, and sciatic nerve was cut off from 1.5 cm distally piriformis muscle and epineurial neurorrhaphy was performed with 10-0 non-trauma thread, with the interval of 1-2 mm. Combination group： Fibrin glue was injected into peripheral muscle of 2 symmetric sutures to produce the gel ring, the mixture curing lead to regeneration division. Suture group： Epineurial neurorrhaphy was performed only.Animal ethology was observed continuously postoperation： movement of lateral hind limb and toes, ulceration, ulcer healing of toes and toe nail, reflection of unfold claws.Four rats from each group were selected for electrophysiologic study and detect the nerve conduction velocity and latency at 8 weeks postoperation. At 2, 4, 6, 8 weeks postoperation, two rats of each group stained with hematoxylin-eosin were observed under light microscope for nerve regeneration. Eight weeks postoperation, four rats of each group was adopted to analyze the number and diameter of axon in toluidine blue-stained nerve tissue slices by means of LUZEX-F color image analyzer, determine the axonal regeneration in slices that were stained with both lead citrate and uranyl acetate using Philip-10 transmission electron microscope, and assay the motor neuron in anterior cornu of medullar spinalis, which.was labeled with horseradish peroxidase （HRP）, respectively. RESULTS： All the 48 rats were involved in the result analysis.（1）Animal ethology： Except a little toe ptosis and flexion 8 weeks postoperation, the gait, reflection of unfold claws and activity of hind limb were found in the combination group, while those of suture group were fair. （2）Electrophysiologic analysis： At 8 weeks postoperation, the nerve conduction velocity of combination group was significantly higher and latency was shorter than that of the suture group [（11.13＋ 0.37）, （9.26＋0.44） m/s, （1.83＋0.18）, （2.17＋0.19） ms, F =27.78, 5.53, P 〈 0.05].（3）Neural regeneration under light microscope： Myelinated nerve fiber presented thick myelin sheath, long diameter, tight arrangement and good regeneration in the combination group, whereas unsatisfactory effect was found in the suture group.（4）Axonal number and diameter was more in the combination group than in the suture group [（2 187＋107）, （1 847＋96） axons/400 field of vision; （2.79＋0.15）, （2.05＋0.17）μm], with the significant difference （F =80.70, 42.92, P 〈 0.05）.（5）Axonal regeneration was observed under transmission electron microscope： At 8 weeks postoperation, the regenerative axon grew well and arranged ordedy in the combination group, additionally both the axonal diameter and myelin sheath thickness were identical, axons were evenly staining, Schwann cellular nucleus in oval shape was also presented. While in the suture group, axonal growth was different, their arrangement was in disorder, and the myelin sheath was thin, accompanying the vascular dilation and partly area of bleeding and edema. （6）HRP-labeled motor neuron in anterior cornu of medullar spinalis： HRP-labeled motor neurons were found in anterior cornu at a greater quantity in the combination group, whereas few in the suture group. CONCLUSION： Fibrin glue can obviously improve the repair and regeneration of injured peripheral nerve, and the effect is superior to that of simple suture.