【作者】 王东生；

【导师】 路来金；

【作者基本信息】 吉林大学 ， 外科学， 2004， 博士

【摘要】 一、本研究的目的、意义及拟要解决的问题由于周围神经损伤后不能早期通过运动及感觉检查或电生理检查来判断伤情及术后恢复的程度，为此，一些作者通过周围神经轴突新生中本身代谢的特点探索了一些方法。在目前所进行的研究中，如何在形态学上较为直观地观察周围神经局部轴突运输的，国内外文献尚未见报导尚未见报导。同时不同程度的神经损伤，其轴突运输的恢复程度或时间是否有某种规律性尚不明确。生物素葡聚糖胺（BDA）是近年来所发现的一种非常有效的双向运输的神经示踪剂。本实验应用神经示踪剂BDA研究局部观察周围神经轴突运输的可行性，同时研究几种不同程度神经损伤修复后的示踪剂轴突运输的恢复程度及规律性，为神经损伤后的早期诊断和判断手术后的转归提供了一种确实有效的方法，同时为研究周围神经轴浆流的运输提供了一种方法学。二、材料和方法1．健康成年白兔4支随机分为2组，麻醉后显露左侧坐骨神经主干，然后分别于坐骨结节远侧2.0cm处用显微镊子给予急性压迫，另一组于坐骨结节远侧2.0cm处用1-0丝线结扎1分钟。两组动物分别于术后第二天取左侧损伤处神经标本进行坐骨神经损伤模型的大体观察及病理检查。 2．健康成年白兔24只随机分为6组，每组4只，麻醉后显露双侧坐骨神经，1至5组于坐骨结节远侧2cm处用微量注射器注射不同浓度、剂量的BDA及生理盐水，1、4、5组右侧坐骨神经于膝部坐骨神经分出腓总神经处近端切断，2、3组双侧切断，近侧断端用4-0线结扎。第6组右<WP=57>侧坐骨神经也于膝部切断，于近侧断端辗挫0.5cm，于断端进针注射试剂，进针深度为0.5cm。各组动物分别于术后6-48小时再次深麻醉、灌注固定。注毕立即取出标本，标本经漂洗、剪除鞘膜外结缔组织、后固定、移入30%蔗糖沉底后，每支神经相隔1cm跳跃性切取标本，标本冰冻切片。神经组织切片在阿维丁-辣根过氧化物酶溶液中孵育、在PBS液中漂洗，然后分别经 DAB显色及FITC标记，用于光镜及共聚焦显微镜观察。3．健康成年白兔50只按手术方法的不同随机分组，各组动物10只。动物麻醉后显露左侧坐骨神经、胫神经及腓总神经。然后，①于坐骨结节远侧2.0cm处用显微镊子给予急性压迫（1组）；②于坐骨结节远侧2.0cm处用1-0丝线结扎1分钟（2组）；③于坐骨结节远侧2.0cm处用显微剪刀剪断形成V度损伤，无创缝合神经外膜（3组）。④于坐骨结节远2.0cm处向远侧用显微剪刀剪取长度分别为0.5cm和1.0cm神经后原位缝合（4、5组）。分别于术后1、2、3、4、6周再次麻醉动物，显露左侧坐骨神经、胫神经及腓总神经，观察损伤神经其外径及色泽。左侧坐骨神经干自损伤处远侧3cm处于腓总神经分出之前切断并用4-0线双结扎，于神经损伤近侧2.0cm处分别注射10%BDA溶液1μl。分别于注射试剂12小时后深麻醉、灌注固定、取标本、后固定、移入蔗糖沉底。取标本冰冻切片，进一步处理后用于光镜下及共聚焦显微镜的观察。三、结果1．坐骨神经损伤模型：（1） 急性压迫损伤模型其损伤介于Sunderland I度至II度损伤之间。（2） 坐骨神经用丝线结扎者损伤介于Sunderland III度至IV度损伤之间。2．BDA用于正常周围神经局部轴突运输的观察：（1）光镜观察：BDA注射点标记阳性的神经纤维呈褐紫色标记，髓鞘染色，轴突亦染色，髓鞘与轴突界限明显。对照组无阳性标记的神经纤维。BDA注射点远侧及近侧1cm、2cm无明显的轴突阳性染色纤维。局部注射10%BDA之坐骨神经远端切断结扎处于注射试剂后6小时即可见阳性标记的神经纤维，其轴<WP=58>突着色明显。至48小时其观察结果类似。（2）共聚焦显微镜观察：在BDA注射点，示踪剂分布的范围位于轴突、髓鞘、神经被膜。对照组观察不到荧光标记的神经纤维。BDA注射点远侧及近侧1cm、2cm看不到轴突的荧光标记。注射10%BDA之坐骨神经，其远端切断结扎处注射BDA后6小时即可发现有荧光物质的积聚，主要位于轴突且标记明显。（3）BDA 注射浓度为0.1%的坐骨神经，无论是通过组织化学方法还是通过共聚焦显微镜的观察，均未发现有阳性标记的神经纤维。3．坐骨神经不同损伤后的观察：（1）大体观察：坐骨神经急性压迫组术后神经外形正常。丝线结扎组、V度损伤以及自体神经移植的坐骨神经损伤处稍粗大，远侧神经干略细，色泽及弹性正常。神经损伤处之神经干与周围组织有不同程度的组织粘连。（2）光镜观察：急性压迫损伤后一周时，在神经切断结扎近侧可见到标记阳性的神经纤维。说明此种损伤至少在损伤后一周时其轴突运输未受明显影响。其它神经损伤术后一周时，于损伤处可见到标记阳性的神经纤维，远侧切断结扎处未见阳性染色。说明伤后一周时示踪剂的轴突运输于损伤处中断，从而引起损伤处示踪剂的积聚。二周时，急性压迫损伤模型的情况同前，其它各组标本于损伤处近侧仍可看到标记阳性的神经纤维，同时神经移植组中于移植的坐骨神经内亦可观察到阳性标记，说明此处有示踪剂的积聚。坐骨神经远侧未发现阳性标记。第三周及以后，所有坐骨神经于远侧切断结扎处均能发现某一局部较密集阳性标记的神经纤维，一些标本损伤处仍有标记阳性的神经纤维。说明此时轴突运输已明确恢复，同时在原损伤处，部分神经纤维轴突运更多还原

【Abstract】 Biotinylated dextran amine(BDA) for observing the local axonal transport in rabbit sciatic nerve : An experimental studyBy now we can’t detect the regeneration quality in earlier period after peripheral nerve injury or therapeutic suture,and it’s responsible for poorly recovery of peripheral nerve. Lots of authors explore some methods involving immunohistochemistry technique and isotopic tracing base on the axonal metabolism behavior of peripheral nerve. However, all of these failed to study the axinal transport of peripheral nerve with different injury severity, and not provide methods to directly visualize the local axinal transport of peripheral nerve. Neural tracer is used in anterograde and retrograde neuroanatomical labelling to elucidate long range anatomical connections within the nervous system. But botinylated dextran, a versatile anterograde and retrograde neuronal tracer, Here we have used it in our experiment to investigate the possibility of evaluating the local axoplasm of peripheral nerve and the changes of axoplasm in traumatic injured sciatic nerve with different injury severity.I、The feasibility of biotinylated dextran amine(BDA) for observing the local axinal transport of peripheral nerveBDA was injected into the sciatic nerve trunk of 24 adult rabbit by a microsyringe with different concentration and different volume,and saline was injected into the matched control nerve. Sampling the sciatic nerve after a survival period of 6 to 48 hours and viewing the sample under the light microscope and confocal microscopy. Result1. Light microscopy :(1) BDA injected region: Following a diaminobenaidine reaction, positive labeled nerve fiber is stained with brown and purpl substance. Both myelin sheath and axis-cylinder were stained, though the boundary of myelin sheath and axis-cylinder is clear. No positive stained fiber can be detected in matched control nerve. (2) Positive stained <WP=61>fiber can be viewed after 6 hours following injection of 10% BDA solution in the proximal to the ligation of sciatic nerve. (3) No definite positive stain can be confirmed in the area proximal to the injection or the region between injection site and proximal to the ligation.2. Confocal microscopy:(1)The distribution of neural tracer is in axis-cylinder,myelin sheath and nerve capsule.(2)To compare the distribution of BDA in the injection region,it is a special pattern in the area proximal to the ligation,which the axis-cylinder was brightly labeled while the myelin sheath is fluorescenced weakly. (3)The accumulation of labeled substances could be detected after 6h following injection of BDA.(4)No definite fluorecence can be confirmed in the area proximal to the injection or the region between injection site and proximal to the ligation.3. No positive labeled nerve fiber could be viewed in the matched control nerve or the nerve injected with 0.1%BDA solution by way of either histochemistry technique(light microscopy) or confocal microscopy.Conclusion (1) The use of neural tracer BDA permits the directly identification of labeled axonal transport of sciatic nerve without difficult processing .(2)Instantaneously,the tracer volume is too minimal in one site of the peripheral nerve trunk to be detected until it reachs some certain extent of accumulation. II、The changes of axoplasm in injured sciatic nerve with different traumatic injury severity Fifty adult rabbit were divided into several experimental group with different injury of sciatic nerve trunk: nerve crush with forceps used in microsurgery,nerve ligation injury with 4-0 silk suture, transected injury,autoplastic transplantation.1,2,3,4,6 weeks after nerve injury,10%BDA solution was injected into the sciatic nerve trunk by a microsyringe proximal to the injury site.Sampling the sciatic nerve following pressure injection of BDA with survival time of 12h, and sections be directly examined undera microscope and confocal microscopy after some proces of sample. Result 1. Pathologiesof different in更多还原