犬化学去细胞神经同种异体移植的实验研究

目的以化学去细胞同种坐骨神经移植修复犬坐骨神经的长段缺损，观察其功能恢复及神经再生。方法15犬分成去细胞同种神经组(实验组)6犬、自体神经组(对照组Ⅰ)6犬、新鲜同种神经组(对照组Ⅱ)3犬。右侧坐骨神经造成5．0cm长缺损，以上述三种移植物桥接修复。术后6个月行步态分析、神经电生理及神经再生观察。结果实验组和对照组Ⅰ在运动功能恢复，踝关节运动步态，小腿二头肌运动诱发电位、感觉诱发电位，移植段内新生轴突、血管及雪旺细胞，远端胫神经内有髓神经纤维及靶肌肉运动终板等方面非常相似。对照组Ⅱ神经功能始终无恢复，移植段被吸收。结论化学去细胞同种神经移植物修复犬粗大长段神经缺损时不会被宿主排斥和吸收，其近期功能恢复及神经再生与自体神经移植无明显差别。     

犬化学去细胞神经同种异体移植的早期观察

目的：以化学去细胞同种异体神经，移植修复犬粗大神经的长段缺损，观察早期功能恢复及神经再生。方法：去细胞神经移植组和自体神经移植组各3犬，分别桥接坐骨神经5.0cm缺损。术后3个月观察其运动功能及神经再生。结果：实验组和对照组在术后功能恢复；移植段内新生神经纤维、新生血管及许旺细胞；吻合口远端有髓神经纤维等方面非常相似。结论：化学去细胞神经作为同种异体神经移植物，在修复粗大和长段神经缺损时不会被宿主排斥和吸收，其早期功能恢复及神经再生与自体神经移植无明显差别。     

犬化学去细胞神经同种异体移植的神经再生研究

目的 观察犬去细胞异体神经移植后近期神经再生的情况。谅15只家犬分成实验组（去细胞神经移植组，6只犬），对照组I（自体神经移植组，6只犬），对照组Ⅱ（新鲜异体神经移植组，3只犬），制成犬坐骨神经缺损5.0cm的模型，以上述3种神经移植物桥接修复。术后6个月行神经再生的组织学观察。结果 实验组移植段内有大量的新生神经纤维，新生血管及雪旺细胞；远端胫神经内出现大量的有髓神经纤维；靶肌肉运动终板的数量，形态及分布均良好。实验组和对照组I非常相似。结论 化学去细胞神经作为同种异体神经移植物，在修复术的粗大和长段神经缺损时不会被宿主排斥和吸收，其神经再生与自体神经移植无明显差别。     

去细胞异种神经复合神经生长因子修复神经缺损的实验研究

目的 应用含有神经生长因子(NGF)的去细胞异种神经基膜管作为神经移植替代物桥接大鼠坐骨神经缺损,观察其对神经再生的作用.方法 选用Wistar大鼠45只,随机分为3组,每组15只,于术制成右后肢坐骨神经长10 mm的神经缺损,取兔胫神经制成去细胞神经基膜管,电镜及HE染色观察神经基膜管超微结构,流式细胞仪检测去细胞前后神经主要组织相容性抗原Ⅱ(MHC Ⅱ)的变化情况.A组以含有NGF的去细胞异种神经基膜管桥接神经缺损,B组单纯采用去细胞异种神经基膜管桥接神经缺损,C组采用自体神经移植修复神经缺损.术后1个月行神经电生理检测即胫后肌群运动诱发电位,用HE染色、免疫组化染色、透射电镜等方法对移植体远端吻个口再生神经纤维进行形态学观察,并对再生有髓神经纤维的数量、密度、直径及雪旺细胞的密度进行量化分析.结果 移植前新鲜神经组MHC-Ⅱ检测值为72.14±19.88,去细胞组MHC-Ⅱ检测值为4.19±3.11,两组比较差异有统计学意义(t=3.817,P＜0.05);透射电镜观察显示为胶原性管道,无细胞成分.术后4周,处死前行运动诱发电位检测,神经传导速度A组为(21.16±2.31)m/s,B组为(13.37±1.89)m/s,C组为(21.43±2.18)m/s,A组与 C组比较差异无统计学意义(P＞0.05),A组与 B组比较差异有统计学意义(P＜0.05).组织学观察见3组移植体远端吻合口横切面再生神经纤维呈微束状,透射电镜观察再生神经纤维具有正常的形态和结构.A、C组再生神纤纤维数量及直径均优于B组,差异有统计学意义(P＜0.05).结论 经化学萃取的去细胞兔胫神经基膜管能够移植于大鼠,成功修复大鼠坐骨神经缺损,而且复合NGF的去细胞基膜管在神经修复质量上优于单纯的去细胞神经基膜管,更加接近自体神经移植的效果.     

化学去细胞异体神经修复神经缺损长度的实验研究

[目的]研究神经长度是否影响化学去细胞神经的质量及化学去细胞异体神经修复犬神经缺损的适当长度范围。[方法]切取犬的坐骨神经全长，截取直径近似段长度12cm，去除神经外膜的脂肪组织及血管，在5．0％Triton X-100和5．0％脱氧胆酸钠中重复消化得到化学去细胞神经；部分神经用于组织学评价，将12cm神经按顺序切割为6段，石蜡包埋制备切片，厚度5μm。HE染色和Fastblue染色，观察去细胞程度、神经细胞外基质结构完整性、髓鞘染色强度，观察每段神经的差异。在体内实验中，用化学去细胞异体神经桥接犬的坐骨神经缺损，缺损长度分别为8、10cm组，每组各有6条成年犬，随访时间12个月。观察动物肢体功能恢复、肌电图变化及再生神经组织学表现，包括HE染色、S-100免疫组织化学染色、乙酰胆碱酯酶染色。[结果]去细胞神经全长各段在去细胞程度、结构完整性及残余髓鞘染色综合评价中无差异。体内移植实验结果显示8cm去细胞异体神经移植组的犬在术后12个月踝关节可直立行走，而10cm去细胞异体神经移植组犬12个月时踝关节不能直立行走。肌电图检查结果显示，两组动物的手术肢体均可记录到诱发的运动和感觉电位。肌电图波幅、时限及运动神经传导速度结果显示，8cm移植组明显高于10cm移植组（P〈0．05）。组织学检查显示8cm移植组再生的神经轴突通过移植段神经到达远侧的神经中，并与所支配的肌肉建立新的运动终板联系，坐骨神经支配的小腿三头肌中有大量新生的运动终板形成。在10cm移植组有部分再生的神经纤维通过移植段神经进入远端的神经，小腿三头肌中新生的运动终板数量和大小明显低于8cm移植组（P〈0．05）。[结论]在化学去细胞神经的制备中，神经的长度对去细胞的质量无影响；化学去细胞异体神经修复神经缺损的长度若不超过8cm可取得较满意的功能康复结果。     

Repair of nerve defect with acellular nerve graft supplemented by bone marrow stromal cells in mice

The acellular nerve graft that can provide internal structure and extracellular matrix components of the nerve is an alternative for repair of peripheral nerve defects. However, results of the acellular nerve grafting for nerve repair still remain inconsistent. This study aimed to investigate if supplementing bone marrow mesenchymal stromal cells (MSCs) could improve the results of nerve repair with the acellular nerve graft in a 10-mm sciatic nerve defect model in mice. Eighteen mice were divided into three groups (n = 6 for each group) for nerve repairs with the nerve autograft, the acellular nerve graft, and the acellular nerve graft by supplemented with MSCs (5 × 10(5)) fibrin glue around the graft. The mouse static sciatic index was evaluated by walking-track testing every 2 weeks. The weight preservation of the triceps surae muscles and histomorphometric assessment of triceps surae muscles and repaired nerves were examined at week 8. The results showed that the nerve repair by the nerve autografting obtained the best functional recovery of limb. The nerve repair with the acellular nerve graft supplemented with MSCs achieved better functional recovery and higher axon number than that with the acellular nerve graft alone at week 8 postoperatively. The results indicated that supplementing MSCs might help to improve nerve regeneration and functional recovery in repair of the nerve defect with the acellular nerve graft.     

兔化学去细胞神经移植修复大鼠坐骨神经缺损

目的：以化学去细胞兔神经移植修复大鼠坐骨神经缺损，从而探索化学去细胞异种神经移植修复神经缺损的可能性；方法：以30％TritonX-100和40%脱氧胆酸钠顺序处理新鲜取材的兔神经(直径15nm左右)，移植修复成年wistar大鼠l.0cm坐骨神经缺损．4个月后行电生理及组织学检查，观察神经再生情况；结果：移植神经未被宿主排斥，大量再生的神经纤维长过移植物，并恢复电传导功能；结论：化学去细胞异种神经可以作为修复周围神经缺损的一种很有前途的方法。     

化学去细胞异体神经复合肝细胞生长因子修复周围神经缺损的研究

目的 研究化学去细胞异体神经复合人肝细胞生长因子(HGF)修复周围神经缺损的作用.方法 体外试验检测携带人肝细胞生长因子基冈的重组腺病毒(Ad-HGF)对小鼠骨骼肌细胞的转染效率以及转染细胞对目的 蛋白的表达.化学玄细胞异体神经修复大鼠坐骨神经10mm缺损后,近、远端吻合口附近肌肉分别注射腺病毒介导的人肝细胞生长因子(Ad-HGF),与自体神经移植组对照,通过步态分析、肌肉湿重测定、轴突生长速率测定、神经电生理、计算机图像分析等指标评价神经移植后再生效果.结果 流式细胞仪结果表明,随着病毒滴度的增加,Ad-HGF对骨骼肌细胞的转染不断增加.骨骼肌细胞对目的 蛋白(HGF)的表达可以持续两周.大鼠动物实验术后16周,去细胞异体神经可以修复周罔神经缺损,同自体神经移植对比,肝细胞生长因子明显增强了去细胞异体神经的神经再生能力.结论 复合肝细胞生K因子的化学去细胞异体神经能促进神经轴突牛长速度,显著增加移植物内新生血管,满意修复一定长度周围神经缺损,可以成为一种有效的周围神经组织工程修复材料.     

Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor

Previous studies have demonstrated the potential of growth factors in peripheral nerve regeneration. A method was developed for sustained delivery of nerve growth factor (NGF) for nerve repair with acellular nerve grafts to augment peripheral nerve regeneration. NGF‐containing polymeric microspheres were fixed with fibrin glue around chemically extracted acellular nerve grafts for prolonged, site‐specific delivery of NGF. A total of 52 Wister rats were randomly divided into four groups for treatment: autografting, NGF‐treated acellular grafting, acellular grafting alone, and acellular grafting with fibrin glue. The model of a 10‐mm sciatic nerve with a 10‐mm gap was used to assess nerve regeneration. At the 2nd week after nerve repair, the length of axonal regeneration was longer with NGF‐treated acellular grafting than acellular grafting alone and acellular grafting with fibrin glue, but shorter than autografting (P < 0.05). Sixteen weeks after nerve repair, nerve regeneration was assessed functionally and histomorphometrically. The percentage tension of the triceps surae muscles in the autograft group was 85.33 ± 5.59%, significantly higher than that of NGF‐treated group, acellular graft group and fibrin‐glue group, at 69.79 ± 5.31%, 64.46 ± 8.48%, and 63.35 ± 6.40%, respectively (P < 0.05). The ratio of conserved muscle‐mass was greater in the NGF‐treated group (53.73 ± 4.56%) than in the acellular graft (46.37 ± 5.68%) and fibrin glue groups (45.78 ± 7.14%) but lower than in the autograft group (62.54 ± 8.25%) (P < 0.05). Image analysis on histological observation revealed axonal diameter, axon number, and myelin thickness better with NGF‐treated acellular grafting than with acellular grafting alone and acellular grafting with fibrin glue (P < 0.05). There were no significant differences between NGF‐treated acellular grafting and autografting. This method of sustained site‐specific delivery of NGF can enhance peripheral nerve regeneration across short nerve gaps repaired with acellular nerve grafts. © 2009 Wiley‐Liss, Inc. Microsurgery, 2009.     

Experimental study of vascularized nerve graft: evaluation of nerve regeneration using choline acetyltransferase activity

A comparative study of nerve regeneration was performed on vascularized nerve graft (VNG) and free nerve graft (FNG) in Fischer strain rats. A segment of the sciatic nerve with vascular pedicle of the femoral artery and vein was harvested from syngeneic donor rat for the VNG group and the sciatic nerve in the same length without vascular pedicle was harvested for the FNG group. They were transplanted to a nerve defect in the sciatic nerve of syngeneic recipient rats. At 2, 4, 6, 8, 12, 16, and 24 weeks after operation, the sciatic nerves were biopsied and processed for evaluation of choline acetyltransferase (CAT) activity, histological studies, and measurement of wet weight of the muscle innervated by the sciatic nerve. Electrophysiological evaluation of the grafted nerve was also performed before sacrifice. The average CAT activity in the distal to the distal suture site was 383 cpm in VNG and 361 cpm in FNG at 2 weeks; 6,189 cpm in VNG and 2,264 cpm in FNG at 4 weeks; and 11,299 cpm in VNG and 9,424 cpm in FNG at 6 weeks postoperatively. The value of the VNG group was statistically higher than that of the FNG group at 4 weeks postoperatively. Electrophysiological and histological findings also suggested that nerve regeneration in the VNG group was superior to that in the FNG group during the same period. However, there was no significant difference between the two groups after 6 weeks postoperatively in any of the evaluations. The CAT measurement was useful in the experiments, because it was highly sensitive and reproducible.     

优化法去细胞大鼠神经同种异体移植修复坐骨神经缺损

[目的]以优化法去细胞大鼠神经移植,修复同种异体坐骨神经缺损,观察术后动物的免疫排斥、早期功能恢复及神经再生情况。[方法]以优化去细胞方法处理新鲜取材的成年SD大鼠坐骨神经,移植修复同种异体1.0cm坐骨神经缺损,以自体神经和新鲜异体神经移植为对照,术后1个月行坐骨神经功能指数评价、神经电生理和组织学检查,观察动物在功能恢复、免疫排斥及神经再生方面的情况。[结果]自体神经和去细胞异体神经移植组动物的坐骨神经功能指数无显著差异(P>0.05),大体观察均可见神经连续性良好。电生理检测表明2组动物移植神经均已恢复电传导能力,在传导速度(CV)上无显著差异(P>0.05),但均未达到正常神经水平(P<0.05)。组织学观察则显示2组再生神经纤维均已长入移植段远端。S-100免疫组化显示两者在雪旺氏细胞数、形态和排列等方面无明显差异。2组在CD8 T细胞和巨噬细胞免疫组化染色阳性面积百分比上差异无统计学意义(P>0.05)。计算移植神经中段轴突密度后表明两者无显著差异(P>0.05),但都比正常神经小(P<0.05),比新鲜异体神经移植组大(P<0.05)。[结论]优化去细胞神经移植组与自体神经移植组在免疫排斥、功能恢复及神经再生方面无显著差异。优化法去细胞神经在移植修复同种异体神经缺损时,可以达到免疫耐受,其早期功能恢复和神经再生情况良好,在修复周围神经缺损时可以作为自体神经移植的一种替代疗法。     

种植脂肪干细胞的去细胞神经修复坐骨神经缺损的实验研究

目的 探讨脂肪干细胞(ADSCs)应用于组织工程化外周神经修复大鼠坐骨神经缺损的效果.方法 48只体重200～220 g的雌性F344大鼠随机分成6组,每组8只,分别用下面6种不同的实验组修复15 mm长坐骨神经缺损.A组:种植ADSCs的去细胞神经;B组:种植诱导ADSCs的去细胞神经;C组:种植许旺细胞(SCs)的去细胞神经;D组:去细胞神经;E组:自体神经移植;F组:空白对照.通过神经电生理检测、荧光金逆行示踪、组织学检测和坐骨神经功能指数测定评价各组修复神经缺损的效果.结果 术后12周,F组未见桥接物,A组和B组的神经电生理等各项指标均分别优于D组(P＜0.05或P＜0.01),与C组和E组间差异无统计学意义(P＞0.05).结论 初步结果显示ADSCs及诱导后ADSCs作为种子细胞,与去细胞神经构建的组织工程化外周神经移植体,能够修复外周神经缺损.     

去细胞异体神经基膜管桥接神经缺损的实验研究

目的 探索修复周围神经缺损的新的有效替代材料。方法 将异体的预变性神经和正常神经经溶血卵磷脂裂解液处理后,得到一种没有细胞及细胞碎片的、空的神经基膜管,将其用来修复大鼠15mm坐骨神经缺损,通过一般观察、肌萎缩测量、电生理检测、连续切片组织学观察和计算机图像为评价神经再生。结果 化学抽提的预变性神经和正常神经桥接物组均获得了密集的神经再生和良好的神经功能恢复,其中前者效果更为优越。结论 这种材料极有可能成为自体神经的替代材料应用于临床较短的神经缺损的修复。     

Inside-out vein graft and inside-out artery graft in rat sciatic nerve repair

Although veins and arteries present similar wall structures, there are differences which may be relevant in peripheral nerve reconstruction. Inside-out vein grafts (IOVG) have been satisfactorily used to repair both motor and sensitive nerves. However, the inside-out artery graft (IOAG) is a new technique and not fully investigated. Our study presents comparative morphological data on nerve regeneration achieved with IOVG and IOAG in the repair of Wistar rat sciatic nerves. Jugular veins and aorta arteries were harvested from donor animals and used "inside-out" to bridge a 10-mm gap. Animals were sacrificed at 10 weeks to evaluate nerve regeneration. Both techniques presented great variability in nervous tissue, though some animals showed satisfactory results. Different intensities of scarring processes might have interfered with nerve regeneration. Although IOVG and IOAG techniques showed similar morphometric results, in general, IOVG presented a closer-to-normal nerve organization than IOAG.     

人脐带间充质干细胞复合去细胞神经基膜管修复大鼠坐骨神经缺损

目的 探讨诱导后的脐带间充质干细胞作为组织工程种子细胞修复大鼠坐骨神经缺损的可行性.方法 从正常足月新生儿脐带中分离培养间充质干细胞并诱导分化为神经样细胞,与去细胞神经基膜管共培养以构建组织工程神经;用30只健康成年雄性SD大鼠建立坐骨神经缺损(10 mm)的动物模型并随机分成3组:A组为脐带间充质干细胞复合去细胞神经基膜管组,B组为单纯去细胞神经基膜管组,C组为自体神经桥接组.术后10周通过神经电生理检测、组织学观察等评测效果.结果 在局部观察和肌肉测量、神经电生理检测、组织学观察等方面,脐带间充质干细胞复合去细胞神经基膜管组(A组)神经再生及肢体功能情况良好,效果接近于自体神经桥接组(C组),明显优于单纯去细胞神经基膜管组(B组).结论 脐带间充质干细胞复合去细胞神经基膜管构建的组织工程神经可有效促进大鼠坐骨神经缺损(10 mm)的修复.     

优化法去细胞兔神经移植修复大鼠坐骨神经缺损

目的 以优化法去细胞兔臂丛神经，移植修复大鼠坐骨神经缺损，观察免疫排斥情况、早期功能恢复及神经再生情况。方法 以优化法处理新鲜取材的新西兰大白兔臂丛神经分支，移植修复成年sD大鼠坐骨神经1．0cm缺损，分别于术后1个月及3个月行功能评价、电生理和组织学检查，观察免疫排斥、功能恢复及神经再生情况。结果 实验组与自体神经移植组在免疫排斥、功能恢复及神经再生方面无显著差异，却明显优于新鲜兔神经移植组。3个月取材时的神经再生及功能恢复情况优于1个月取材时。结论 优化法去细胞神经在移植修复异种神经缺损时，可以达到免疫耐受，其早期功能恢复和神经再生情况良好，在修复周围神经缺损时有望作为自体神经移植的一种替代疗法。     

无细胞的异体神经修复鼠坐骨神经缺损

目的 通过化学萃取同种异体神经,去除髓鞘和雪旺细胞,形成无细胞基膜管后桥接鼠坐骨神经缺损,研究神经再生效果。方法 正常鼠坐骨神经用非变性生物剂处理后得到无细胞的基膜管,桥接鼠坐骨神经20mm缺损。实验分3组:无细胞基膜管移植组(A组),自体神经移植组(B组)和异体神经移植组(C组)。术后进行肌电图、光镜、电镜及图象分析仪检查。结果 A组再生神经有大量轴突通过移植体,术后2个月电生理检测再生神经的潜伏期及波幅低于B组(P<0.05),术后3个月2组差异无显著意义。髓鞘厚度在术后3个月时亦低于B组,差异有显著意义(P<0.05)。轴突直径及数目两组无差异。C组因无神经再生,结果无法测量。结论 这种无细胞基膜管移植体能支持轴突的生长和雪旺细胞的迁移,是一种良好的神经移植替代材料。     

Cavernous nerve regeneration using acellular nerve grafts

INTRODUCTION: The restoration of erectile function following complete transection of nerve tissue during surgery remains challenging. Recently, graft procedures using sural nerve grafts during radical prostatectomy have had favorable outcomes, and this has rekindled interest in the applications of neural repair in a urologic setting. Although nerve repair using autologous donor graft is the gold standard of treatment currently, donor nerve availability and the associated donor site morbidity remain a problem. In this study, we investigated whether an "off-the-shelf" acellular nerve graft would serve as a viable substitute. We examined the capacity of acellular nerve scaffolds to facilitate the regeneration of cavernous nerve in a rodent model. MATERIALS AND METHODS: Acellular nerve matrices, processed from donor rat corporal nerves, were interposed across nerve gaps. A total of 80 adult male Sprague-Dawley rats were divided into four groups. A 0.5-cm segment of cavernosal nerve was excised bilaterally in three of the four groups. In the first group, acellular nerve segments were inserted bilaterally at the defect site. The second group underwent autologous genitofemoral nerve grafts at the same site, and the third group had no repair. The fourth group underwent a sham procedure. Serial cavernosal nerve function assessment was performed using electromyography (EMG) at 1 and 3 months following initial surgery. Histological and immunocytochemical analyses were performed to identify the extent of nerve regeneration. RESULTS: Animals implanted with acellular nerve grafts demonstrated a significant recovery in erectile function when compared with the group that received no repair, both at 1 and 3 months. EMG of the acellular nerve grafts demonstrated adequate intracavernosal pressures by 3 months (87.6% of the normal non-injured nerves). Histologically, the retrieved regenerated nerve grafts demonstrated the presence of host cell infiltration within the nerve sheaths. Immunohistochemically, antibodies specific to axons and Schwann cells demonstrated an increase in nerve regeneration across the grafts over time. No organized nerve regeneration was observed when the cavernous nerve was not repaired. CONCLUSION: These findings show that the use of nerve guidance channel systems allow for accelerated and precise cavernosal nerve regeneration. Acellular nerve grafts represent a viable alternative to fresh autologous grafts in a rodent model of erectile dysfunction.     

Effect of platelet-rich plasma on peripheral nerve regeneration

Activated platelets release various growth factors, some of which are recognized to improve nerve regeneration. This study evaluated the effect of platelet-rich plasma (PRP) in end-to-end neurorrhaphy. A total of 45 Wistar rats were used, with the initial five used for PRP preparation. The right hind limbs were used as experimental, with the left as control. The animals were treated in five groups. Group A (n = 4): The right sciatic nerve was dissected only from the sciatic notch to the bifurcation. In all other groups, the nerve was sharply transected and repaired with: group B (n = 8): two sutures; group C (n = 8): six sutures; group D (n = 10): two sutures and PRP; and group E (n = 10): six sutures and PRP. Groups D and E were compared with groups B and C, respectively. Group E had a shorter latency time in electromyography ( P < 0.01) and a thicker myelin layer in the histological evaluation ( P < 0.003) in comparison with group C. These positive effects of PRP were not detected in the nerves were repaired with two sutures. In this animal model, the application of PRP to the repair site helped to improve remyelinization of the sciatic nerve in rats when the epineural repair was done with six sutures.     

Vein graft for repair of peripheral nerve gap

Ten millimeter gaps in sciatic nerves of rats were repaired by vein grafting (n = 30) and by nerve grafting (n = 30). Electrophysiologic evaluation demonstrated that the nerve-graft group had a significantly higher percentage of re-innervation and shorter delay of terminal latency in myoelectrical response evoked by electrical stimulation of the sciatic nerve, compared with the vein-graft group at two and three months. But there were no differences between the two grafted groups after six months. In both grafted groups, the numbers of myelinated nerve fibers increased substantially over time, although the vein-grafted segments had smaller numbers of fibers than the nerve-grafted segments at both three and 12 months. Microangiograms also demonstrated different patterns of revascularization between the two groups. Results suggest that, although autologous vein grafting requires a longer period of time for nerve regeneration, compared with autologous nerve grafting, its ability to repair nerve is considerable.