小肠黏膜下层复合雪旺细胞构建组织工程化人工神经修复周围神经缺损的研究

目的小肠黏膜下层（small intestinal submucosa，SIS）复合雪旺细胞构建组织工程化人工神经并探讨其修复周围神经缺损的效果。方法SD大鼠60只随机分成三组，每组20只。构建右侧坐骨神经14mm缺损，A组：单纯SIS修复组；B组：复合雪旺细胞的SIS修复组；C组：自体神经移植对照组。术后不同时间段通过大体观察、电生理检测、组织学、透射电镜、图像分析、逆行示踪和小腿三头肌称重等方法分析评价修复效果。结果术后16周，B组移植段与近远段神经外观相似，未有神经瘤形成。组织学和透射电镜检查见B组再生神经组织可顺利通过缺损区，再生神经纤维排列整齐呈束状，含有大量的有髓神经纤维，与C组相似。电生理检测见第16周B组神经传导速度和复合动作电位的波幅均较第12周有所提高，与C组相比差异无统计学意义。真蓝逆行示踪证实B组和C组再生神经轴突轴浆运输功能恢复良好。图像分析证实B组再生神经组织面积百分比、有髓神经纤维密度和髓鞘厚度与C组相比差异无统计学意义，优于A组。B组和C组患侧小腿三头肌肌肉重量和恢复率均优于A组，C组优于B组，且差异有统计学意义。结论SIS复合雪旺细胞构建组织工程化人工神经能有效修复大鼠长距离坐骨神经缺损，有望成为自体神经的替代材料应用于周围神经缺损的修复。     

胸腺内注射异基因抗原诱导鼠神经移植免疫耐受的实验研究

目的探讨小鼠胸腺内注射异基因抗原在同种异体异基因坐骨神经移植免疫耐受中的作用。方法自供体小鼠C57BL／6的脾细胞中提取MHC抗原注人受体鼠Balb／c小鼠胸腺内，于2周后移植供体鼠坐骨神经。48只Balb／c小鼠随机分为4组，A组（胸腺内注射组）、B组（自体神经移植组）、C组（冷冻异体神经移植组）、D组（异体神经移植加用免疫抑制剂组）。于3周后进行电生理学、组织学、免疫学检测。结果A组运动神经传导速度（38．23m／s）与D组（36．39m／s）相比无显著性差异（P〉0．05），组织学、电镜、免疫学（混合淋巴细胞培养及迟发性超敏反应）检测结果均证实B组分别优于A组、D组和C组。结论胸腺内注射异基因MHC抗原可诱导大鼠对异体坐骨神经移植的特异性免疫耐受。     

辐照和绿茶多酚处理同种异体神经移植体的实验研究

[目的]比较经绿茶多酚溶液及辐照预处理的同种异体神经修复大鼠坐骨神经缺损的效果.[方法] 48只成年雄性Wista大鼠,将坐骨神经在梨状肌孔下5 mm处切除1.0 cm,随机分为4组,每组12只,神经缺损分别用4种移植物桥接.A组:自体神经移植;B组:新鲜异体神经移植;C组:经辐照处理的异体神经移植;D组:绿茶多酚溶液保存的异体神经移植.术后6、12周通过大体、电生理学、组织学、透射电镜观察评价各组修复神经缺损的效果.[结果]A、D组间差异无统计学意义,A、D组的各项指标均优于B、C组.[结论]大鼠坐骨神经缺损模型中,绿茶多酚溶液保存的同种异体神经是良好的神经移植替代材料,移植后神经再生情况优于辐照预处理的异体神经.     

FK506缓释膜应用于同种异体神经移植的实验研究

目的研究普乐可复(prograf,FK506)缓释膜在同种异体神经移植中的作用。方法应用异体神经桥接大鼠坐骨神经缺损,术中局部应用FK506缓释膜,分别于术后4、8、12周对移植物行大体和光镜观察,及轴突图像分析、移植神经电镜检查、小腿三头肌肌湿重比较、患肢坐骨神经电生理检查。结果C组(应用FK506缓释膜)的神经生长最好,基本与D组(自体神经移植)相同,B组(经预处理异体神经移植)次之,A组(新鲜异体神经移植)最差。经过对肌电图、轴突计数、肌湿重的统计学分析,C、B、A组的差异有统计学意义(P<0.05),C、D组之间差异无统计学意义。结论应用FK506缓释膜有助于减轻同种异体神经的免疫排斥反应,为神经再生创造良好条件;在同种异体神经移植中应用FK506缓释膜有助于促进神经再生。     

超低温冷冻保存后同种异体神经移植的实验研究

目的　探索大鼠同种异体神经移植的可行性。方法　取Wistar大鼠坐骨神经 ,经超低温冷冻保存后移植于SD大鼠坐骨神经缺损处。分成超低温冷冻同种神经移植组 (A)、新鲜同种神经移植组(B)、及自体神经移植组 (C)。 3组均在术后 3、8、12、16周行大体观察 ,检测形态学、电生理变化及血清IL 2、TNF水平。结果　A、C组的腓肠肌萎缩 ;足无明显畸形 ,趾无缺损、无溃疡。B组的腓肠肌萎缩 ;足部溃疡伴趾部分缺损。A、C组在术后 8周刺激神经移植段近端有动作电位出现 ,B组在术后 12周出现。A组动作电位的波幅较B组高。血清IL 2 ,C组与B组差别有显著性 (P <0 .0 5 ) ,A组与C组比较差别无显著性 (P >0 .0 5 )。光镜下A组空泡变性、炎性细胞浸润少。电镜下见髓鞘厚薄基本相同 ,轴突密度高 ,雪旺细胞发育较完善 ,明显优于B组。结论　超低温冷冻保存能降低同种异体神经的抗原性 ,在不用免疫抑制剂情况下 ,动物用同种异体神经移植是可行的     

化学去细胞同种异体神经复合缓释神经生长因子修复周围神经缺损

目的 探讨化学去细胞异体神经复合缓释神经生长因子(nerve growth factor,NGF)修复周围神经缺损的效果. 方法采用药物微球技术制备NGF微球,与生物纤维蛋白胶混合形成NGF复合缓释制剂.取健康雄性SD大鼠20只,体重280～300 g,制备化学去细胞异体神经.取健康雄性Wismr大鼠52只,体重250～300 g,制备大鼠左侧世骨神经10 mm缺损模型.根据修复缺损材料不同,随机分成4组(n=13):自体神经移植组(A组)、化学去细胞异体神经移植加NGF复合缓释制剂组(B组)、化学去细胞异体神经移植组(C组)和化学去细胞同种异体神经移植和纤维蛋白胶组(D组).右侧不作处理作为空白埘照组.术后行大体观察;术后2周测鼍神经轴突生长距离;术后16周行电生理检测,计算术侧坐骨神经运动传导速度恢复率、术侧小腿三头肌收缩力恢复率及湿重恢复率,并行移植神经吻合中段HE和半薄切片甲苯胺蓝染色观察. 结果 所有动物均存活至实验完成,切口愈合良好.术后2周A组神经再生距离较其余3组长(P＜0.05),B组优于C、D组(P＜0.05),C、D组间比较差异无统计学意义(P＞0.05).术后16周,A、B、C、D组术侧坐骨神经运动传导速度恢复率分别为73.37%±7.82%、70.39%±8.45%、53.51%±6.31%、55.28%±5.37%;A、B组与C、D组比较,差异有统计学意义(P＜0.05).A、B、C、D组术侧小腿三头肌收缩力恢复率分别为85.33%±5.59%、69.79%±5.31%、64.46%±8.49%、63.35%±6.40%:A组与B、C、D组比较筹异有统计学意义(P＜0.05),B、C、D组间比较差异无统计学意义(P＞0.05).A、B、C、D组术侧小腿三头肌湿重恢复率分别为62.54%±8.25%、53.73%±4.56%、46.37%±5.68%、45.78%±7.14%;A组与B、C、D组比较筹异有统计学意义(P＜0.05),B组与C、D组比较差异有统计学意义(P＜0.05),C、D组间比较差异无统计学意义(P＞0.05).组织学图像分析示B组有髓神经纤维数量、轴突直径及髓鞘厚度均优于C、D组(P＜0.05),B组轴突直径小于A组(P＜0.05). 结论 复合缓释NGF的化学去细胞同种异体神经能满意修复一定长度的周围神经缺损,是一种有效的周围神经组织工程修复材料.     

大鼠失神经靶肌肉注射异体不同细胞对神经再生影响的研究

目的研究注射异体不同细胞于大鼠失神经靶肌肉对神经再生的影响.方法 SD成年大鼠36只,雌性,体重120～150 g,随机分为4组,每组9只.无菌条件下切断左侧坐骨神经,一期神经外膜缝合.术后即于小腿三头肌处注射相应细胞,7天注射1次,共4次.A组注射单纯雪旺细胞1×106/ml 1 ml,B组注射雪旺细胞加成肌细胞(雪旺细胞∶成肌细胞为1∶1)1×106/ml 1 ml,C组注射肾内皮细胞提取液1 ml,D组注射无血清培养液1 ml作对照.术后3个月取手术侧坐骨神经及坐骨神经所支配的小腿三头肌,进行大体和组织形态学观察、神经鞘细胞密度及单位面积靶肌肉(小腿三头肌)运动终板计数.结果术后3个月,各组近端神经鞘细胞均数为A组0.134 5±0.029 8,B组0.093 1±0.025 6,C组0.072 4±0.023 7,D组0.187 7±0.054 2;A组∶D组P值<0.05,B组∶D组、C组∶D组及A组∶B组P值均<0.01,B组∶C组P值＞0.05.术后3个月各组远端神经鞘细胞密度均数为A组0.186 0±0.042 5,B组0.155 1±0.032 1,C组0.104 7±0.013 3,D组0.240 9±0.056 8;A组∶D组P值<0.05,B组∶D组、C组∶D组及B组∶C组P值<0.01,A组∶B组P值＞0.05.术后3个月各组靶肌肉运动终板个数均数为A组6.000±0.866,B组9.000±2.291,C组12.780±1.394,D组3.110±0.782;A组∶D组、B组∶D组及C组∶D组P值<0.01,A组∶B组、A组∶C组及B组∶C组P值<0.01.结论雪旺细胞、混合细胞、肾内皮细胞提取液均有促进神经再生作用,肾内皮细胞提取液优于雪旺细胞及混合细胞.     

冻干去细胞同种异体神经种植类许旺细胞修复坐骨神经缺损的实验研究

目的研究冻干去细胞异体神经修复大鼠坐骨神经缺损的效果。方法50只成年雌性DA大鼠随机分为5组，每组10只，分别用5种移植物桥接大鼠1．5cm坐骨神经缺损。A组：冻干去细胞异体神经种植类许旺细胞移植组；B组：冻干去细胞异体神经移植组；C组：去细胞异体神经移植组；D组：新鲜异体神经移植组；E组：自体神经移植组。术后4、24周通过大体观察、神经电生理、肌肉湿重及组织学指标评价各组修复神经缺损的效果。结果术后24周A、E组间差异无统计学意义（P〉0．05），A、E组的各项指标均优于B、C、D组（P〈0．05或P〈0．01）。结论冻干化学去细胞神经是良好的神经移植替代材料。     

细胞治疗促进周围神经再生的实验研究

目的研究注射异体不同细胞于大鼠失神经靶肌肉,对神经再生的促进及防止运动终板变性、促进肌肉功能恢复的作用。方法取1周龄SD乳鼠400只,体重20.0±2.3g,制备雪旺细胞、成肌细胞和肾内皮细胞。将SD成年雌性大鼠36只,体重120～150g,随机分为四组,每组9只。无菌条件下切断左侧坐骨神经,一期神经外膜缝合。术后即于小腿三头肌处注射相应细胞,每7天注射1次,共4次。A组注射1×106/ml单纯雪旺细胞1ml,B组注射1×106/ml雪旺细胞加成肌细胞1ml（雪旺细胞∶成肌细胞为1∶1）,C组注射1×106/ml混合细胞提取液1ml（雪旺细胞∶成肌细胞∶肾内皮细胞为1∶1∶1）,D组注射无血清培养液1ml作对照。术后行大体观察,术后3个月取材行靶肌肉的组织形态学观察,行单位面积运动终板个数检测、单位面积靶肌肉Actin阳性肌纤维个数检测,以及酶组织化学观察。于神经缝合口近、远端检测神经鞘细胞密度和再生神经纤维面积的观察。结果术后1~3个月A、B、C组大鼠患肢由不同程度的足踝部溃疡逐渐愈合,肿胀消退,恢复正常行走;D组患侧下肢肌肉萎缩,足踝溃疡、肿胀,足趾挛缩明显,拖行。术后3个月,单位面积靶肌肉运动终板个数、Actin阳性细胞数、失神经肌肉各种酶活性和再生神经的组织学改变,C组优于A、B组（P〈0.01）。A、B、C组实验结果均明显优于D组（P〈0.01）。结论雪旺细胞、雪旺细胞加成肌细胞、混合细胞提取液均有促进神经再生及防止运动终板变性、促进肌肉功能恢复的作用,混合细胞提取液作用优于雪旺细胞及雪旺细胞加成肌细胞移植。     

犬化学去细胞神经同种异体移植的神经电生理研究

目的：以化学去细胞同种异体坐骨神经，移植修复犬坐骨神经的粗大和长段缺损，观察其近期神经电生理恢复。方法：12只犬随机分成去细胞神经移植组（实验组）和自体神经移植组（对照组）各6只。右侧坐骨神经造成5.0cm长缺损，以两种神经移植物桥接修复。术后6个月行神经电生理观察，包括小腿三头肌运动诱发电痊、神经移植段运动传导速度、感觉诱发电位等。结果：（1）方波（1.0mA-2.0mA，0.1ms，1.0Hz）刺激移植段近侧神经，均在小腿三头肌上记录到运动诱发电位曲线。（2）神经移植段运动传导速度，实验组平均为47.2m/s、对照组为60.9m/s、正常值为122.0m/s。（3）方波（5.0mA-10.0mA，0.2ms，1.9Hz）刺激胫神经远端，均在颅顶部记录到感觉诱发电位曲线；两组动物的感觉恢复程度相似，但均不及正常侧。结论：化学去细胞神经同种异体移植修复犬坐骨神经长段缺损，术后6个月近期感觉及运动传导功能恢复与自体神经移植相似。     

Quantification of nerve tension after nerve repair: correlations with nerve defects and nerve regeneration

This study tested the validity of a quantitative in vitro nerve-tension-measuring technique, by correlating the tension measurements with functional and morphologic assessments of nerve regeneration. Initially, harvested nerves were used in vitro to determine a K value for lateral displacement in this tissue. Next, this value was used to calculate the tension of nerve repair, following 0-, 3-, 6-, and 9-mm resections of nerves in groups of rats. After quantifying the nerve tensions following excision and repair, the authors determined a sciatic function index to evaluate functional recovery and axon diameter in the animals. Functional recovery was significantly impaired in animals with elevated measurable tension (9.04 +/- 0.74 g in a 6-mm defect, 27.76 +/- 8.86 g in a 9-mm defect), compared to animals with no or 3-mm excision and measured tension of 3.3 +/- 1.09 g or less. Increased tension was also associated with a significant decrease in axon diameter. This study succeeded, therefore, in quantitatively relating the elements of measured nerve tension, nerve gaps, functional nerve recovery, and morphologic regeneration. Quantification of nerve tension by lateral displacement in vivo offers a possible solution to clinical management of nerve gaps, when the choice between primary repair and nerve grafting is not a clear one.     

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.     

内脏神经-体神经端侧吻合后神经纤维再生研究

目的 观察大鼠内脏神经-体神经端侧吻合后神经纤维的再生.方法 24只成年SD大鼠随机分为实验组(n=12)和正常对照组(n=12),实验组大鼠通过内脏神经-体神经端侧吻合建立人工体神经-内脏神经反射弧6个月后,在吻合口近端和远端分别截取10 mm的供体神经(L4VR)和受体神经(L6VR),在L6VR延续的盆副交感神经(PPN)和阴部神经(PN)分别截取10 mm的神经.正常对照组大鼠分别取相应节段的L4VR、L6VR、PPN和PN神经.标本经石蜡包埋切片并行甲苯胺蓝染色,比较实验组和对照组大鼠L6VR、PPN、PN神经纤维数量.结果 实验组大鼠横断面可见新生的有髓神经纤维,L4VR、L6VR、PPN和PN的神经纤维数量分别为1602.2±75.7、1037.9±123.6、817.0 ±52.2、510.4±29.1,吻合口远近端神经纤维通过率为64.8%,实验组和对照组大鼠相应的L6VR、PPN、PN神经纤维数目比率分别为70.2%、68.9%和62.2%.结论 大鼠内脏神经-体神经端侧吻合后体神经能够长入并替代内脏神经.     

End-to-side nerve repair in peripheral nerve injury

In peripheral nerve injury, end-to-side neurorrhaphy has been reported as an alternative in cases that the proximal nerve stump is not accessible. Several hypotheses have been proposed to explain peripheral nerve regeneration after end-to-side neurorrhaphy. Recent evidence suggests that nerve regeneration occurs by collateral sprouting. Although a great number of humoral factors have been identified, molecular mechanism of nerve regeneration after end-to-side neurorrhaphy has not been completely clarified yet. The goal of this technique is to provide satisfactory functional recovery for the recipient nerve, without any deterioration of the donor nerve function. End-to-side technique has been investigated in detail in both experimental and clinical studies. Only a limited number of reported cases in clinical practice, until today, can reveal that end-to-side technique may become a viable means of repairing peripheral nerves in certain clinical situations.     

End-to-side nerve graft for facial nerve reconstruction

Reconstruction of multiple branches of the facial nerve by sural nerve graft using end-to-side nerve suture was performed successfully on a patient with advanced parotid tumor. In this technique, one end of the grafted nerve is sutured with the stump of the facial nerve trunk in an end-to-end manner. Epineural windows are made on the nerve graft, and the distal stumps of the facial nerve branches (temporal, zygomatic, and buccal branches) are sutured with the graft in an end-to-side manner. Functional recovery of all branches and satisfactory facial expression were obtained within 2 years postoperatively. Axonal regeneration through the graft was confirmed by electrodiagnosis. Regeneration through the anastomosis at the stump of the facial nerve trunk using this technique is more efficient than conventional cable grafting, and the length of the nerve required is minimal. This technique may be a useful option for facial nerve reconstruction managing multiple branches.