周围神经移植修复脊髓损伤的实验研究

目的：探讨自体坐骨神经移植修复脊髓损伤的可行性。方法：将58只雌性Wistar大鼠分为二组，实验组：采用显微外科技术，将50只大鼠于T13水平切除左半侧脊髓10mm，再取右侧坐骨神经10mm移植到脊髓缺损处，近端接脊髓，远端接马尾，分别于术后2、4、6、8、12、22周在光镜和电镜下观察移植处坐骨神经、吻合口远端马尾神经、左后肢坐骨神经再生情况，并用摄像机记录患肢功能恢复情况。对照组：8只大鼠，于13水平切除左半侧脊髓10mm，不移植坐骨神经，观察脊髓缺损远端马尾神经和左右肢坐骨神经再生情况。结果：对照组坐骨神经的轴突及髓鞘部分崩解，密度降低，无再生轴突形成。实验组术后4周电镜下偶见移植处坐骨神经髓鞘及轴突形成，术后8周光镜及电镜下可见较多细的有髓神经纤维，22周时接近正常；同时观察到左后肢坐骨神经再生；大鼠后肢功能部分恢复，肌力达3级。结论：大鼠脊髓损伤后有再生能力，周围神经移植修复脊髓损伤是可行的。     

体神经-内脏神经吻合后神经纤维再生过程的光镜电镜观察

目的：观察大鼠体神经-内脏神经吻合后神经纤维的再生。方法：人工建立体神经-内脏神经反射弧大鼠模型，用电镜配合光镜观察12只大鼠术后1、4、8、24周神经变性与再生。结果：术后8、24周大体观察见神经吻合口位置稍许膨大，光镜观察发现术后8周吻合口位置可见新生的轴突，电镜观察见术后1周吻合口及其远近段神经纤维发生Waller变性，术后4周吻合口部位有再生的有髓和无髓纤维，术后8周新生的髓鞘进一步增厚，板层结构清晰可见，术后24周，髓鞘成熟，轴浆富含微管、微丝、线粒体。结论：体神经运动纤维能够再生长入并替代内脏神经节前纤维；再生的神经纤维具备基本正常的周围神经超微结构。     

神经端侧缝合与带血供的自体神经游离移植修复神经缺损的比较研究

目的通过神经端侧缝合与带血供的自体神经游离移植的对比观察，分析神经端侧缝合的修复效果。方法选用SD大鼠20只，每只大鼠随机选择一侧下肢行神经端侧缝合，另一侧下肢建立带血运的神经游离移植模型。术后分笼饲养，观察肢体瘫痪及感觉恢复情况。术后6个月取缝合处以远腓总神经段标本切半薄切片，分别做甲胺蓝、变色酸2R-亮绿髓鞘染色和HE染色，测量神经横切面轴突数目、密度、直径等。超薄切片做透射电镜观察，测髓鞘厚度和有髓神经纤维面积。结果所有大鼠伤口一期愈合，足部无溃疡形成，外观红润饱满。于术后3个月左右胫前肌肌力恢复，趾展反射出现。光镜检查见两种手术方式均有有髓神经纤维生长，再生纤维排列整齐密集，呈线形平行排列。电镜观察见髓鞘形态正常。两种术式轴突数目、密度、直径、髓鞘厚度差异均无统计学意义（P〉0．05）。结论神经端侧缝合后有有髓神经纤维生长，修复效果与带血供的自体神经游离移植无显著性差异。     

细胞外ATP对外周神经再生作用的实验研究

目的：研究细胞外ATP对外周神经再生的影响。方法：采用大鼠坐骨神经硅胶管再生室模型,左侧再生室中加入1mmol/LATP作为实验组,右侧再生室中加入生理盐水对照。术后1、2、4、6和12周取材,每次取材除作肉眼观察外,第6、12地行光镜及电镜观察,并采用图像分析检测再生轴突数目和髓鞘厚度。结果：ATP实验组再生神经干的直径,神经干内有髓纤维的数目以及髓鞘的厚度同对照组相比 ,差异非常显著。电镜观察表明,实验组再生随鞘的厚度及成熟情况明显优于对照组。结论：细胞外ATP具有较强的促进神经再生的作用。     

人羊膜上皮细胞对大鼠坐骨神经再生影响的实验研究

目的 通过实验研究了解人羊膜上皮细胞(human amniotic epithelial cells,HAECs)对大鼠坐骨神经再生的促进作用.方法 取60只SD大鼠随机分为两组,羊膜细胞组和对照组,各组再分为2周和4周组,每组15只.制作大鼠周围神经损伤再生室模型,HAECs组局部应用培养的HAECs,对照组局部使用等量的生理盐水.术后分别于2周、4周检测神经传导功能和HE染色观察神经纤维形态学变化.结果 术后2周两组的神经电生理及组织学检测比较差异无统计学意义;4周HAECs组的大鼠坐骨神经传导功能明显恢复,HE染色显示术后坐骨神经手术部位出现大量炎性肉芽组织,呈现纤维性修复,吻合口以远HAECs组神经纤维形态结构较对照组完整,神经纤维与髓鞘直径均较对照组大.结论 HAECs移植可加速大鼠坐骨神经损伤后神经传导功能恢复,有助于有髓神经纤维损伤后的轴突与髓鞘的再生修复.     

人羊膜上皮细胞对大鼠坐骨神经再生影响的实验研究

目的 通过实验研究了解人羊膜上皮细胞(human amniotic epithelial cells,HAECs)对大鼠坐骨神经再生的促进作用.方法 取60只SD大鼠随机分为两组,羊膜细胞组和对照组,各组再分为2周和4周组,每组15只.制作大鼠周围神经损伤再生室模型,HAECs组局部应用培养的HAECs,对照组局部使用等量的生理盐水.术后分别于2周、4周检测神经传导功能和HE染色观察神经纤维形态学变化.结果 术后2周两组的神经电生理及组织学检测比较差异无统计学意义;4周HAECs组的大鼠坐骨神经传导功能明显恢复,HE染色显示术后坐骨神经手术部位出现大量炎性肉芽组织,呈现纤维性修复,吻合口以远HAECs组神经纤维形态结构较对照组完整,神经纤维与髓鞘直径均较对照组大.结论 HAECs移植可加速大鼠坐骨神经损伤后神经传导功能恢复,有助于有髓神经纤维损伤后的轴突与髓鞘的再生修复.     

同种异体神经复合体修复兔坐骨神经缺损的研究

目的 用种植胎兔雪旺细胞的去细胞同种异体神经复合体修复兔缺损的坐骨神经,观察坐骨神经再生及功能恢复.方法 健康成年新西兰白兔48只,体质量1.5-2.0 kg,随机分成2组.两组动物均切除一段坐骨神经,造成2.0 cm长的缺损.实验组:用种植胎兔雪旺细胞的同种异体神经复合体修复坐骨神经.对照组:仅用去细胞同种异体神经修复.术后4、8、16周进行大体观察、标本光镜和电镜观察且进行量化分析、肌湿重检测.结果 手术区局部均未出现明显的排斥反应,实验组足部溃疡愈合情况优于对照组,实验组再生神经纤维数目、有髓神经纤维轴突直径、髓鞘厚度4、8、16周分别为(906.25±30.68,1726.25±51.89,2825.13±22.79)、(5.35±0.62,5.46±0.38,5.59±0.80),(1.65±0.37,1.75±0.41,1.83±0.49)均优于对照组,差异有统计学意义(P<0.05).小腿三头肌湿重于术后4周两组间差异无统计学意义(P>0.05),实验组术后8、16周分别为(5.62±0.99,7.38±0.26)恢复优于对照组,差异有统计学意义(P<0.05).结论 种植雪旺细胞的去细胞同种异体神经复合体对神经再生及功能恢复有更好的促进作用.     

柠檬酸锂对大鼠周围神经损伤修复作用的实验研究

目的探讨柠檬酸锂对大鼠坐骨神经损伤后神经再生的影响及潜在机制。方法将36只雄性SD大鼠随机分为对照组和柠檬酸锂组, 每组18只。均行右侧坐骨神经挤压术, 建立周围神经损伤修复模型。柠檬酸锂组在损伤点以远神经外膜下显微注射10.0 g/L柠檬酸锂20 μl。对照组显微注射等体积的生理盐水。随后于各对应时间点检测两组大鼠行为学、神经电生理、小腿三头肌恢复率、再生神经纤维定量与形态学、远端坐骨神经髓鞘碱性蛋白及巨噬细胞浸润变化。结果术后3周, 相较于对照组, 行为学结果显示柠檬酸锂组坐骨神经功能指数显著增加(P<0.05);神经电生理结果显示柠檬酸锂组复合肌肉动作电位波幅显著升高(P<0.05);三头肌恢复率柠檬酸锂组显著升高(P<0.05);再生神经纤维定量与形态学结果显示柠檬酸锂组部分髓鞘及轴突近似圆形, 髓鞘厚薄均匀、板层清晰, 雪旺细胞形态饱满, 髓鞘外可见少量胞质, 核清晰, 且柠檬酸锂组有髓神经数量显著增加(P<0.05);免疫荧光结果显示柠檬酸锂组新生神经纤维较多, 再生纤维较粗, 新生髓鞘包绕轴突结构规整紧密。术后1、2周, 各组上述检测指标之间差异无...     

神经旁路移植的实验研究

目的 探讨并设计神经旁路移植治疗神经瘤性不全损伤的术式及其疗效。方法 SD大鼠20只。实验组：10只大鼠建立神经旁路移植模型。麻醉后显露右侧坐骨神经干，用显微持针钳钳夹其中点10s，于钳夹点近、远端3mm处神经外膜上各开一直径为1．5mm的窗口，取前肢8mm长桡神经与近、远端窗口作端侧缝合。对照组：10只大鼠右侧坐骨神经仅作钳夹。结果 术后8周，实验组的运动神经传导速度(MNCV)、再生有髓神经纤维数及截面积、腓肠肌肌湿重及肌纤维截面积均优于对照组。电镜观察见旁路移植神经中有多量大小不一的再生有髓神经纤维。结论 神经旁路移植具有保留原有神经的功能、不牺牲动力神经的优点，可为临床应用提供实验依据。     

睫状神经营养因子对周围神经损伤后再生的影响

目的 研究睫状神经营养因子（ＣＮＴＦ）对周转神经损伤后再生的影响。方法 用硅胶管桥接大鼠双侧坐骨神经缺损,形成神经再生室。左侧室内注入基因重组人ＣＮＴＦ,右侧注入生理盐水（ＮＳ）。术后１４、３０、６０和９０天取材,行大体、光镜、电镜观察。术后９０天行轴突图像分析、电生理检测及霍乱毒素－辣根过氧化物酶（ＣＢ－ＨＲＰ）逆行追踪。结果 与对照侧相比,实验侧再生有髓神经纤维数目较多、轴突较粗且髓鞘较厚,复合肌肉动作电位（ＣＭＡＰ）的潜伏期较短、神经传导速度较快且波幅较高。实验侧ＣＢ－ＨＲＰ标记的脊髓前角运动细胞数明显多于对照侧。结论 外源性ｈＣＮＴＦ有明显促进周围神经再生作用。     

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。     

Behavioral and morphologic studies of the chronically compressed cauda equina. Experimental model of lumbar spinal stenosis in the rat.

STUDY DESIGN: An experimental model in rats of chronically compressed cauda equina was produced, and behavioral and morphologic changes were examined. OBJECTIVES: To provide a useful model for analyzing the pathophysiologic changes of the cauda equina by chronic compression and to examine behavioral and morphologic changes in this model. SUMMARY OF BACKGROUND DATA: Several animal models have been reported in which various materials were used to compress the cauda equina. However, the pathophysiology of the cauda equina by chronic compression is not yet well understood. Studies in which rats were used are scarce. METHODS: A silicone sheet was applied to the spinal canal at L4 in the rat. Walking durations on treadmill tests and paw-withdrawal latencies to thermal stimuli were measured before and after the operation for 24 weeks. Histologic changes also were examined. RESULTS: Walking durations decreased after chronic compression. However, paw-withdrawal latencies were not significantly changed. Histologically, the number of large-diameter myelinated axons decreased after compression, whereas the number of small-diameter myelinated axons increased. Electron microscopic observation indicated that the continuous degeneration and regeneration of axons occurred throughout the chronic compression experiment. CONCLUSIONS: The current model and behavioral assessments may be useful in analyzing the pathophysiology of chronically compressed cauda equina.     

丙戊酸钠充填导管促进大鼠外周神经再生的实验研究

目的 观察丙戊酸钠(VPA)充填导管对缺损外周神经再生的促进作用.方法 通过建立大鼠坐骨神经缺损模型.用硅胶管(1 cm)进行缺损神经段(0.8 cm)的桥接,局部应用8%VPA注射液10ul,观察VPA对神经再生和运动神经功能恢复的影响.30只大鼠随机分成2组,实验组在硅胶管局部注射VPA注射液;对照组在硅胶管局部注入生理盐水. 结果 术后每只大鼠每2周进行坐骨神经功能指数(SFI)检测,每4周做电生理检查,最后术后12周处死所有大鼠,对坐骨神经进行组织形态学分析.用数字图像分析软件检测有髓神经纤维髓鞘厚度.并对再生神经纤维轴突计数.通过统计软件分析发现SFI,电生理检查,神经组织性形态上两组差异均有统计学意义(P＜0.05). 结论 局部使用VPA于神经缺损的大鼠,可以促进损伤神经的轴突再生和运动功能恢复.因此VPA有望在临床上应用于外周神经损伤病例的治疗.     

Nutritional supply to the cauda equina in lumbar adhesive arachnoiditis in rats

Laminectomy-induced cauda equina adhesion has been proved by rat experiments and postoperative serial MRI in humans. A degenerative change of the cauda equina has been proved when cauda equina adhesion has been prolonged. Since it has not been reported how the nutritional supply is changed in such a condition, we evaluated the glucose supply to the adhered cauda equina in rats. Wistar rats were divided into the following three groups: the control group which received no operation, the laminectomy group which underwent L5-L6 laminectomy only, and the koalin group which received 5 mg of kaolin on the dorsal extradural space following L5-L6 laminectomy. Based on ³H-methyl-glucose uptake study, we analyzed (1) glucose transport from the intraneural vessels to the nerve tissue, and (2) glucose transport from the cerebrospinal fluid to the nerve tissue. We evaluated the relation between the severity of cauda equina adhesion and ³H uptake into the cauda equina. Cauda equina adhesion was observed in 2 of 12 rats in the control group, in 3 of 12 rats in the laminectomy group, and in 18 of 20 rats in the kaolin group. In the ³H-methyl-glucose uptake study, at 12 weeks the glucose transport to the cauda equina from the vessels increased by 44%, and that from the cerebrospinal fluid decreased by 64% in the kaolin group compared with thecontrol group. In the condition of complete cauda equina adhesion, the glucose transport to the cauda equina from the vessels increased by 53% and that from the cerebrospinal fluid remarkably decreased by 72% compared with the normal cauda equina. Considering the greater nutritional importance of the cerebrospinal fluid in the cauda equina, it is most likely that the impairment of nutritional supply to adhered cauda equina may lead to eventual neural degeneration. Received: 8 September 1998 Revised: 28 December 1998 Accepted: 10 February 1999     

Functional recovery of sciatic nerve through inside-out vein graft in rats

Objective: Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model.Methods: The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery.Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments.Results: Functional analysis ofmyelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves.Conclusion: Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis     

神经再生室中神经再生过程和微环境的研究

目的 探讨再生室中神经再生过程、微环境成份及作用。方法 取SD大鼠70只,坐骨神经于股中部切断,用硅胶管套接,神经断端间间距10mm。于术后3d,1、2、3、4、5、6周取材,将10mm再生段由近向远分为S1,S2,S3,S4,S5,共5段,每段2mm,行组织学和电镜观察。结果 术后3d,再生充满棕黄色浑浊液体。术后1周,再生室中央形成条状连接,巨嗜细胞迁移至S3会合。术后2周成纤维细胞、雪旺细胞、内皮细胞迁移到S3会合。有髓神经纤维长入S1,无髓神经纤维长入S3。术后3周无髓纤维通过再生室,有髓纤维长入S3。术后4周有髓纤维通过再生聚集成束。术后5、6周神经纤维数量增多,髓鞘增厚,束膜形成。结论 再生室中再生过程可分为：（1）液体育孕期（1周内）。（2）纤维连接期（1－2周）。（3）神经连接期（2－4周）。（4）神经成熟期（术后4周以上）。神经再生微环境包括再生条件液、纤维基质带和巨嗜细胞、成纤维细胞、雪旺细胞、内皮细胞等成份。     

Enhanced rat sciatic nerve regeneration through silicon tubes implanted with valproic acid

Valproic acid (VPA) is an effective antiepileptic drug and mood stabilizer. It has recently been demonstrated that VPA could promote neurite outgrowth, activate the extracellular signal-regulated kinase pathway, and increase B-cell lymphoma/leukemia-2 (bcl-2)and growth cone-associated protein 43 (GAP-43) levels in spinal cord. We hypothesized that VPA could enhance axonal regeneration in the rat. In the present research, we demonstrate the effect of VPA on peripheral nerve regeneration and recovery of motor function through a silicon tube implanted with VPA. The left sciatic nerves were exposed through dorsal-splitting incisions, and 8-mm nerve sections were excised at the middle of the thigh. Then, a 1.0-cm-long silicone tube (internal diameter,1.0 mm; exterior diameter, 2.0 mm) was used to bridge the nerve deficit, anchored to the proximal and distal terminals of the excised deficit of sciatic nerves with 9-0 nylon epineural suture. Sterile petroleum jelly was used to seal the ends of the tubes to avoid leakage. The rats in the VPA group and control group were locally delivered 10 muL VPA injection (400 mg/5 mL) and normal saline, respectively, after the operation. The sciatic nerve index (SFI) was observed in each animal at 2-week intervals and electrophysiology was studied at 4-week intervals for 12 weeks. Histological and morphometrical analyses were performed at the end of the experiment (12 weeks after the operation). Using the digital image-analysis system, the thickness of the myelin sheath was measured, and total numbers of regenerated axons were counted. There was a significant difference in SFI, electrophysiological index (motor-nerve conduct velocity, amplitude of activity potential), and morphometrical results (regenerated axon number and thickness of myelin sheath) in nerve regeneration between the VPA group and controls ( P < 0.05). The results demonstrated that VPA is able to enhance sciatic nerve regeneration in rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.     

Implantation of cauda equina nerve roots through a biodegradable scaffold at the conus medullaris in rat

Background contextTraumatic injuries occurring at the conus medullaris of the spinal cord cause permanent damage both to the central nervous system and to the cauda equina nerve roots.PurposeThis proof-of-concept study was to determine whether implanting the nerve roots into a biodegradable scaffold would improve regeneration after injury.MethodsAll experimental works involving rats were performed according to the approved guidelines by the Mayo Clinic Institutional Animal Care and Use Committee. Surgical procedures were performed on 32 Sprague-Dawley rats. Four ventral cauda equina nerve roots were reimplanted either directly into the ventral cord stump or through a poly(lactic-co-glycolic acid) (PLGA) scaffold. These experimental groups were compared with a control group in which the nerves were inserted into a muscle fascia barrier that was placed between the spinal cord and the nerve roots. Animals were sacrificed at 4 weeks.ResultsThere was no difference in motor neuron counts in the spinal cord rostral to the injury in all treatment groups, implying equal potential for the regeneration into implanted nerve roots. One-way analysis of variance testing, with Tukey post hoc test, showed a statistically significant improvement in axon regeneration through the injury in the PLGA scaffold treatment group compared with the control (p<.05, scaffold n=11, control n=11).ConclusionsThis pilot study demonstrated that a PLGA scaffold improved regeneration of axons into peripheral nerve roots. However, the number of regenerating axons observed was limited and did not lead to functional recovery. Future experiments will employ a different scaffold material and possible growth factors or enzymes to increase axon populations.