兔坐骨神经损伤修复后骶交感神经节内c-ret mRNA表达的变化

目的 探讨兔坐骨神经损伤修复后骶交感神经节内c-ret mRNA表达的变化。方法 用组织原位杂交方法观察兔坐骨神经损伤修复1，3，7，14，30，60d后c-ret mRNA在骶交感神经节内的表达。结果 兔坐骨神经损伤修复3d后，在损伤侧骶交感神经节内c-ret mRNA表达明显上调，在损伤后7d达高峰，损伤后14d与30d仍维持较高水平，损伤后60d基本恢复到正常水平。结论 C—ret mRNA在兔坐骨神经损伤修复后骶交感神经节内表达的变化，提示在坐骨神经损伤修复后给予外源性胶质源性神经营养因子(GDNF)可能有利于交感神经纤维的再生。     

小间隙桥接周围神经损伤Gap-43表达变化的实验研究

目的 观察采用小间隙桥接法修复大鼠坐骨神经损伤时套管内Gap-43 mRNA含量的变化.方法 SD 大鼠78只,随机分为13组,每组6只.其中6组切断右侧坐骨神经并原位缝合,另6组切断右侧坐骨神经采用小间隙桥接法修复.1组为正常对照.分别于术后1 d、3 d、5 d、7 d、14 d、28 d以吻合口为中心,取上下各5 mm共1 cm坐骨神经提取总RNA,采取实时荧光定量反转录聚合酶链(Real-time RT-PCR)技术检测组织中Gap-43 mRNA含量变化.结果 鼠周围神经中Gap-43 mRNA含量在正常组织有低水平表达,在损伤后1 d即升高,在损伤后28 d降低,但仍高于正常坐骨神经内表达水平.桥接缝合组Gap-43表达高于外膜缝合组.结论 大鼠坐骨神经损伤后采用不同修复方法其Gap-43基因表趋势相似,但由套管形成的封闭空间蕴含了较高的神经再生相关因子,有利于神经再生.     

大鼠坐骨神经损伤后Spastin表达变化的实验研究

目的观察内源性Spastin蛋白在大鼠坐骨神经损伤后再生过程中的表达变化,探讨其在周围神经再生过程中的作用和意义。方法取成年雄性SD大鼠36只,随机分为实验组(n=30)和假手术对照组(n=6),实验组建立坐骨神经挤压损伤模型,对照组仅暴露坐骨神经。实验组分别于术后1、3、7、14、28 d(n=6),假手术组于术后7 d取大鼠坐骨神经相应节段的L4~6脊髓组织,采用实时荧光定量PCR检测Spastin m RNA相对表达量,Western blot检测Spastin蛋白表达;并取损伤远端5 mm处神经组织,通过透射电镜观察坐骨神经远端轴突的超微结构变化。结果两组大鼠L4~6节段脊髓组织中Spastin蛋白表达和基因表达变化趋势基本一致。实验组Spastin蛋白和基因表达量呈先下降后逐渐上升的趋势,于术后7 d降至最低,28 d时达初始水平。其中实验组术后3、7、14 d Spastin蛋白和基因表达量显著低于假手术对照组(P0.05);术后1、28 d与假手术对照组比较差异无统计学意义(P0.05)。实验组术后3、7、14 d Spastin蛋白和基因表达量显著低于术后1 d和28 d(P0.05),术后1 d和28 d比较差异无统计学意义(P0.05)。透射电镜观察发现实验组术后1、3、7 d,坐骨神经损伤远端髓鞘严重破坏;术后14 d雪旺细胞增生;术后28 d可见大量有髓神经纤维,接近正常形态。结论内源性Spastin蛋白在坐骨神经损伤后再生过程中出现了表达变化,提示Spastin可能在周围神经损伤后的再生过程中起着一定作用。     

银杏酮酯对大鼠坐骨神经损伤后神经生长因子基因表达的影响

目的 观察银杏酮酯（EGb50）对大鼠坐骨神经损伤后神经生长因子（NGF）mRNA表达的影响。方法 取SD大鼠78只，其中72只切断右侧坐骨神经并缝合，随机分成损伤对照组与实验组，另6只为正常对照组。实验组给予EGb50200mg／kg／d溶于1．0ml生理盐水中灌胃，损伤对照组每天给予生理盐水1．0ml灌胃，正常对照组不作任何处理。分别于术后1、3、7、14、21及28d取吻合口远段的神经、相应节段的脊神经节及脊髓，采用逆转录-多聚酶链反应技术检测所取组织中NGFmRNA的表达水平。结果 实验组坐骨神经中NGFmRNA的表达在术后7、14和21d明显高于对照组（P〈0．05）。术后7d和14d，实验组脊神经节及脊髓中NGFmRNA的表达高于对照组（P〈0．05）。结论 大鼠坐骨神经损伤后用银杏酮酯治疗，在早期可促使坐骨神经及相应节段脊神经节和脊髓组织中的NGFmRNA的表达增加。     

小间隙桥接周围神经对缝合口神经调节蛋白-1表达变化的影响

目的 观察采用小间隙桥接法修复大鼠坐骨神经损伤时套管内神经调节蛋白-1(NRG-1)mRNA含量的变化.方法 取SD大鼠78只,体质量200～250 g.随机分为3组,其中实验组A和实验组B各有大鼠36只,正常对照组有大鼠6只.2组实验组根据观察时间的不同再分为6组,每组有大鼠6只.实验组A:切断并原位缝合右侧坐骨神经.实验组B:切断并采用小间隙桥接法修复右侧坐骨神经.分别于术后1、3、5、7、14、28 d以缝合口为中心,取上下各5 m共1 cm坐骨神经提取总RNA,采取实时荧光定量反转录聚合酶联(Real-time RT-PCR)技术检测组织中NRG-1mRNA的含量变化.结果 大鼠周围神经中NRG-1 mRNA含量在正常组织有低水平表达,在损伤后1 d即明显升高,在损伤后14 d降低,随即恢复较高水平,持续至伤后28 d.在部分时间点高于外膜缝合组.结论 大鼠坐骨神经损伤后采用不同修复方法NRG-1基因表达变化不同步,提示套管形成的封闭空间蕴含了较高的神经再生相关因子,有利于神经再生.     

坐骨神经损伤脊髓神经元退变与组织型纤溶酶原激活物及其抑制物表达的关系研究

目的 探讨坐骨神经离断后脊髓内组织型纤溶酶原激活物(tissue plasminogen activator,tPA)及其抑制物纤溶酶原激活物抑制物1(type-1 plasminogen activator inhibitor,PAI-1)、神经丝氨酸蛋白酶抑制剂(neuroserpin,NSP)的表达与神经元退变的关系.方法 将56只雄性SD大鼠随机分为实验组和对照组.对实验组大鼠行坐骨神经离断术,于术后各时间点取材伤侧脊髓L4～6节段,经Nissl染色后,运用透射电镜观察神经元退变及死亡情况;运用免疫组化染色和半定量RT-PCR检测tPA、PAI-1及NSP的表达变化.结果 坐骨神经离断术后7d,伤侧相应脊髓节段前角外侧核神经元存活率显著下降,术后21 d神经元存活率为61.6％.电镜观察显示,术后7d开始,脊髓前角可见处于不同凋亡阶段的神经元及神经胶质细胞,术后14 d开始脊髓后角也可见少数凋亡样变的神经元及胶质细胞.免疫组化结果显示,坐骨神经损伤后ld,伤侧脊髓Ⅴ～Ⅸ板层内tPA表达水平开始上调(P＜0.05),第7天时达到高峰后下降,至术后21 d仍未恢复正常水平(P＞0.05);PAI-1则在正常脊髓及损伤后脊髓均未能检测到.半定量RT-PCR结果显示,tPA的mRNA变化趋势与蛋白表达基本相同,略早于后者;NSP的mRNA术后1d内迅速上调,随后2周都处于较高水平,21 d才基本回复正常.结论 坐骨神经离断后同侧相应脊髓节段近50％的前角运动神经元死亡可能与损伤刺激脊髓灰质内神经元和小胶质细胞合成、释放tPA增加有关,同时损伤也促使tPA抑制物NSP表达上调,后者可能发挥神经保护作用.     

细胞外ATP对失神经支配后骨骼肌和脊髓前角运动神经元ATPase活性的影响

目的了解细胞外三磷酸腺苷（adenosine triphosphate，ATP）对坐骨神经损伤后腓肠肌和相应脊髓节段ATPase活性的影响。方法SD大鼠168只，切断其右侧坐骨神经后，随机分为3组，每组56只。损伤组：坐骨神经离断后不修复。对照组和实验组在坐骨神经离断修复后，于腓肠肌内注射同体积的生理盐水和ATP。术中和术后每日用药1次至取材为止。3组分别于手术后12h、1d、3d、7d、14d、28d、56d时每组各取8只大鼠，测定腓肠肌和L4-6水平脊髓前角运动神经元Na-K-ATPase及Ca-ATPase活性。术后7d、14d、28d、56d取双侧腓肠肌称肌湿重。结果实验组术后能显著改善ATPase的活性，这种改变与肌湿重的变化相一致，与损伤组和对照组相比差异有统计学意义（P〈0．05）。结论细胞外ATP可通过对ATPase的影响，对失神经骨骼肌和脊髓前角运动神经元具有一定的保护和促进功能恢复作用。     

大鼠脊髓损伤后坐骨神经GDNFmRNA表达变化及意义

目的：探讨大鼠脊髓损伤后GDNFmRNA在坐骨神经的表达变化及其意义。方法：Allen's方法致大鼠T13段脊髓不完全损伤后，以β-Actin为内参照物，应用半定量RT-PCR方法，观察大鼠坐骨神经在脊髓损伤前、后不同时间GDFNmRNA表达变化。结果：脊髓损伤前GDNFmRNA在大鼠坐骨神经仅有微量表达，脊髓损伤后24h表达增加4倍，72h增加15倍，7d增加3倍，10d仍高于正常水平。结论：脊髓损伤后坐骨神经高表达GDNFmRNA，是神经元通过“胞体--轴突--靶器官”途径自我保护的一种反应形式，GDNF治疗脊髓损伤时应早期用药以获得最佳效果。     

大鼠坐骨神经切断后相应脊髓节段中P-S473-AKT表达的变化

目的 探讨成年Wister大鼠在坐骨神经切断后P-S473-Akt于相应脊髓节段前角运动神经元内的表达变化.方法 选取健康成年雄性Wister大鼠50只,随机分为坐骨神经切断组和对照组,分别于术后1、2、4、6、8周5个时相处死后取其L4～L6脊髓,利用免疫组织化学技术检测P-S473-Akt在相应脊髓节段中的表达变化,并利用影像分析系统进行统计学分析.结果 在对照组中P-S473-Akt表达呈阴性,随着时间的变化无明显改变.坐骨神经切断组与对照组在5个时相中的变化:1周时两组比较差异无统计学意义;2周时坐骨神经切断组运动神经元胞体内P-S473-Akt有明显表达,4周时达到高峰,6～8周时逐渐下调,8周时仍可见细胞核内有少量表达.结论 坐骨神经切断可导致成年大鼠相应脊髓节段中前角运动神经元P-S473-Akt表达明显增加,可能对损伤的运动神经元发挥保护作用.     

补阳还五汤和腰交感神经节切除对大鼠坐骨神经损伤的影响

目的 探讨补阳还五汤和腰交感神经节切除对大鼠坐骨神经损伤的影响.方法： 60 只SD大鼠损伤左侧坐骨神经,随机分为2组,对照组钳夹并切除腰交感神经节（LSG）,实验组在钳夹并切除腰交感神经节后加用补阳还五汤口服及药浴治疗.观察对照组和实验组大鼠左足皮肤温度、坐骨神经功能指数（SFI）和坐骨神经传导速度（SNCV）的差异.结果：实验开始后1、2周实验组皮肤温度较对照组有明显升高（P〈 0.01）.实验组的坐骨神经功能指数2、4、6周恢复均快于对照组（P〈0.01）.分别测实验组、对照组的坐骨神经传导速度.实验组2、4、6周坐骨神经传导速度恢复快于对照组（P〈 0.01）.结论：补阳还五汤口服加药浴并切除腰交感神经节,对大鼠周围神经损伤后再生有明显的促进作用.     

银杏叶提取物对大鼠坐骨神经切断后HSP 70在脊髓与脊神经节中表达的影响

目的 研究周围神经损伤后脊髓热休克蛋白(heat shock proteirns,HSPs)的表达变化,及银杏叶提取物EGb 761对其影响,探讨银杏叶提取物对周围神经损伤的保护机制.方法 取成年雄性SD大鼠144只,随机分成三组:对照组、坐骨神经切断组、坐骨神经切断+银杏叶提取物干预组[术后每天用EGb 761(100 mg·kg-1*d-1,溶于2 ml SAL)灌胃,直到取材],每组48只.术后6 h、12 h、1 d.2 d,4 d、7 d、14 d、28 d取L4～6节段脊髓节段,采用免疫组织化学方法检测HSP 70在脊髓前角及脊神经节中的表达.结果 正常大鼠脊髓与神经节中均有少量HSP 70表达,在坐骨神经切断后表达迅速增加,持续一段时间后又回到正常水平,用EGb 761干预后,HSP 70表达早期较未干预组表达低,后期表达增高,且表达持续时间长.结论 银杏叶提取物EGb 761可以增加神经损伤后HSP 70表达,可能是银杏叶提取物保护神经,促进神经再生作用的机制.     

周围神经损伤后急性期NGF、BDNF基因表达变化的实验研究

目的 观察大鼠坐骨神经损伤急性期神经生长因子(NGF)、脑源性神经生长因子(BDNF)mRNA含量的变化.方法 取SD大鼠48只,随机均分为8组.其中7组切断右侧坐骨神经并原位缝合,另1组为健康对照组.分别于术后1,2,3,4,5,6,7 d以吻合口为中心,取上下各5 mm共1cm坐骨神经提取总RNA,采取反转录-多聚酶链反应(RT-PCR)技术检测组织中NGF、BDNF mRNA含量变化.结果 大鼠周围神经中NGF mRNA含量在正常组织有低水平表达.在损伤后1 d即明显升高,在损伤后5 d降低,随即恢复较高水平.BDNF在正常组织亦低水平表达,在损伤后4 d明显增高,5,6 d低至正常组织水平,7 d恢复较高水平.结论 大鼠坐骨神经损伤急性期NGF、BDNF基因表达变化不同步,提示不同因素在损伤后急性期NGF、BDNF含量变化中起作用.     

Adenoviral gene transfer in the peripheral nervous system

Background Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 10⁸ pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves.     

Vasoactive intestinal peptide and nerve regeneration.

The role of vasoactive intestinal peptide (V.I.P.) in nerve regeneration was investigated by assessing the changes in immunoreactive V.I.P. levels in rat sciatic nerves following injury and repair. 60 rats were divided into three surgical groups and one control group: In group I (primary repair), sciatic nerves were divided and immediately repaired; in group II (secondary repair), sciatic nerves were divided and repaired two weeks later; in group III (no repair), sciatic nerves were divided and not repaired; and in group IV (controls), sciatic nerves were exposed but not divided. Animals were sacrificed at three days and at weekly intervals. Their sciatic nerves were extracted and assayed for V.I.P. concentrations by a specific radioimmunoassay. The mean V.I.P. concentration varied between 22 and 46 pg./mg. protein in the control nerves and between 60 and 529 pg./mg. protein in all other groups. In the three surgical groups the levels were significantly higher in proximal than in distal stumps. Following nerve injury, there was an increase in V.I.P. concentration in the injured and repaired areas. This increase was greater in injured non-repaired areas and was highest in the first 48 hours, but continued during regeneration. The accumulation of V.I.P. in divided nerves occurred in response to nerve injury.     

腺病毒介导的NT—3基因在大鼠从骨神经的表达

目的观察腺病毒介导的神经营养素 - 3(neurotrophin- 3,NT- 3)基因在大鼠坐骨神 经雪旺细胞 ( Schwann cells,SCs) 的表达.方法 NT- 3重组腺病毒在 293细胞中培养繁殖并 测定滴度后,直接注入大鼠损伤修复的坐骨神经内,不同时间点取材,采用免疫组织化学染 色检测 NT- 3蛋白的表达,并用 LEICA M550型图像分析仪对坐骨神经切片 NT- 3免疫组化染色 强弱进行定量评价.结果坐骨神经损伤修复后直接注射 Ad- NT- 3,2 d后出现 NT- 3免疫组化 染色阳性产物,主要位于吻合口附近,阳性产物呈平行条纹状排列,7 d时显著增加 ( 与 2 d组相比,P< 0.01),14 d和 28 d时有所下降 ( 与 7 d组相比,P< 0.01),两者差异无显著 性意义 ( P >0.05),但与 2 d组相比,仍维持在较高的水平 ( P< 0.01).而正常坐骨神经、 损伤修复后注射 Ad- LacZ或生理盐水的坐骨神经 NT- 3免疫组化染色结果为阴性.结论 NT- 3基因能通过腺病毒介导转入损伤修复后周围神经的 SCs并表达 NT- 3蛋白,为腺病毒介导神经 营养因子基因治疗促进周围神经损伤再生提供了初步的理论和实验依据.     

腺病毒介导的 NT- 3基因在大鼠坐骨神经的表达

目的观察腺病毒介导的神经营养素-3(neurotrophin-3,NT-3)基因在大鼠坐骨神经雪旺细胞(Schwanncells,SCs)的表达。方法NT-3重组腺病毒在293细胞中培养繁殖并测定滴度后,直接注入大鼠损伤修复的坐骨神经内,不同时间点取材,采用免疫组织化学染色检测NT-3蛋白的表达,并用LEICAM550型图像分析仪对坐骨神经切片NT-3免疫组化染色强弱进行定量评价。结果坐骨神经损伤修复后直接注射Ad-NT-3,2d后出现NT-3免疫组化染色阳性产物,主要位于吻合口附近,阳性产物呈平行条纹状排列,7d时显著增加(与2d组相比,P<0.01),14d和28d时有所下降(与7d组相比,P<0.01),两者差异无显著性意义(P>0.05),但与2d组相比,仍维持在较高的水平(P<0.01)。而正常坐骨神经、损伤修复后注射Ad-LacZ或生理盐水的坐骨神经NT-3免疫组化染色结果为阴性。结论NT-3基因能通过腺病毒介导转入损伤修复后周围神经的SCs并表达NT-3蛋白,为腺病毒介导神经营养因子基因治疗促进周围神经损伤再生提供了初步的理论和实验依据。     

Local administration of neurotrophic growth factor in subcutaneous silicon chambers enhances the regeneration of the sensory component of the rat sciatic nerve.

An experimental model for local administration of neurotrophic growth factor (NGF) in peripheral nerve lesions is tested. The model consists of a subcutaneous reservoir connected to the sciatic nerve neurorrhaphy. The right sciatic nerves were exposed, severed, and repaired at a level 1.5 cm proximal to their trifurcation. Then, a dome-shaped silicone reservoir connected to the proximal end of a silicone tube was placed subcutaneously in the dorsum of the experimental animal. The distal end of the connecting tube was located in the nerve neurorrhaphy. Two experimental groups were made: Group A (n = 90) received daily doses of a solution containing NGF-7S during the first 4 weeks after surgery and a single weekly dose thereafter. Within this group, three subgroups of 30 rats each were made: A-4 sacrificed 4 weeks after surgery, A-8 sacrificed after 8 weeks, and A-12 after 12 weeks. Group B (n = 90) received the same vehicle solution without NGF under the same schedule and volume as in Group A. Three subgroups were also made as in Group A depending on the survival period. In order to locate the neurons in the dorsal root ganglia, the retrograde tracer horseradish peroxidase was administered at the proximal stump of the sciatic nerve (tibialis branch), which was severed 1 cm distal to the sciatic trifurcation. In respect of the nonoperated side, the percentage between the number of dorsal root ganglia neurons in the NGF-treated group was significantly higher than in the control group (P < 0.001). These results demonstrate that percutaneous administration of multiple doses of NGF in this model enhances sensory nerve regeneration after sciatic lesions evaluated by horseradish peroxidase labeling of dorsal root ganglia neurons.     

Expression of vascular endothelial growth factor and its fetal liver kinase-1 receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats

egenerationofinjured peripheralnervesiscloselyrelatedtosomegrowthfactorssecretedbycentralneurons .Vascularendothelialgrowthfactor (VEGF)isapotentangiogenicfactor ,which possessesspecificmitogenicactivityforvascularendothelialcells ,stimulatestheformationofnascentbloodvesselsandenhancesvascularpermeability .1RecentevidencesindicatethatVEGFcanalsoactdirectlyonneuronstoinduceneurotrophiceffects.Forexample ,VEGF promotestheproliferationofculturedcerebralcorticalneurons ,stimulatestheaxonaloutg…