Roundaboutl对大鼠周围神经再生轴突导向的影响

目的:探讨Roundaboutl(Robo1)对周围神经再生轴突的导向作用.方法:利用坐骨神经压榨和切断后立即缝合模型,将可溶性Robo1-Fc融合蛋白注入到压榨或缝合部位,形成压榨组和缝合组;以人的Fc蛋白注入上述模型形成两对照组.对术后不同时间点行再生纤维半薄切片、图像分析、步态分析.结果:术后21 d两实验组再生纤维数均明显少于对照组;至术后42 d,与对照组比较差异无统计学意义.术后14、21 d,压榨实验组坐骨神经功能指数(SFI)明显不及压榨对照组.术后14、21 d,缝合实验组与缝合对照组比较,SFI差异无统计学意义;而在术后35、42 d明显少于缝合对照组.结论:Robo1可能促进周围神经再生并参与调控再生轴突生长方向.     

大鼠坐骨神经横断后背根节Robo1表达的时空模式

目的:观察周围神经损伤对脊神经节(DRG)Robo1表达的影响,初步了解Robo1在周围神经再生中的作用.方法:横断成年SD大鼠坐骨神经或脊神经后根,术后3、 7、 14、 21、 28d取其腰4～6DRG,用RT-PCR和免疫组织化学方法检测Robo1表达,免疫荧光双标法检测Robo1与Robo2有否共表达.结果:坐骨神经横断能上调DRG Robo1的表达,术后7～14d达高峰,而脊神经后根切断对其表达无影响;DRG感觉神经元不共表达Robo1和Robo2.结论:Robo1在DRG内的表达存在细胞特异性,周围突受损能上调Robo1在DRG中的表达,其表达模式与多数神经营养因子类似,提示Robo1可能参与感觉神经元再生过程.     

大鼠坐骨神经损伤后诱导型一氧化氮合酶表达的变化

为了探讨大鼠坐骨神经损伤后再生过程中诱导型一氧化氮合酶(iNOS)表达的变化及意义,本研究采用大鼠坐骨神经切断缝合模型,分别于术后1、3、7、14、21及28d取吻合口远端的神经,采用免疫组织化学和实时荧光定量聚合酶链反应(RT-PCR)方法检测损伤神经远端iNOSmRNA及其蛋白的表达水平。结果显示:假手术对照组坐骨神经中未见明显的iNOS阳性产物,iNOSmRNA表达极低。实验组神经损伤后iNOSmRNA及其蛋白的表达水平均明显增高(P<0.01),iNOS阳性产物的吸光度(A)值在术后7d达高峰。iNOSmRNA表达在术后1、3、7d维持较高水平,此后则明显下降。上述结果说明大鼠坐骨神经损伤后神经纤维中iNOS的表达增加,iNOS可能在周围神经损伤后的再生过程中起着一定的作用。     

靶肌肉注射促红细胞生成素对大鼠周围神经再生的作用

目的探讨靶肌肉注射人重组促红细胞生成素（recombinant human erythropoietin，rh-EP0）对大鼠坐骨神经损伤后神经再生的作用。方法选用健康雄性SD大鼠12只，制备大鼠右侧坐骨神经钳夹损伤模型。实验动物随机分为2组，每组6只，EPO组：靶肌肉注射rh-EPO2500U／kg；对照组：注射同体积的生理盐水。术后第7d、14d、21d观察坐骨神经功能指数（SFI），第21d组织学观察脊髓腰膨大（L4～L6）、夹伤远端坐骨神经、损伤侧腓肠肌组织并作图象分析测定脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标。结果术后第7d两组SFI无显著性差异，术后第14d、21dEPO组SFI恢复程度明显大于对照组，差别有显著性意义（P〈0．05）；术后第21d损伤侧脊髓前角运动神经元数、再生有髓神经纤维数、髓鞘厚度、轴突直径和腓肠肌肌细胞横截面积等指标，EPO组均优于对照组（P〈0．01，P〈0．05）。结论靶肌肉注射rh-EPO能促进周围神经再生和功能恢复。     

银杏酮酯对大鼠坐骨神经损伤后生长相关蛋白43表达的影响

目的:研究银杏酮酯对大鼠坐骨神经损伤后生长相关蛋白43(GAP-43)表达的影响。方法:SD大鼠78只,随机分成正常组、损伤对照组与实验组,给予不同处理,后两组切断右侧坐骨神经并缝合。实验组给予银杏酮酯200mg·kg-1.d-1溶于1ml生理盐水中灌胃,损伤对照组给予生理盐水1ml灌胃,正常组不做处理。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,应用免疫组织化学和图像分析的方法研究所取组织中GAP-43蛋白的表达并进行定量分析。结果:实验组坐骨神经、脊神经节及脊髓中GAP-43蛋白免疫阳性区域面积和平均光密度值在术后7、14和21d明显高于对照组。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GAP-43蛋白表达增加。     

大鼠坐骨神经切断后Robo2在脊髓及背根节的表达变化

目的:研究坐骨神经损伤后Roundabout 2(Robo2)在成年大鼠背根节和脊髓的表达变化。方法:健康成年雌性SD大鼠坐骨神经切断后分别存活3～28d,取其L_(4～6)背根节(DRG)和脊髓;利用RT-PCR和免疫组织化学技术检测Robo2在上述组织中的表达变化。图像分析技术对阳性细胞的灰度值进行测定。结果:正常DRG感觉神经元表达Robo2 mRNA和蛋白质,脊髓前角运动神经元不表达。坐骨神经切断后3 d DRG内Robo2表达增加,7～14 d达高峰,21～28 d恢复到正常水平。结论:坐骨神经切断可导致DRG内Robo2的表达上调,可能与早期的感觉轴突再生有关。     

脑源性神经营养因子对出生后大鼠周围神经髓鞘化的作用

目的:探讨脑源性神经营养因子(BDNF)对出生后大鼠周围神经髓鞘化的作用及其作用机制.方法:用光镜和电镜方法检测坐骨神经纤维的髓鞘化情况;用免疫印迹方法检测坐骨神经p75的表达;用免疫荧光方法检测坐骨神经雪旺细胞内核转录因子(NF-kB)的表达.结果:出生后3 d,与对照组相比,实验组坐骨神经髓鞘化的神经纤维数目减少,p75表达下调,雪旺细胞内NF-KB转移率下调;出生后14 d,与对照组相比,实验组坐骨神经出现较多的髓鞘化异常.结论:内源性BDNF影响出生后大鼠周围神经的髓鞘化,尤其在早期作用明显;内源性BDNF主要通过p75和NF-kB信号途径调节出生后大鼠周围神经的髓鞘化.     

坐骨神经选择性损伤痛模型小鼠脊髓背角线粒体解偶连蛋白UCP4的表达变化

目的:观察线粒体保护蛋白解偶联蛋白4(uncoupling protein 4,UCP4)在坐骨神经选择性损伤(sparednerve injury,SNI)模型小鼠脊髓背角中的表达变化。方法:健康C57BL/6小鼠分为假手术对照组(n=21)和坐骨神经分支选择性损伤SNI组(n=21),实验组损伤后饲养3,7,14 d。行为学采用测定小鼠热痛阈和Von Frey机械性痛阈;用免疫荧光组织化学染色法检测对比小鼠脊髓L3-6节段背角内UCP4免疫阳性细胞的数量。结果:SNI术后3 d,小鼠手术侧热痛阈和机械性痛阈明显低于假手术组,术后14 d达最低值。UCP4分布于正常小鼠脊髓背角,SNI后3 d损伤组小鼠脊髓背角中的UCP4表达降低,图像分析表明UCP4的光密度与对照组比较,差异有统计学意义(P<0.05);脊髓背角中UCP4的表达在14 d时其降低程度最明显,图像分析表明光密度与对照组、3d和7 d比较,差异均有统计学意义(P<0.05)。结论:SNI后脊髓背角线粒体保护蛋白UCP4表达降低可能参与神经病理性疼痛的中枢敏化过程。     

牛膝活性提取物对大鼠坐骨神经横断的修复作用

目的:研究牛膝活性提取物(ABPPk)对大鼠坐骨神经横断的修复作用.方法:SD大鼠随机分为5组,ABPPk低、中、高剂量组(剂量分别为2.5、5.0、10.0 mg/kg),阳性对照组(弥可保,0.13mg/kg),生理盐水组(阴性对照).行大鼠左侧坐骨神经横断缝合术,术后每日腹腔注射给药.于术前1d和术后1、7、14、21、28 d行热痛阈实验,测定大鼠热痛阈值;术后7、14、21、28d行足迹实验,测定大鼠坐骨神经功能指数;术后28 d行电生理检测,测定大鼠复合肌动作电位;透射电镜观察大鼠再生有髓神经纤维的髓鞘厚度和髓鞘板层数目;行Masson三色染色观察大鼠腓肠肌肌纤维横截面.结果:与生理盐水组相比,ABPPk中、高剂量组大鼠热痛阈值、坐骨神经功能指数、复合肌动作电位幅度、再生有髓神经纤维髓鞘厚度、板层数目及腓肠肌肌纤维横截面积均增高,各剂量组之间呈量效关系.结论:ABPPk有利于轴突再生和髓鞘形成,能促进大鼠周围神经损伤后功能和形态的恢复.     

bFGF对同种异体神经移植后周围神经再生的影响

目的 :探讨bFGF对同种异体神经移植后周围神经再生的影响。方法 :将反复冻融的大鼠神经移植于另一大鼠的坐骨神经 ,实验组注射bFGF 1 0 0u/d共 1 0d ,对照组注射生理盐水 1 0d。术后大鼠存活 1 2周 ,光镜下用体视学方法测试再生神经纤维的面数密度 (NA)、面积密度 (AA)、横切面面积 (AE)、脊髓前角运动细胞和脊神经节细胞的体密度 (VV)、数密度 (NV)。结果 :两组均可见再生神经纤维长入异体移植神经并向远段延伸。实验组再生神经纤维的NA、AA、脊髓前角运动细胞和脊神经节细胞的VV、NV 与对照组的比较 ,有显著性差异。结论 :bFGF能促进周围神经再生 ,对脊髓前角运动细胞和脊神经节细胞的存活有保护作用。     

Erythropoietin promotes peripheral nerve regeneration in rats by upregulating expression of insulin-like growth factor-1

Introduction

Erythropoietin (EPO) has been shown to have beneficial effects on peripheral nerve damage, but its mechanism of action remains incompletely understood. In this study we hypothesized that EPO promotes peripheral nerve repair via neurotrophic factor upregulation.

Material and methods

Thirty adult male Wistar rats were employed to establish a sciatic nerve injury model. They were then randomly divided into two groups to be subjected to different treatment: 0.9% saline (group A) and 5000 U/kg EPO (group B). The walking behavior of rats was evaluated by footprint analysis, and the nerve regeneration was assessed by electron microscopy. The expression of insulin-like growth factor-1 (IGF-1) in the injured sciatic nerves was detected by immunohistochemical analysis.

Results

Compared to saline treatment, EPO treatment led to the growth of myelin sheath, the recovery of normal morphology of axons and Schwann cells, and higher density of myelinated nerve fibers. Erythropoietin treatment promoted the recovery of SFI in the injured sciatic nerves. In addition, EPO treatment led to increased IGF-1 expression in the injured sciatic nerves.

Conclusions

Erythropoietin may promote peripheral nerve repair in a rat model of sciatic nerve injury through the upregulation of IGF-1 expression. These findings reveal a novel mechanism underlying the neurotrophic effects of EPO.     

坐骨神经瓦勒变性大鼠许旺细胞生物学特性及分泌功能变化

背景：研究表明外周神经损伤后,许旺细胞在基底膜管内形成Bunger带,引导再生轴突延伸,但具体作用机制目前尚不清楚。 目的：观察大鼠坐骨神经损伤后瓦勒变性对许旺细胞生物学特性及分泌功能的影响。 方法：建立大鼠坐骨神经横切模型,分为坐骨神经瓦勒变性组(坐骨神经横断组)和手术对照组。采用神经段单酶消化法分离培养许旺细胞,光镜下观察细胞形态变化,S-100免疫荧光鉴定。取第1代许旺细胞,利用计数法绘制14 d内许旺细胞的生长曲线,MTT法检测14 d内许旺细胞增殖活性,酸性磷酸酶法检测许旺细胞黏附能力,ELISA法检测神经生长因子浓度。 结果与结论：坐骨神经段培养第14天,坐骨神经横断组神经段边缘可见大量许旺细胞,呈线形排列;手术对照组许旺细胞数量少,呈散在分布,两组许旺细胞S-100均呈阳性表达。许旺细胞传代培养第3天,两组许旺细胞均进入对数增长期,随时间延长,细胞数及细胞增殖吸光度值均呈上升趋势,坐骨神经横断组细胞数及增殖吸光度值明显高于手术对照组(P < 0.05);坐骨神经横断组许旺细胞黏附能力明显高于手术对照组(P < 0.05);ELISA法检测示,坐骨神经横断组神经生长因子浓度在培养第4,6,8,10,12,14天时均高于手术对照组(P < 0.05)。结果表明大鼠坐骨神经损伤后两三周,瓦勒变性对许旺细胞生物学功能具有显著影响,可诱导许旺细胞幼稚化,促使许旺细胞在短期内迅速分裂增殖,并分泌大量神经营养因子及细胞外黏附成分,为再生轴突的延伸提供适宜的神经微环境;并增加细胞黏附能力,为外周神经损伤修复提供适宜的神经微环境。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Dynamic changes in Robo2 and Slit1 expression in adult rat dorsal root ganglion and sciatic nerve after peripheral and central axonal injury

Robos are transmembrane receptors that mediate Slit signaling to repel growth cone outgrowth and neural migration in the developing central nervous system. Their distribution and function in the peripheral nervous system remains unclear. In the present study, we examined expression of Slit1 and Robo2 in adult rat dorsal root ganglion (DRG), spinal cord and sciatic nerve after peripheral nerve injury (axotomy). In control rats, Slit1 and Robo2 mRNA and protein were expressed at basic levels in the L5 and L6 DRGs. Sciatic transection resulted in a significant up-regulation of both Robo2 and Slit1 mRNA and protein (p<0.05 versus control). The peak of Slit1 and Robo2 expression occurred at days 7 and 14, respectively, and returned to control levels at days 28 and 21 post-axotomy, respectively. By contrast, injury to the central axons of the DRG by dorsal rhizotomy did not up-regulate Slit1 and Robo2 expression. Robo2 staining was stronger in small diameter neurons than in large diameter neurons in control DRG. Interestingly, post-axotomy, Robo2 immunostaining increased in the large diameter neurons and the number of Robo2 positive large diameter neurons increased significantly relative to controls. Non-neuronal cells surrounding the primary sensory neurons, including the satellite cells, were Slit1-positive, and Slit1 protein was expressed in the myelin sheath and non-neural cells in both intact and degenerating sciatic nerve axons. Sciatic nerve transection also led to an accumulation of Slit1 protein in peripheral region of the traumatic neuroma. In conclusion, we report an altered expression and redistribution of Robo2 and Slit1 in the DRG and sciatic nerve trunk after peripheral axotomy. Our results indicate that Slit1 and Robo2 likely play an important role in regeneration after peripheral nerve injury.     

成年大鼠脊髓背角神经元受损轴突在外周神经移植物中的再生速度

用抗神经丝(anti-neurofilament)单克隆抗体免疫组织化学技术,研究大鼠脊髓背角神经元受损轴突在外周神经移植物中的再生速度,发现脊髓背角神经元受损轴突在外周神经移植物中的最短初始延搁时间为4d;其后在一定时期内不断有再生神经纤维长入移植物,即不同神经纤维初始延搁时间不一致;再生轴突在移植物中的生长速度也不一致,最快再生速度为2.14mm/d。     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

Axonal regrowth downregulates the synthesis of glial cell line-derived neurotrophic factor in the lesioned rat sciatic nerve

The effect of axonal regeneration on de novo synthesis of glial cell line-derived neurotrophic factor (GDNF) in rat sciatic nerves was examined. Transection of the sciatic nerve caused a prominent increase in the GDNF content in the distal segments within 1 week. The high level was sustained until 4 weeks in the animal model in which the nerve ends were ligated with thread (non-regeneration group); however, it was reduced to the original level within 2 or 4 weeks after the transection only in the segments invaded by regenerating axons in the models in which the nerve ends were coaptated (regeneration group). Expression of both GDNF protein and mRNA was decreased with a reciprocal increase in the density of neurofilaments, used as a marker of axonal ingrowth in distal segments of the regeneration group, suggesting that axonal contact turned off the GDNF-mediated nerve regeneration activity.     

神经溃变及再生的组织化学法评价

本文观察了鼠坐骨神经切断后溃变及再生过程中的乙酰胆碱酯酶(AChE)组织化学染色特征。结果表明:鼠坐骨神经切断后15天,远端酶组织化学染色阴性,而近端酶反应正常。再生神经纤维一开始便具有AChE酶活性反应。这种酶活性反应在溃变与再生神经纤维中的显著差别,为用该酶组织化学法评价神经再生提供了依据。     

骨髓神经组织定向干细胞性组织工程化神经移植修复坐骨神经损伤

目的:为临床周围神经损伤的组织工程化神经替代治疗提供可靠的实验依据。方法:采用体外成功构建的骨髓神经组织定向干细胞(NTCSCs)组织工程化神经,桥接缺损10mm的大鼠坐骨神经。通过术后行为学、神经功能指数分析(SFI)、神经电生理功能检查和HRP逆行追踪,以及对移植至支架的标记细胞在体内的存活、增殖及分化的形态学观察,综合评价损伤神经的功能恢复情况。结果:(1)SFI及神经电生理功能检查显示,NTCSCs组织工程化神经移植能明显促进神经传导功能及运动功能的恢复。(2)HRP逆行追踪结果显示NTCSCs组织工程化神经移植能明显促进神经纤维再生。(3)免疫荧光双标结果显示移植到体内的NTCSCs,从4周到12周,其细胞数显著增加,体内的NTCSCs细胞能分化成少量NSE阳性细胞,但大部分分化成S-100阳性细胞。结论:该骨髓神经组织定向干细胞性组织工程化神经能够有效的促进损伤坐骨神经的功能恢复。