大鼠坐骨神经压榨损伤后trkB、trkC在雪旺氏细胞内表达的变化

<正> 周围神经损伤后雪旺氏细胞可产生多种神经营养因子以促进受损神经元的存活;轴突再生与功能恢复.最近Ohnishi、Meyer等的研究表明雪旺氏细胞本身也受一些神经营养因子的影响:如BDNF可调节雪旺氏细胞的活性,促进其对新生轴突的髓鞘化;NT3、CNTF等对雪旺氏细胞的存活有一定的影响.本实验采用免疫组织化学的方法检测了BDNF与NT-3的高亲和性受体trkB、trkC在成年大鼠坐骨神经压榨损伤后的不同时期在雪旺氏细胞内表达的变化.动物右侧坐骨神经压榨损伤后分别存活5天、7天、14天、21天.经心灌注固定.在压榨损伤处近侧端与远侧端各取0.1cm长的神经,经后固定、浸糖后纵行冰冻切片.按ABC法做trkB、trkC受体的免疫组化反应.结果显示:在正常的坐骨神经雪旺氏细胞内trkB与trkC均有较强的表达.trkB在损伤后5天、7天在雪旺氏细胞内的表达稍有下降,在损伤后14天恢复到正常水平,远侧段与近侧段无明显差异.在损伤后21天雪旺氏细胞内trkB的表达明显增加,尤以远侧段为重.trkC在雪旺氏细胞内的表达在损伤后5天、7天与正常相比无明显差异,在损伤后14天表达明显增高,在损伤后21天远侧段的表达进一步增加     

p27kip1和Skp2在损伤后大鼠坐骨神经中的表达变化

为了探讨损伤后周围神经p27kip1和S期激酶相关蛋白2(Skp2)的定位表达和变化,本实验将成年SD大鼠随机分为正常对照组、夹伤组和切断组,运用Western blot结合免疫组织化学及免疫荧光双标,在大鼠坐骨神经损伤时,对p27kip1和Skp2表达的影响进行了研究。结果表明:(1)坐骨神经夹伤后,p27kip1蛋白表达先逐渐下降,后又逐渐上升;坐骨神经切断后,远侧段p27kip1蛋白表达持续下降,而近侧段p27kip1蛋白表达在切断后6h明显下降,后又逐渐升高至正常水平,而Skp2表达变化与之相反;(2)免疫组织化学染色结果显示,坐骨神经切断后1w,远侧段从断端到末端,p27kip1阳性信号逐渐增加,而Skp2阳性信号逐渐减弱;(3)免疫荧光双标显示,正常和损伤坐骨神经的雪旺氏细胞中都有p27kip1和Skp2表达。以上结果提示:周围神经损伤后影响雪旺氏细胞中p27kip1和Skp2的表达变化,为进一步研究它们在周围神经损伤和修复中的作用机制奠定基础。     

坐骨神经生后发育过程中CNTF基因表达的研究

目的 观察大鼠生长发育过程中外周神经组织CNTF基因表达的变化.方法 采用RT-PCR的方法,半定量分析正常大鼠坐骨神经发育过程中CNTF mRNA表达量.结果 正常大鼠生后仅1d,其坐骨神经中即可见CNTF mRNA表达,但表达量较低;出生14d后,表达量明显增加;30d时表达量达高峰,60d时的表达量稍有下降趋势.结论 大鼠出生后其坐骨神经中CNTF基因表达量随着周围神经的发育过程而有相应的变化.提示CNTF在周围神经生后发育过程中具有重要作用.     

大鼠坐骨神经髓鞘相关蛋白随年龄的变化

目的:研究大鼠坐骨神经髓鞘相关蛋白随年龄的变化及与髓鞘板层数之间的相互关系.方法:SD大鼠分别于饲养1周、1月、3月、9月、15月和21月后取坐骨神经,利用透射电镜观察计数髓鞘板层;采用免疫印迹检测髓鞘相关蛋白—髓鞘碱性蛋白(MBP)、髓鞘相关糖蛋白(MAG)和P0蛋白(P0)的表达变化,并分析与板层数之间的相关性;采用荧光免疫组织化学显色观察MBP的表达变化.结果:透射电镜观察显示,大鼠坐骨神经髓鞘板层数从1周到15月逐渐增加,直到21月无显著变化.免疫印迹显示,MBP的表达随年龄增长而增加,21月时达到高峰,与髓鞘板层数呈正性直线相关;MAG在出生后1周表达最高,而P0在1月时达峰值,后皆随年龄增加而下降,MAG与髓鞘板层数呈负性直线相关,而P0与髓鞘板层数之间呈非直线相关.荧光免疫组织化学显色显示,随着年龄增长,MBP在大鼠坐骨神经的髓鞘表达的荧光强度逐渐增强.结论:大鼠坐骨神经髓鞘相关蛋白随年龄改变呈现不同的表达模式,并与髓鞘板层数之间存在一定相关性.     

成年大鼠坐骨神经早期横断后近侧段proBDNF的表达和分布

目的:研究成年大鼠坐骨神经横断后早期近侧段脑源性神经营养因子前体(proBDNF)的表达和分布.方法:建立成年大鼠坐骨神经横断模型,30只SD大鼠随机分为假手术组和手术实验组,每组的每个时间点(0、2、3、4d)各3只.通过免疫荧光染色检测横断神经近段proBDNF的表达和分布.结果:成年大鼠坐骨神经横断后2-4d,p...     

白藜芦醇对糖尿病大鼠坐骨神经MnSOD的作用

探讨白藜芦醇对糖尿病大鼠坐骨神经锰超氧化物歧化酶（MnSOD）的作用。50只SD大鼠随机分为5组：正常组、糖尿病对照组、α-硫辛酸组、白藜芦醇小剂量组、白藜芦醇大剂量组,进行相应药物或生理盐水灌胃干预。12周后处死大鼠,检测各组坐骨神经抗氧化酶活力和MnSOD表达量,并观察其超微结构。结果显示,糖尿病对照组大鼠抗氧化酶活力降低,白藜芦醇能显著提高MnSOD的表达量和活性,坐骨神经髓鞘和雪旺细胞核出现退行性改变。以上数据表明,糖尿病大鼠坐骨神经中MnSOD活性下降;白藜芦醇能提高MnSOD表达和活性,改善糖尿病神经病变。     

miR-124在大鼠坐骨神经损伤后的表达及对雪旺细胞增殖与迁移的影响

目的探讨miR-124在大鼠坐骨神经损伤后的表达及对雪旺细胞增殖与迁移的影响。方法建立大鼠坐骨神经损伤模型,实时PCR方法检测坐骨神经损伤后0 d、1 d、3 d、7 d神经近侧断端miR-124的表达水平。设计合成miR-124模拟物,转染雪旺细胞;MTT实验检测过表达miR-124对雪旺细胞增殖的影响;Transwell实验检测过表达miR-124对雪旺细胞迁移能力的影响。结果坐骨神经损伤后,与0d相比,miR-124在1 d、3 d、7 d的表达水平均明显增加(P0.01)。转染miR-124模拟物能够显著上调雪旺细胞miR-124的表达水平,促进雪旺细胞增殖与迁移(P0.01)。结论 miR-124在坐骨神经损伤后表达水平逐渐升高,可能参与坐骨神经损伤后神经再生过程。     

丙烯酰胺染毒对大鼠坐骨神经MBP和MAG表达的影响

目的:通过检测染毒大鼠坐骨神经中髓鞘碱性蛋白(MBP)和髓磷脂相关糖蛋白(MAG)表达量的变化,探讨丙烯酰胺(ACR)对大鼠周围神经系统的毒性影响。方法:成年雄性SD大鼠32只,随机分为对照组、9、18、36 mg/kg ACR组,每组8只,灌胃染毒21 d后取材。每周一次步态评分,记录大鼠步态改变;利用HE染色和劳克坚牢蓝染色,观察坐骨神经及其髓鞘的改变;通过免疫组织化学方法检测蛋白MBP和MAG表达量的变化。结果:大鼠步态得分与染毒剂量和染毒时间呈正相关; HE染色显示随染毒剂量增大,坐骨神经神经纤维排列紊乱、髓鞘结构发生改变、轴突数目减少;劳克坚牢蓝染色结果显示与对照组相比,染毒组髓鞘染色较浅、着色不均,髓鞘变细;免疫组化结果显示,MBP和MAG在坐骨神经中的表达量均随染毒剂量增大而下降。结论:ACR染毒对大鼠坐骨神经髓鞘的毒性损伤,可能与MBP和MAG蛋白表达量的下降有关。     

雪旺氏细胞在周围神经损伤修复中的作用及其分子机制

雪旺氏细胞是周围神经系统中特有的胶质细胞,在周围神经损伤后的变性和再生中有着非常重要的作用。周围神经的再生主要依赖于雪旺氏细胞提供了适宜的微环境,如分泌多种神经营养因子和其它相关因子,位于轴突和雪旺氏细胞之间的紧密连接加强信息传递,雪旺氏细胞形成Büngner带为轴突生长的通道,并形成髓鞘等。本文阐述了雪旺氏细胞在周围神经再生中的重要功能以及相关机制,展望围绕雪旺氏细胞的未来研究方向,临床应用的潜在价值。     

大鼠坐骨神经切断后IGF-Ⅰ、BDNF和TrkB在背根节表达的实验研究

目的：观察大鼠坐骨神经切断后IGF-Ⅰ、BDNF及TrkB在背根节（DRG）表达变化规律，探讨三者在神经保护和再生中的作用机制。方法：健康成年雄性Wister大鼠随机分为对照组和实验组，实验组切断右侧大腿坐骨神经后存活3～28d，荧光定量RT-PCR方法检测L4-6背根节（DRG）中IGF-Ⅰ、BDNF和TrkB基因mRNA的表达。结果：坐骨神经损伤后3d，L4-6 DRGIGF-Ⅰ、BDNF和TrkB基因mRNA表达均有不同程度的升高，到7-14d达高峰，后逐渐下降到正常水平。结论：坐骨神经损伤后，IGF-Ⅰ、BDNF和TrkB基因mRNA表达同步变化提示它们在外周神经损伤后神经保护及再生中可能存在相互作用。     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L_3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。     

Differential effects of endogenous brain-derived neurotrophic factor on the survival of axotomized sensory neurons in dorsal root ganglia: a possible role for the p75 neurotrophin receptor

After peripheral nerve injury, axotomized sensory neurons in dorsal root ganglia (DRG) undergo apoptosis and up-regulate brain-derived neurotrophic factor (BDNF). We tested whether endogenous BDNF plays any role in the survival of axotomized sensory neurons using in vitro and in vivo models. In the in vitro model, treatment with BDNF antibody significantly reduced apoptosis of sensory neurons in DRG explants from both adult and neonate rats and adult mice cultured for 48 h. Consistently, exogenous BDNF increased the percentage of apoptotic neurons in the DRGs from mice. The effects of the BDNF antibody and BDNF were not seen in DRGs from p75NTR(-/-) mice. In the in vivo model, sciatic nerve transection in neonatal rats decreased the total number of neurons in the injured DRG and treatment with antiserum to BDNF significantly exaggerated the loss of DRG neurons. Numbers of sensory neurons expressing BDNF and p75NTR in cultured DRGs increased but that expressing TrkB decreased. In contrast, sciatic nerve transection in vivo reduced the numbers of neurons expressing both p75NTR and TrkB but increased the numbers of cells expressing BDNF, 1 and 7 days after the surgery. These results suggest that BDNF may have differential effects on the survival of sensory neurons depending on the expression of p75NTR. While endogenous BDNF induced apoptosis of axotomized sensory neurons through p75NTR in vitro where more neurons expressed p75NTR, it prevented apoptosis in vivo where fewer neurons expressed p75NTR after sciatic nerve transection.     

坐骨神经损伤大鼠模型鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达

背景：坐骨神经损伤模型可测试伤害性的热刺激和机械刺激所引发的痛觉过敏及冷、触觉异常。 目的：观察坐骨神经损伤模型大鼠鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达。 方法：72只SD大鼠随机均分为假手术组、对照组和实验组。对照组和实验组制作坐骨神经损伤模型,假手术组仅暴露坐骨神经,不结扎。分别于造模后第3,10天进行鞘内移植,实验组注入30 μL的神经干细胞悬液,空白组和对照组注入30 μL的细胞培养液。 结果与结论：与假手术组相比,对照组和实验组移植后3 d机械痛阈和热痛阈逐渐降低,至移植后7 d降低至最低点(P < 0.01),于移植后21 d恢复至移植前水平;实验组移植后7,14 d机械痛阈和热痛阈较对照组明显上升(P < 0.01)。与对照组相比,假手术组移植后7,14,21 d各组大鼠脑源性神经营养因子的表达呈低水平(P < 0.05);移植后14,21 d,实验组脑源性神经营养因子的表达量高于对照组(P < 0.05)。提示鞘内移植神经干细胞可提高脊髓背角和背根神经节中脑源性神经营养因子的表达。从而抑制了周围神经损伤产生的神经病理性疼痛。 关键词：脑源性神经营养因子;神经干细胞;慢性限制损伤;脊髓背角;背根神经节 doi:10.3969/j.issn.1673-8225.2012.10.031     

Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins

Summary Rabbit antisera to the rat myelin proteins P₀ and P₁ were used to assay for the presence of these components by both immunochemical and immunofluorescence methods. The antiserum to P₀ did not react detectably with polyacrylamide gels containing central myelin, or with P₁ and P₂ in peripheral myelin; it did react with P₀ in peripheral myelin, and in extracts of adult and neonatal sciatic nerve. When reacted with frozen tissue sections using indirect immunofluorescence, it did not stain central myelin but did stain myelin in adult sciatic nerve, the myelinated fibres in cervical sympathetic trunk and occasional areas in neonatal sciatic nerve where Schwann cells had presumably begun to form myelin. Antiserum to basic protein reacted with both of the basic protein bands in central and peripheral myelin, but not P₀; P₁ and P₂ were detectable in adult and neonatal sciatic nerve. In indirect immunofluorescence assays, the antiserum stained both central and peripheral myelin, the few myelinated fibres of sympathetic trunk and myelinating regions of neonatal sciatic nerve.Cultured secondary rat Schwann cells showed no detectable reaction with either reagent, using either technique. We conclude that these three proteins are probably expressed as a consequence of the neuron-Schwann cell interaction that initiates myelination.     

MMP-9和TIMP-1在高血压合并糖尿病周围神经病大鼠坐骨神经的表达

目的探讨高血压(HTN)合并糖尿病周围神经病(DPN)大鼠模型坐骨神经中基质金属蛋白酶-9(MMP-9)和组织金属蛋白酶抑制剂-1(TIMP-1)的表达。方法实验大鼠分为正常组、HTN组、DPN组和DPN+HTN组。造模后4w检测各组血糖、血压及坐骨神经传导速度,RT-PCR检测坐骨神经MMP-9mRNA和TIMP-1mRNA表达。结果与正常组和HTN组比较,DPN组和HTN+DPN组血糖显著增高,神经传导速度显著降低(0.01),坐骨神经MMP-9mRNA和TIMP-1mRNA表达显著上调(0.01)。与DPN组比较,HTN+DPN组血压显著增高,神经传导速度显著降低,坐骨神经MMP-9mRNA表达较DPN组上调,TIMP-1 mRNA表达显著下降(0.01)。。结论高血压合并糖尿病周围神经病坐骨神经MMP-9mRNA表达上调,TIMP-1mRNA表达下调,可能与抑制施万细胞和髓鞘形成加剧周围神经损伤相关。     

N-Acetylated alpha-linked acidic dipeptidase may be involved in axon-Schwann cell signalling

Summary N-Acetylated alpha-linked acidic dipeptidase is a membrane-bound peptidase that cleaves the neuropeptide N-acetylaspartyl-glutamate to N-acetyl-aspartate and glutamate. Previously, we have shown that in adult rat this enzyme is expressed by the non-myelinating Schwann cells in peripheral nerve. In the present study, we have determined the expression pattern of this peptidase in rat sciatic nerve during late embryonal and early postnatal development, using double-label immunofluorescence, enzyme assays and immunoblotting. We demonstrate that N-acethylated alpha-linked acidic dipeptidase is expressed by all Schwann cell precursor cells on embryonal day 14/15 and by all undifferentiated Schwann cells on embryonal days 16/17 and 20/21 and postnatal day 1. Moreover, we show that during the first postnatal week, the peptidase expression is down-regulated in the myelinating Schwann cells while the total enzyme activity levels and the enzyme amounts present in the nerve are transiently increased. To determine whether Schwann cell peptidase expression is dependent on axonal contact, we performed immunofluorescence experiments in cultured Schwann cells. Thesein vitro experiments demonstrate that the expression of this enzyme is maintained in culture for several weeks without axonal contact. Furthermore, they confirm previous suggestions that this peptidase is expressed on the extracellular side of the Schwann cell membrane. These findings support the notion that N-acetylated alpha-linked acidic dipeptidase takes part in signaling between peripheral axons and Schwann cells. The temporary increase in peptidase activity during the first postnatal week strongly implicates a role for this enzyme in the process of axon ensheathment and/or axon myelination.     

周围神经髓化的主要影响因素及可能的作用机制

髓鞘是周围神经的重要结构,也是有髓神经纤维发育成熟的标志。Neu基因调节素-1（NRG-1）、神经营养因子和P0蛋白等是调节周围神经发育期和损伤后再生过程中髓鞘形成（髓化）的重要因素,它们以多种形式参与对髓化不同阶段的调节。总结影响周围神经髓化的主要因素及其可能的作用机制,将为目前临床脱髓鞘疾病的治疗提供新的思路。     

Dexamethasone and vitamin B12 synergistically promote peripheral nerve regeneration in rats by upregulating the expression of brain-derived neurotrophic factor

Introduction

Dexamethasone and vitamin B₁₂ are currently used in the clinic to treat peripheral nerve damage but their mechanisms of action remain incompletely understood. In this study we hypothesized that dexamethasone and vitamin B₁₂ promote the production of endogenous neurotrophic factors, thereby enhancing peripheral nerve repair.

Material and methods

Ninety-six adult male Wistar rats were employed to establish a sciatic nerve injury model. They were then randomly divided into 4 groups to be subjected to different treatment: saline (group A), dexamethasone (group B), vitamin B₁₂ (group C), and dexamethasone combined with vitamin B₁₂ (group D). The walking behavior of rats was evaluated by footprint analysis, and the nerve regeneration was assessed by electrophysiological analysis and ultrastructural examination. The expression of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor, NT-3 and IL-6 in the injured sciatic nerves was detected by immunohistochemical and RT-PCR analysis.

Results

Dexamethasone and vitamin B₁₂ promoted the regeneration of myelinated nerve fibers and the proliferation of Schwann cells. Furthermore, dexamethasone and vitamin B₁₂ promoted the recovery of sciatic functional index and sensory nerve conduction velocity, and upregulated BDNF expression in the injured sciatic nerves.

Conclusions

Dexamethasone and vitamin B₁₂ promote peripheral nerve repair in a rat model of sciatic nerve injury through the upregulation of BDNF expression. These findings provide new insight into the neurotrophic effects of dexamethasone and vitamin B₁₂ and support the application of these agents in clinical treatment of peripheral nerve injury.     

血管紧张素Ⅱ对人内皮细胞转录因子NF－kB的激活机制及其对血小板源生长因子B链基因转录的影响

目的：研究血管紧张素Ⅱ对ECV304细胞中转录因子NF－kB的作用和对血小板源生长因子B链（PDGF－B）基因表达的影响。方法：采用电泳迁移率移动分析（EMSA）,免疫组织化学方法,共聚焦显微镜及金颗粒标记免疫电镜技术;荧光素酶报告基因与变异型激酶质粒共转染的方法及Northern印迹法研究血管紧张素Ⅱ激活ECV304细胞NK－kB的信号传递路径和检测了血管紧张素Ⅱ刺激前后ECV304细胞PGF－BmRNA的表达水平。结果：血管紧张素Ⅱ刺激后,在ECV304细胞内有NK－kB的激活及核易位过程,应用免疫荧光聚集显微镜,免疫镜及Northern印迹等方法均可观察到PDGF－B或其基因表达增高。变异型激酶质粒IKKa-KM,IKKβ－KM及NIK－Km可抑制经血管紧张素Ⅱ刺激的转染细胞内与NF－kB启动相连的荧光素酶的表达。结论：血管紧张素Ⅱ可激活胞质内NK－kB并出现核易位,激酶NIK、IKKα和IKKβ参与了此信号传递路径,血管紧张素Ⅱ刺激后PDGF－B链mRNA水平增高。     

Immunohistochemical localization of the neural cell adhesion molecule in the rat sciatic nerve.

The neural cell adhesion molecule (NCAM) has been widely studied in the early embryonal development of the nervous system. The data about NCAM distribution in the peripheral nerves during postnatal life are scant and some controversial. In the present study, the NCAM localization in the sciatic nerves of 15-day-old Wistar rats has been studied. Semi-thin sections of the nerves were immunotested with a polyclonal antibody (Santa Cruz Biotechnology) that recognizes rat NCAM. The antibody was visualized with donkey anti-goat IgG, conjugated to 12 nm colloidal gold, and silver amplification. In the myelinated nerve fibres, the immunoreactivity was associated with the axons, mainly with their plasma membrane, which was unstained in the nodes of Ranvier. The myelin sheaths and the myelinating Schwann cells were negative. The extracellular matrix and the bundles of non-myelinated nerve fibres were immunopositive.