缺血缺氧对体外培养星形胶质细胞细胞活化和细胞周期的影响

目的观察缺血缺氧损伤对星形胶质细胞细胞活化和细胞周期的影响。方法用流式细胞仪及BrdU掺入法检测缺血缺氧后不同时间点星形胶质细胞细胞周期变化和细胞的增殖活力;用荧光免疫细胞化学技术测定胶质细胞纤维酸性蛋白(GFAP)及细胞周期蛋白cyclinD1的表达水平。结果体外缺血缺氧损伤后星形胶质细胞S期较正常组明显增高,6h达高峰,BrdU掺入法显示损伤后6h星形胶质细胞的增殖活力最高,而随后S期细胞数目及细胞增殖活力都呈下降趋势。在缺血缺氧早期,GFAP阳性染色增强,6h最高;缺血缺氧12h后GFAP阳性染色变弱,而cyclinD1的表达在损伤后逐渐增加,在24h时达高峰。结论缺血缺氧损伤激活星形胶质细胞,使其进入新的细胞周期,出现细胞的增殖反应;cyclinD1参与了损伤后星形胶质细胞的修复和增殖;细胞周期事件与星形胶质细胞的增殖活化密切相关。     

电针对脊髓损伤星形胶质细胞增生及其NGF表达的影响

目的研究脊髓损伤后电针治疗对星形胶质细胞增生及其内源性神经生长因子(nerve growth factor,NGF)表达的影响.方法选用成年雌性Wistar大鼠,随机分为3组.A组为正常对照组,B组、C组为下胸段脊髓不完全损伤.B组损伤后不治疗,C组损伤后给予督脉电针治疗.损伤后3 d、1 、2或4周应用免疫组化染色分别观察损伤脊髓胶质原纤维酸性蛋白(glial fibroblast acid protein,GFAP)和NGF表达的变化.结果 B组术后3 d,GFAP阳性细胞明显增多, 2周后开始减少,4周时仍有较多的阳性细胞;C组GFAP阳性细胞明显少于B组,1周时达高峰.脊髓损伤后NGF表达呈逐渐增加的趋势.C组NGF的表达明显高于B组,且一直保持在较高水平.NGF阳性细胞大部分与GFAP阳性细胞形态相似.结论电针治疗能减少星形胶质细胞增生,促进内源性NGF的合成,从而创造了有利于神经再生的微环境.     

正加速度暴露对大鼠海马星形胶质细胞GFAP表达的影响

目的探讨正加速度( Gz)重复暴露后不同时间海马星形胶质细胞GFAP表达的变化.方法 SD大鼠60只,随机分成对照组、 Gz重复暴露后1h、6h、12h、24h和48h组,每组10只.采用动物离心机,建立 Gz引发急性脑缺血模型;应用免疫组织化学技术,分别检测 Gz重复暴露后不同时间,海马星形胶质细胞GFAP的表达状况.结果海马星形胶质细胞GFAP阳性细胞数,在 Gz暴露后1h即显著增加,于12h达到高峰,而后逐渐下降,48h仍维持在较高水平,实验组与对照组比较,有显著性差异.结论 Gz重复暴露导致海马星形胶质细胞GFAP表达上调,可能对神经元的缺血损伤起保护作用.     

马桑内酯慢性致痫大鼠海马星形胶质细胞的激活

目的：研究慢性癫痛大鼠点时海马星形胶细胞的激活情况。方法：采用马桑内酯慢性癫痫大鼠模型,观察大鼠点燃后海马NF－kBp65和胶质原纤维酸性蛋白（glial fibrillary acidic protein, GFAP）免疫细胞化学反应（immunoreactivity,IR)的变化。结果：点燃后1h,海马CA1区GFAR－IR开始增强,4－8h可观察到GFAP－IR阳性细胞数量增多并明显浓染。这种强GFAP－IR持续至点燃点24h点燃后1h,NF－kBp65即可在海马CA1区神经元和胶质细胞内表达,主要位于胞核内,至8h阳性神经元细胞核基本消失而可见大量NF－kBp65－IR阳性的胶质细胞,双重免疫细胞化学方法显示GFAP／NF－kBp65－IR阳性细胞在点燃后1h即可观察到,4h表达最高峰,24h恢复对照组水平。结论：马桑内酯慢性致痫大鼠点燃时星形胶质细胞表现一种早期而持续的激活,提示反复激活的星形胶质细胞对癫痫的复发可能起重要的作用。     

创伤性脑损伤后IL-1?茁在神经胶质细胞中经时定位表达的研究*

目的:探讨创伤性脑损伤(TBI)后白细胞介素-1β(IL-1β)在神经胶质细胞中的经时定位表达情况。方法:选择72只SPF级雄性小鼠分为假手术组(sham组)、TBI 6h组、TBI 12h组、TBI 1d组、TBI 4d组与TBI 7d组,每组12只,分别在脑损伤后6h、12h、1d、4d、7d时获取血清和脑组织并且制作切片。ELISA检测损伤后炎症因子IL-1β、白细胞介素-6 (IL-6)和肿瘤坏死因子-α(TNF-α)的表达。Western blot检测钙离子结合蛋白-1(IBA-1)和胶质纤维酸性蛋白(GFAP)的表达。应用免疫荧光双重染色技术观察炎症因子IL-1β在小胶质细胞和星形胶质细胞中的定位表达情况。结果:TBI后6h-7d时炎症因子IL-1β、IL-6和TNF-α的表达量均高于sham组(P0.05)。Western blot结果显示,IBA-1的表达在损伤后6h-7d时高于sham组,GFAP的表达在损伤后1d-7d时高于sham组(P0.05)。免疫荧光双重染色技术显示,6h、12h时IL-1β主要表达在小胶质细胞中,IL-1β和IBA-1共表达细胞数量多于sham组(P0.05);1d、4d、7d时IL-1β主要表达在星形胶质细胞中,IL-1β和GFAP共表达细胞数量多于sham组(P0.05)。结论:TBI诱导了胶质细胞和炎症因子的表达,其表达随脑损伤的时间而变化,IL-1β早期定位表达于小胶质细胞,后期定位表达于星形胶质细胞中。     

PD大鼠胼胝体和扣带胶质原纤维酸性蛋白的表达

目的观察不同病程帕金森病(PD)大鼠胼胝体和扣带胶质原纤维酸性蛋白(GFAP)的表达。方法大鼠右侧前脑内侧束注射六羟基多巴胺制备PD模型。大鼠分为正常对照组,2周、4周和6周模型组。以酪氨酸羟化酶(TH)和GFAP抗体免疫组化阳性分别显示黑质的多巴胺能神经元、胼胝体与扣带处的星形胶质细胞。结果 2、4、6周模型组右侧黑质TH阳性细胞数较正常对照组均显著降低,而2、4、6周模型组之间无显著差异。正常对照组和4周模型组胼胝体和扣带处星形胶质细胞呈未活化状态,两组之间的GFAP表达强度和细胞密度均无显著差异。2周和6周模型组两部位星形胶质细胞呈活化状态,其表达强度和细胞密度均显著高于正常对照组和4周模型组。结论急性完全损伤PD模型大鼠注射侧胼胝体和扣带处GFAP表达随病程呈增高、降低和增高趋势。     

活化星形胶质细胞NGF、IL-6表达的时间特性

目的:研究星形胶质细胞活化后神经生长因子(nerve growth factor,NGF)、白细胞介素-6(interleukin-6,IL-6)表达的时间规律性,探讨星形胶质细胞活化后启动保护性机制与损伤性机制的时间特性.方法:体外分离培养星形胶质细胞,分为对照组、活化组、抑制组.通过光学显微镜及免疫荧光化学观察各组细胞的形态变化;应用半定量RT-PCR方法分析各组细胞间胶原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)mRNA及NGF mRNA、IL-6 mRNA表达变化;用ELISA法检测各组细胞上清液中不同时间点(6h,24h,48h,72h)NGF、IL-6的含量.结果:活化组与对照组比较,细胞胞体变大,GFAP荧光增强;RT-PCR示GFAP mRNA、NGF mRNA、IL-6 mRNA表达均明显增高,与对照组比较差异有显著性(P＜0.01);ELISA法检测示星形胶质细胞活化后NGF分泌量在活化后24小时达到高峰,与对照组比较差异有显著性(P＜0.01);活化后48小时IL-6的含量达到高峰,与对照组比较差异有显著性(P＜0.01);应用抑制剂Genistein干预后,与活化组相比,抑制组细胞胞体变小,星形胶质细胞活化被抑制,GFAP mRNA表达下降,NGF mRNA、IL-6 mMRA表达亦下降,与活化组比较差异有显著性(P＜0.01).结论:星形胶质细胞活化后NGF、IL-6表达均上调,但NGF表达时间早于IL-6表达时间,表明在星形胶质细胞活化的早期,可能其神经保护作用占主导,而后期其神经毒性作用逐渐明显;Genistein能抑制星形胶质细胞活化,使NGF、IL-6表达下调.     

NDRG2与GFAP在不同脑区星形胶质细胞的表达与分布

目的:观察NDRG2(N-myc下游调节基因2)与GFAP(胶质纤维酸性蛋白)在不同脑区星形胶质细胞的表达与分布。方法:利用免疫荧光NDRG2与GFAP双标技术以及Western Blot技术观察皮层、海马及纹状体等不同脑区星形胶质细胞NDRG2和GFAP的表达与分布。结果:免疫荧光结果显示NDRG2阳性细胞广泛而均匀地分布于不同脑区,并与GFAP存在较好的共定位;NDRG2与GFAP标记的星形胶质细胞形态不尽相同。Western Blot结果显示NDRG2在皮层中表达比海马和纹状体多,而GFAP在海马中表达比皮层和纹状体多。结论:NDRG2广泛表达于不同脑区星形胶质细胞,并于GFAP存在较好的共定位。     

异丙酚对局灶性脑缺血／再灌注星形胶质细胞GFAP表达的影响

目的：探讨异丙酚对局灶性脑缺血／再灌注后星形胶质细胞胶质纤维酸性蛋白（GFAP）表达的影响。方法：大脑中动脉插线法制作大鼠局灶性脑缺血／再灌注模型。观察脑缺血2h再灌注24h后神经功能损害改变并评分,并采用免疫荧光组织化学法检测大鼠齿状回GFAP蛋白的表达。结果：缺血／再灌注后可诱导大鼠齿状回GFAP表达明显增强,异丙酚可抑制缺血／再灌注后GFAP的表达,明显改善大鼠神经功能损害（P〈0．05或0.01）。结论：异丙酚通过抑制脑缺血后星形胶质细胞GFAP的过度表达发挥抗脑缺血损伤保护神经元作用。     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化北大核心CSCD

尽管miR-144与细胞活化、增殖有关,但其对脊髓星形胶质细胞的作用尚不明确。本文旨在探究正常和脊髓损伤(spinal cord injury,SCI)组织和细胞中miR-144表达,以及miR-144能否通过靶向调节G蛋白偶联受体激酶5(GRK5)上调脊髓星形胶质细胞活化。实时定量PCR(RT-q PCR)和Western印迹结果揭示,与正常的组织/细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低,而GRK5的表达升高;脊髓损伤大鼠的星形胶质细胞中胶质原纤维酸性蛋白(GFAP)的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示,转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性分析发现,miR-144显著提高SOD和GSH活性;萤光素酶报告基因检测结果证明,miR-144能靶向结合GRK5,下调GRK5表达。miR-144 mimic转染或miR-144 mimic与pc DNA-GRK5共转染脊髓损伤的星形胶质细胞后,我们发现,miR-144转染可通过激活NF-κB通路消除GRK5对细胞活化的抑制作用。综上所述,miR-144通过靶向抑制GRK5促进脊髓星形胶质细胞的活化。     

NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

Background

A major class of axon growth-repulsive molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). Experimental spinal cord injury (SCI) has demonstrated rapid re-expression of CSPGs at and around the lesion site. The pharmacological digestion of CSPGs in such lesion models results in substantially enhanced axonal regeneration and a significant functional recovery. The potential therapeutic relevance of interfering with CSPG expression or function following experimental injuries seems clear, however, the spatio-temporal pattern of expression of individual members of the CSPG family following human spinal cord injury is only poorly defined. In the present correlative investigation, the expression pattern of CSPG family members NG2, neurocan, versican and phosphacan was studied in the human spinal cord.

Methods

An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.

Results

In sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. Furthermore, phosphacan, neurocan and versican were present in the myelin sheaths of ventral and dorsal nerve roots axons. After human SCI, NG2 and phosphacan were both detected in the evolving astroglial scar. Neurocan and versican were detected exclusively in the lesion epicentre, being associated with infiltrating Schwann cells in the myelin sheaths of invading peripheral nerve fibres from lesioned dorsal roots.

Conclusion

NG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might play an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. The present data points to the importance of such correlative investigations for demonstrating the clinical relevance of experimental data.     

The molecular cloning of glial fibrillary acidic protein in <Emphasis Type="Italic">Gekko japonicus</Emphasis> and its expression changes after spinal cord transection

The glial fibrillary acidic protein (GFAP) is an astrocyte-specific member of the class III intermediate filament proteins. It is generally used as a specific marker of astrocytes in the central nervous system (CNS). We isolated a GFAP cDNA from the brain and spinal cord cDNA library of Gekko japonicus, and prepared polyclonal antibodies against gecko GFAP to provide useful tools for further immunochemistry studies. Both the real-time quantitative PCR and western blot results revealed that the expression of GFAP in the spinal cord after transection increased, reaching its maximum level after 3 days, and then gradually decreased over the rest of the 2 weeks of the experiment. Immunohistochemical analyses demonstrated that the increase in GFAP-positive labeling was restricted to the white matter rather than the gray matter. In particular, a slight increase in the number of GFAP positive star-shaped astrocytes was detected in the ventral and lateral regions of the white matter. Our results indicate that reactive astrogliosis in the gecko spinal cord took place primarily in the white matter during a short time interval, suggesting that the specific astrogliosis evaluated by GFAP expression might be advantageous in spinal cord regeneration.     

Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.     

Glial fibrillary acidic protein in regenerating teleost spinal cord

Immunohistological and ultrastructural studies were carried out on normal and regenerating spinal cord of the gymnotid Sternarchus albifrons, and in the brain and spinal cord of the goldfish Carassius auratus, to examine the distribution of glial fibrillary acidic protein (GFAP) in these tissues. Sections of normal goldfish brain and spinal cord exhibited positive staining for GFAP. In normal Sternarchus spinal cord, electron microscopy has revealed filament-filled astrocytic processes; however, such astrocytic profiles were more numerous in regenerated cord. Likewise, while normal Sternarchus spinal cord showed only a small amount of GFAP staining, regenerated cords were strongly positive for GFAP. Positive staining with anti-GFAP was observed along the entire length of the regenerated cord in Sternarchus, and was especially strong in the transition zone between regenerated and unregenerated cord. Both regeneration of neurites and production of new neuronal cell bodies occur readily in such regenerating Sternarchus spinal cords (Anderson MJ, Waxman SG: J Hirnforsch 24: 371, 1983). These results demonstrate that the presence of GFAP and reactive astrocytes in Sternarchus spinal cord does not prevent neuronal regeneration in this species.     

Spinal cord regeneration in a tail autotomizing urodele

Adult urodele amphibians possess extensive regenerative abilities, including lens, jaws, limbs, and tails. In this study, we examined the cellular events and time course of spinal cord regeneration in a species, Plethodon cinereus, that has the ability to autotomize its tail as an antipredator strategy. We propose that this species may have enhanced regenerative abilities as further coadaptations with this antipredator strategy. We examined the expression of nestin, vimentin, and glial fibrillary acidic protein (GFAP) after autotomy as markers of neural precursor cells and astroglia; we also traced the appearance of new neurons using 5‐bromo‐2′‐deoxyuridine/neuronal nuclei (BrdU/NeuN) double labeling. As expected, the regenerating ependymal tube was a major source of new neurons; however, the spinal cord cranial to the plane of autotomy showed significant mitotic activity, more extensive than what is reported for other urodeles that cannot autotomize their tails. In addition, this species shows upregulation of nestin, vimentin, and GFAP within days after tail autotomy; further, this expression is upregulated within the spinal cord cranial to the plane of autotomy, not just within the extending ependymal tube, as reported in other urodeles. We suggest that enhanced survival of the spinal cord cranial to autotomy allows this portion to participate in the enhanced recovery and regeneration of the spinal cord. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Transplanted olfactory ensheathing cells modulate the inflammatory response in the injured spinal cord

Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to exert neuroprotective effects and promote functional recovery. In the present study, we investigated the potential modulatory effects of OECs on the inflammatory reaction developed after photochemical injury to the spinal cord. OEC cultures were obtained from olfactory bulbs of adult Sprague-Dawley rats. Photochemical spinal cord injury was induced in adult rats at T8. Thirty minutes after the insult, either a suspension of OECs (180 000 cells in 12 microl DMEM) or DMEM alone was injected into the lesioned spinal cord.At 3, 7 and 14 days post-operation (dpo), five animals from each group were processed for histology. Double-fluorescent labeling of transverse sections of the cord were made by combination of immunohistochemistry for inflammatory markers, interleukin 1b(IL-1b) and inducible nitric oxide synthase (iNOS), and for selective markers of astrocytes (glial fibrillar acidic protein; GFAP)and microglia/macrophages (tomato lectin; LEC). Differences in the intensity and time course of glial response, and IL-1band iNOS expression were found between the two groups of rats. The reactivity grade against IL-1beta, iNOS, GFAP and LEC in OEC-transplanted rats was higher at 7 dpo and lower at 14 dpo compared with DMEM-injected rats. These results indicate that the mechanisms underlying neuroprotection by OECs might be caused by earlier, higher and shorter duration of microglia/macrophage and astrocyte responses after injury.     

Expression of Glial Fibrillary Acidic Protein and Neurofilament mRNA in Gliosis Induced by Experimental Autoimmune Encephalomyelitis

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5-2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 +/- 35 micrograms (mean +/- SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11-14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11-14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to approximately 50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

CREB 和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用

目的:探讨CREB和NF-κB在p38MAPK所致脊髓星形胶质细胞活化中的作用,明确脊髓星形胶质细胞活化中p38MAPK细胞信号转导途径的作用。方法:分离培养SPF大鼠脊髓星形胶质细胞,设正常组、SP刺激组(SP组,10-7mol/L)、SP刺激+SB203580(10μmol/L)阻断p38MAPK组(SP+SB组)、SP刺激+PD98059(10μmol/L)阻断CREB组(SP+PD组)、SP刺激+SN50(10μmol/L)阻断NF-κB(SP+SN组)。WB法、免疫荧光法、ELISA法检测12 h和24 h时p-p38、p-CREB、NF-κBp65水平及GFAP、TNF-、IL-1β水平变化。结果:SP组脊髓星形胶质细胞p-p38、p-CREB、NF-κBp65显著升高,GFAP水平显著增高,同时TNF-和IL-1β水平显著增高。与SP组比较,用SB203580阻断p38MAPK通路后,SP+SB组p-p38、p-CREB、NF-κBp65显著降低,GFAP、TNF-和IL-1β水平显著降低。用PD98059阻断CREB通路后,SP+PD组p-p38、NF-κBp65无显著变化,p-CREB显著降...     

miR-31 promotes neural stem cell proliferation and restores motor function after spinal cord injury

This study aims to examine whether miR-31 promotes endogenous NSC proliferation and be used for spinal cord injury management. In the present study, the morpholino knockdown of miR-31 induced abnormal neuronal apoptosis in zebrafish, resulting in impaired development of the tail. miR-31 agomir transfection in NSCs increased Nestin expression and decreased ChAT and GFAP expression levels. miR-31 induced the proliferation of mouse NSCs by upregulating the Notch signaling pathway, and more NSCs entered G1; Notch was inhibited by miR-31 inactivation. Injection of a miR-31 agomir into mouse models of spinal cord injury could effectively restore motor functions after spinal cord injury, which was achieved by promoting the proliferation of endogenous NSCs. After the injection of a miR-31 agomir in spinal cord injury mice, the expression of Nestin and GFAP increased, while GFAP expression decreased. In conclusion, the zebrafish experiments prove that a lack of miR-31 will block nervous system development. In spinal cord injury mouse models, miR-31 overexpression might promote spinal cord injury repair.