甲基强的松龙对伽玛刀照射后胶质细胞Cx43和GFAP表达的影响

目的 研究甲基强的松龙（MP）对伽玛刀照射后胶质细胞缝隙连接蛋白43（Cx43）和胶质纤维酸蛋白（GFAP）表达的影响。方法体外培养原代星形胶质细胞,经伽玛刀照射（边缘剂量32Gy）并培养36h后随机分为1μg／ml、5μg／ml、10μg／ml、20μg／ml、30μg／ml、40μg/ml组及实验对照组,各组进一步随机分为48h、72h、96h、120h4个亚组。将各组胶质细胞与相应剂量MP共培养,于48、72、96、120h各时间点采用免疫组化方法检测GFAP及Cx43的表达。结果伽玛刀照射后,胶质细胞Cx43表达降低,GFAp表达增高。添加MP后,20μg／ml、30μg／ml组Cx43呈时间依赖性上调;GFAP表达虽呈时间依赖性上升,但其峰值低于实验对照组,峰值出现时间亦晚于实验对照组。结论MP可上凋伽玛刀照射后胶质细胞Cx43的表达,抑制GFAP表达的上升趋势。     

X线照射后体外培养星形胶质细胞VEGF和GFAP表达变化及意义

目的 研究X线照射体外培养的星形胶质细胞后血管内皮生长因子(VEGF)和胶质原纤维酸性蛋白(GFAP)表达随时间及剂量的变化,探讨星形胶质细胞与放射性脑损伤(RBI)的关系. 方法 以5、10、15、20 Gy剂量的X线照射体外原代培养的星形胶质细胞后继续培养48 h,或以20 Gy剂量照射后分别培养4、12、24、48 h,实验均设正常对照组即未照射组.免疫荧光染色检测GFAP观察各组细胞形态学的变化;DAPI染色观察星形胶质细胞的凋亡;Western blotting检测细胞GFAP、VEGF蛋白的表达情况. 结果 与正常对照组比较,不同剂量照射组星形胶质细胞增生、增多,变形,胞体肥大肿胀,分支增多,突起增粗,GFAP染色加深,且这种变化随照射剂量和时间增加而表现更明显;与正常对照组相比,各剂量照射组星形胶质细胞凋亡率差异无统计学差异(P＞0.05); Western blotting检测显示不同剂量组、20Gy剂量照射不同时间组星形胶质细胞GFAP、VEGF蛋白的表达均不同,差异有统计学意义(P＜0.05).与正常对照组相比,5、10、15、20 Gy照射组和20 Gy剂量照射后各时间组GFAP、VEGF的蛋白表达均增高,且随着照射剂量的增加GFAP、VEGF的蛋白表达亦增高,差异有统计学意义(P＜0.05).20 Gy剂量照射后4～48 h内GFAP的表达呈时间依赖性,20 Gy剂量照射后4～24 h内VEGF的表达呈时间依赖性. 结论 X线能诱导体外培养的星形胶质细胞活化,GFAP及VEGF的表达呈时间及剂量依赖性增高,VEGF异常高表达可能是造成RBI的重要原因.     

伽玛刀照射后猫脑运动区皮质GFAP、S-100蛋白变化的研究

目的研究伽玛刀照射猫脑额叶运动区皮质后该区星形胶质细胞(Astrocyte,AST)中胶质原纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)和S-100蛋白表达的变化,为临床上伽玛刀治疗癫痫等疾病选择合适剂量,减少不良反应,提供敏感监测指标。方法健康家猫45只,随机分为对照组、伽玛刀10Gy和25Gy组,于照射后24小时、7天、14天,对靶区取材,进行GFAP、S-100检测。结果伽玛刀照射猫额叶运动区皮质后14天内GFAP和S-100蛋白均有表达增强,与照射剂量和时间正相关。结论伽玛刀照射猫额叶运动区皮质后14天内GFAP和S-100蛋白均有表达增强,与剂量和时间成正相关。S-100蛋白可能是放射性脑损伤急性期更敏感的指标。     

甘珀酸对癫痫大鼠前脑connexin-32和GFAP表达研究

目的研究大鼠在脑室埋管注射甘珀酸(CBX)预处理后戊四氮导致癫痫发作时前脑内胶质纤维酸性蛋白(GFAP)和连接蛋白-32(Cx32)表达及其相互关系. 方法动物分为生理盐水(NS)对照组(NS组)、CBX预处理对照组(CBX组)、戊四氮(PTZ)致痫组(PTZ组)、CBX预处理后再PTZ致痫组(CBX PTZ组),应用免疫荧光组织化学双重标记显示GFAP和Cx32在前脑的表达及其相互关系.结果 CBX PTZ组的大鼠癫痫发作的行为表现比PTZ组显著加重,星形胶质细胞GFAP的表达也比PTZ组明显升高,值得注意的是CBX组的星形胶质细胞GFAP的表达比NS组明显升高.Cx32在PTZ引起的癫痫大鼠的大脑皮层、海马和杏仁核内是增加的,而CBX预处理后的癫痫模型中Cx32却降低了.在Cx32与GFAP双标的切片上发现,Cx32与GFAP阳性的星形胶质细胞非常接近,在CBX预处理后的癫痫模型中GFAP阳性的星形胶质细胞显著增加的同时Cx32却明显降低. 结论在整体动物CBX预处理可以导致癫痫发作增强,这可能与星形胶质细胞增生及其Cx32表达降低有关.     

大鼠脑伽玛刀不同剂量损伤研究的初步结果

目的　应用伽玛刀观察不同剂量照射大鼠脑后不同时间的效应。方法　根据大鼠脑立体定位图谱 ,选择正常大鼠右侧尾状核头部为照射靶点 ,依据伽玛刀最大照射剂量分为 2 0、5 0、75、16 0Gy四组 ,使用准直器直径4mm。采用伊文思蓝 (EB)灌注和HE染色、胶质细胞原纤维酸性蛋白 (GFAP)抗体免疫组化染色方法 ,观察伽玛刀照射后不同时间发生的形态学变化。结果　在不同时期内 ,各剂量组动物靶区均出现微血管系统改变和星形胶质细胞肥大、增生 ,5 0、75、16 0Gy组发现坏死灶。 结论　形态学变化呈现时间和剂量依赖性。     

大鼠正常脑组织伽玛刀照射后GFAP表达的实验研究

目的研究伽玛刀（Gamma knife）立体定向放射外科照射大鼠正常脑组织后亚急性期胶原纤维酸性蛋白（GFAP）的表达随时间的变化,探讨星形胶质细胞（AS）的增殖与放射损伤的关系。方法30只成年雌性Wistar大鼠,随机分为6组,每组5只。其中1组为假照射组,其余5组行伽玛刀照射。运用Leksell 23004B型伽玛刀4mm准直器以50Gy照射大鼠右侧尾壳核。不同组别的大鼠分别在照射后1、2、4、8、12周深度麻醉下断头取出脑组织,行免疫组织化学染色观察GFAP的表达。结果照射后4周靶区内GFAP阳性细胞数目开始增多,细胞形态变得不规则。至8周时GFAP阳性细胞进一步增多,胞体变大,胞浆染色较深。照射后12周GFAP阳性细胞数目最多,胞体明显肥大,形态各异,细胞突起粗大不规则,胞浆呈深棕色。结论伽玛刀放射外科以50Gy照射大鼠单侧尾壳核后,照射靶区GFAP阳性细胞数不断增加,细胞形态肥大、形状不规则。     

正常大鼠脑组织伽玛刀照射后胶质细胞原纤维酸性蛋白的表达及变化

目的:观察正常大鼠脑受伽玛刀(γ-刀)照射后星形胶质细胞的反应。方法:45只正常成年大鼠脑接受γ-刀100Gy量的照射,分别成活0.5h、1h、3h、6h、12h、24h、3d、7d、14d、30d和3个月后被处死,固定取脑,冰冻切片,进行抗胶质细胞原纤维酸性蛋白(GFAP)的免疫组织化学反应。结果:①γ-刀照射后在靶区和海马等处的GFAP免疫反应(GFAPir)阳性细胞增多;②可见到2种阳性细胞,一种是胞体小、突起细短的细小型,另一种是胞体肥大、突起粗长、染色深的肥大型,存活时间越长,越靠近靶中央区,肥大型GFAPir细胞越多,肿胀越明显,反之则少;③GFAPir表达有2次高峰,第一次在照射后24h以内,第二次在照射后14d以后;④从照射后14d起海马内GFAPir细胞有的部位脱失,形成“斑秃”样分布。结论:以上结果表明GFAPir细胞在脑内的数量、形态及分布特点可以作为脑损伤的一种标志。     

缝隙连接蛋白43与创伤后脑水肿相关性研究

目的探讨缝隙连接蛋白43（Cx43）与创伤后脑水肿的相关性。方法大鼠脑损伤前2h经侧脑室注射Cx43特异性阻断剂反义寡核苷酸（ODNs）或生理盐水，用Feeney＇s自由落体法制作局灶性脑损伤动物模型，通过星形胶质细胞的特异性标记物-胶质纤维酸性蛋白（GFAP）免疫荧光染色，观察脑皮质区星形胶质细胞的形态、数量的变化，同时观察脑组织的水肿情况。结果脑损伤后生理盐水对照组GFAP标记的星形胶质细胞数量较ODNs预处理组明显增多（P〈0．05），细胞胞体较肥大；ODNs预处理组脑水肿较生理盐水对照组明显减轻（P〈0．05）。结论阻断Cx43可减轻脑创伤后脑水肿的程度，Cx43参与了脑损伤后的脑水肿的形成。     

脑白质低灌注损伤后星形胶质细胞缝隙连接蛋白43的表达变化

目的研究慢性脑白质缺血后星形胶质细胞和缝隙连接蛋白Connexin43(Cx43)的变化。方法原代培养星形胶质细胞,建立体外慢性缺氧模型;双侧颈总动脉狭窄法,建立慢性低灌注脑白质损伤小鼠模型;免疫荧光共染观察星形胶质细胞活化与Cx43表达。Western蛋白定量分析髓鞘相关指标髓鞘相关糖蛋白MAG,星形胶质细胞标记物GFAP和Cx43的表达。结果与对照组相比,细胞慢性缺氧7d后,星形胶质细胞明显增生活化,伴随Cx43表达水平明显上调。Western blot发现,在慢性脑白质缺血过程中,MAG的表达逐渐降低,GFAP持续增高,Cx43表达明显上调。免疫荧光共标记可见,星形胶质细胞中Cx43表达上调,主要分布于胼胝体中央区。结论慢性脑白质缺血损伤过程伴随星形胶质细胞Cx43表达增加,Cx43可能成为临床治疗血管性认知障碍的新靶点。     

大剂量伽玛刀照射后脑组织一氧化氮合酶亚型表达改变及其与急性脑水肿的关系

目的探讨大剂量伽玛刀(γ刀)照射后脑组织一氧化氮合酶(NOS)亚型表达改变及其与急性脑水肿的关系。方法200Gy量γ刀照射正常大鼠脑,采用光镜、电镜、免疫组化及原位杂交技术研究照射后急性脑水肿的发生发展及脑组织NOS亚型表达变化。结果①照射后30min出现急性脑水肿病理改变,照射后2h出现明显血管源性脑水肿,照射后6h出现明显细胞性脑水肿,照射后3d急性脑水肿达高峰。②脑组织NOS亚型表达在照射后30min开始增高,内皮型一氧化氮合酶(eNOS)及诱导型一氧化氮合酶(iNOS)表达分别于照射后2h及6h显著增高,神经元型一氧化氮合酶(nNOS)表达亦增高,但nNOS阳性表达细胞数量较少。3种NOS亚型表达增高持续时间长,伴行于脑水肿的急性发展阶段。结论大剂量γ刀照射后脑组织NOS亚型表达增高与急性脑水肿的发生发展有关。     

反式激活应答DNA结合蛋白-43及其相关疾病的研究进展

反式激活应答DNA结合蛋白-43(TDP-43)是近年发现的一种病理沉积蛋白,常见于多种神经变性疾病,如肌萎缩侧索硬化、额颞叶变性、阿尔茨海默病等,这一类以病理性TDP-43沉积为主的神经变性疾病统称为TDP-43蛋白病。研究发现TDP-43在这些疾病中既有相同(均有TDP-43异常沉积),又有差异(不同的形态、分布而导致不同的症状),其作用机制至今仍有许多未明。这一异常蛋白的发现不仅为研究TDP-43蛋白病的发病机制及相关疾病间的联系提供了新途径,同时为探索新的诊断方法、治疗方案提供了方向。     

Acute ethanol administration decreases GAP-43 and phosphorylated-GAP-43 in the rat hippocampus

Acute alcohol ingestion is well known to have deleterious effects on memory and also known to inhibit long-term potentiation, a putative cellular substrate of memory. In this study, we for the first time revealed that growth-associated protein 43 (GAP-43), which is well known as a presynaptic substrate of protein kinase C and one of the major synaptic plasticity-related genes, was down regulated by single ethanol administration (2.5 g/kg, 15% in saline, i.p.) in the rat hippocampus. Using real-time PCR, we confirmed that GAP-43 mRNA level is significantly decreased 2 h after ethanol administration. GAP-43 and p-GAP-43 (Ser41) immunoreactivities in the hippocampus were also reduced 4 h after ethanol administration. Immunohistochemical study showed that the reduction of GAP-43 and p-GAP-43 expression was associated with the perforant and mossy fibers pathways. These results suggest that the reduction of GAP-43 in the hippocampus might be, at least in part, a cause of memory impairment after acute ethanol ingestion.     

TDP-43 and frontotemporal dementia

TAR DNA-binding protein of about 43 kDa (TDP-43) is the main ubiquitinated peptide in tau-negative frontotemporal lobar degeneration (FTLD). TDP-43 is typically a nuclear protein, and its aggregation and cytoplasmic translocation are thought to represent major steps in the pathogenesis of FTLD due to TDP-43 proteinopathy (FTLD-TDP). Certain clinical syndromes of frontotemporal dementia are preferentially associated with pathologic findings of FTLD-TDP, and TDP-43 pathology represents the connection between FTLD-TDP and amyotrophic lateral sclerosis. Recent advances in clinical, genetic, and pathologic studies of FTLD-TDP and amyotrophic lateral sclerosis have shed light on the potentially pathogenic role of TDP-43 and identified TDP-43 itself as a candidate biomarker for antemortem diagnosis of FTLD-TDP.     

Lack of TAR-DNA binding protein-43 (TDP-43) pathology in human prion diseases

Aims: TAR-DNA binding protein-43 (TDP-43) is the major ubiquitinated protein in the aggregates in frontotemporal dementia with ubiquitin-positive, tau-negative inclusions and motor neurone disease. Abnormal TDP-43 immunoreactivity has also been described in Alzheimer's disease, Lewy body diseases and Guam parkinsonism–dementia complex. We therefore aimed to determine whether there is TDP-43 pathology in human prion diseases, which are characterised by variable deposition of prion protein (PrP) aggregates in the brain as amyloid plaques or more diffuse deposits. Material and methods: TDP-43, ubiquitin and PrP were analysed by immunohistochemistry and double-labelling immunofluorescence, in sporadic, acquired and inherited forms of human prion disease. Results: Most PrP plaques contained ubiquitin, while synaptic PrP deposits were not associated with ubiquitin. No abnormal TDP-43 inclusions were identified in any type of prion disease case, and TDP-43 did not co-localize with ubiquitin-positive PrP plaques or with diffuse PrP aggregates. Conclusions: These data do not support a role for TDP-43 in prion disease pathogenesis and argue that TDP-43 inclusions define a distinct group of neurodegenerative disorders.     

Rodent Models of TDP-43 Proteinopathy: Investigating the Mechanisms of TDP-43-Mediated Neurodegeneration

Since the identification of phosphorylated and truncated transactive response DNA-binding protein 43 (TDP-43) as a primary component of ubiquitinated inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions, much effort has been directed towards ascertaining how TDP-43 contributes to the pathogenesis of disease. As with other protein misfolding disorders, TDP-43-mediated neuronal death is likely caused by both a toxic gain and loss of TDP-43 function. Indeed, the presence of cytoplasmic TDP-43 inclusions is associated with loss of nuclear TDP-43. Moreover, post-translational modifications of TDP-43, including phosphorylation, ubiquitination, and cleavage into C-terminal fragments, may bestow toxic properties upon TDP-43 and cause TDP-43 dysfunction. However, the exact neurotoxic TDP-43 species remain unclear, as do the mechanism(s) by which they cause neurotoxicity. Additionally, given our incomplete understanding of the roles of TDP-43, both in the nucleus and the cytoplasm, it is difficult to truly appreciate the detrimental consequences of aberrant TDP-43 function. The development of TDP-43 transgenic animal models is expected to narrow these gaps in our knowledge. The aim of this review is to highlight the key findings emerging from TDP-43 transgenic animal models and the insight they provide into the mechanisms driving TDP-43-mediated neurodegeneration.     

An ELISA assay for GAP-43

An ELISA assay for the growth associated protein GAP-43 was developed to determine rapidly its relative abundance in neuronal tissue. The assay was performed with affinity-purified anti-GAP-43 antibody that detected a single band of Mr = 42,000-45,000 on Western blots of rat brain homogenates but no bands on blots of liver homogenates. GAP-43 was determined by ELISA assay in as little as 0.6 microgram protein of brain homogenate. The assay was highly reproducible; the standard error of the mean of sample to sample variation was less than 5%. When ELISA development time was held constant, the standard error of the mean of inter-assay variation was between 2 and 7%. Using this method, GAP-43 immunoreactivity was examined in developing rat brain. At post-natal day 1, GAP-43 immunoreactivity was 3-4 times greater than that observed in the adult, remained elevated for several weeks, and decreased by the end of the first month of life. These results are in accord with previous studies on the expression or synthesis of GAP-43 during neuronal development.