朊病毒病实验动物脑组织PrPSc样本库的建立

目的 建立朊病毒病实验动物脑组织检测样本库,检测其在特定保存条件下的稳定性,以用于动物和人朊病毒病诊断技术的评估和考核.方法 选用30只经颅内注射感染羊瘙痒因子263K的仓鼠和30只正常仓鼠,每只分别制备10%、1%和0.5%3种浓度的脑组织匀浆,分装冷冻保存.以Western Blot方法 确定脑组织匀浆中PrPSc的存在情况,并分别在建库半年和3年检测PrPSc的稳定性.结果 30只感染仓鼠10%脑组织匀浆全部可检出PrPSc,1%脑组织匀浆有26份为PrPSc阳性,0.5%脑组织匀浆有19份为PrPSc阳性.半年及3年后复检,PrPSc阳性检出率基本不变.所有正常仓鼠脑匀浆均为PrPSc阴性.结论 建立了稳定性良好的含有90份PrPSc阳性标本和90份PrPSc阴性样本的朊病毒病实验动物脑组织检测样本库.     

卡马西平对大鼠海马星形胶质细胞形态和GFAP表达的影响

目的观察治疗剂量卡马西平对SD大鼠海马星形胶质细胞形态和胶质纤维酸性蛋白(GFAP)表达的影响。方法治疗剂量卡马西平每24 h单次灌胃给药后,于不同时间点经心脏灌注,取脑;GFAP免疫组化染色测量海马GFAP阳性细胞数量及形态。结果1周组海马CA1区星形胶质细胞的形态及GFAP表达无变化,1个月组星形胶质细胞数量及GFAP表达量无明显增加,3个月组GFAP阳性细胞数显著增加,达对照组的1.74倍(P<0.05);并伴细胞体积增大,侧支增多。结论治疗剂量的卡马西平对星形胶质细胞形态和GFAP表达的影响呈时间依赖性。     

免疫组织化学和电镜观察成年大鼠大脑皮质损伤后星形胶质细胞的反应

<正>实验用成年SD大鼠15只,经切除双侧大脑皮质顶叶正中部分(3×3×1mm),术后第四天取脑组织,其中10只用于胶质原纤维酸性蛋白(GFAP)抗体免疫染色,用以标记星形神经胶质细胞的形态结构,其余5只用于电镜常规制作标本.每只动物在损伤区旁左右四壁各边取lmm~3脑组织块,进行超薄切片、电镜观察.实验结果:GFAP免疫染色:在损伤区旁边缘～300μm范围内,可见星形胶质细胞明显增加及细胞增大.其细胞增生程度在各层并不一致.在皮质的深层,v～Ⅵ层,星形胶质细胞增生最明显,在浅层Ⅰ～Ⅱ层增生也较明显,其次是浅层与深层之间增生的细胞稍为稀疏,并见星形胶质细胞各期分裂像.星形胶质细胞增大、胞质丰     

小鼠流行性乙型脑炎疾病进展中反应性星形胶质细胞活化模式的变化及干预

目的 明确星形胶质细胞活化模式与流行性乙型脑炎疾病进展的关系。方法 首先通过足垫注射乙型脑炎病毒(JEV)构建流行性乙型脑炎小鼠模型,采用免疫荧光技术(IFA)检测JEV非结构蛋白3(NS3)在小鼠全脑的表达情况;进一步利用IFA、 RNA测序技术(RNA-seq)、实时定量PCR明确流行性乙型脑炎发病不同阶段星形胶质细胞活化模式的变化;最后,通过侧脑室注射鸢尾素(irisin)调控补体C3阳性A1型星形胶质细胞、 S100钙结合蛋白A10(S100A10)阳性的A2型星形胶质细胞活化比例,探索其是否可改善乙型脑炎模型小鼠的体质量、行为学评分及脑组织损伤情况。结果 流行性乙型脑炎模型组小鼠的M1、 M2等运动皮层脑区和海马组织中NS3蛋白显著增加。上述区域胶质原纤维酸性蛋白(GFAP)阳性星形胶质细胞的数目和体积较对照组也显著增加,其中JEV感染后与A1/A2型星形胶质细胞活化相关的基因均显著升高。侧脑室或者腹腔注射irisin虽不能改善流行性乙型脑炎预后,但可在一定程度上抑制A1型星形胶质细胞活化,减轻神经炎症。结论 JEV主要感染小鼠的运动皮质和海马神经元,且星形胶质细胞异常激活导...     

辐射诱导大鼠脑损伤后星形胶质细胞活性及其自噬的变化

目的:探究辐射诱导大鼠脑损伤后星型胶质细胞活性及其自噬的相关变化。方法:取36只体质量为180～200 g的Sprague-Dawley大鼠,进行4 d的Morris水迷宫训练后,随机分为假手术(sham)组、模型(model)组及3-甲基腺嘌呤(3-MA)组,其中model组及3-MA组大鼠经腹腔麻醉后给予单次全脑X线照射,剂量为20 Gy。照射完成后,model组侧脑室注射5μL Na Cl,3-MA组侧脑室注射600 nmol 3-MA,饲养8周,用Morris水迷宫进行学习和记忆能力测评后,断头取脑,HE染色观察海马区病理变化。用GFAP的免疫组化和Western blot检测星形胶质细胞活性;用GFAP及LC3的双重荧光染色评定星形胶质细胞自噬的变化; Western blot法测定cleaved caspase-3蛋白水平的变化,TUNEL检测同侧海马区细胞凋亡情况; ELISA法检测肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平以评估海马区炎症反应。结果:辐射可抑制星形胶质细胞活性,激活星形胶质细胞自噬,加重脑组织损伤。3-MA可促进星形胶质细胞的活化,促进脑组织损伤的修复。结论:大鼠放射性脑损伤后海马区损伤明显,星形胶质细胞数量明显减少,3-MA可显著缓解受损程度。该发现可能会为放射性脑损伤的治疗提供新途径。     

β-淀粉样蛋白诱导大鼠行为学改变及星形胶质细胞变化

为了探讨不同剂量β-淀粉样蛋白 2 5 -3 5片段 ( Aβ2 5- 35)诱导老年性痴呆 ( AD)大鼠动物模型中星形胶质细胞变化与大鼠行为学改变之间的关系 ,在大鼠双侧海马内注射不同剂量 Aβ2 5- 35( 2 .5、5、10和 2 0 μg)制作大鼠 AD模型 ,注射一周后采用胶质原纤维酸性蛋白 ( GFAP)免疫组化染色、NOS组化染色及 NOS组化和 GFAP双重染色分析大鼠海马 GFAP与 NOS的表达。结果发现海马内注射 Aβ2 5- 35剂量≥ 10 μg时 ,模型组大鼠出现学习记忆减退 ,海马星形胶质细胞增生、肥大 ,数目明显多于对照组 ( P<0 .0 5 ) ;海马 NOS阳性神经元数较对照组显著减少 ( P<0 .0 5 ) ;并出现 NOS阳性星形胶质细胞。结论 :星形胶质细胞参与Aβ神经毒性导致 NOS阳性神经元损伤 ,间接导致大鼠学习记忆能力下降 ,在 AD模型大鼠早期学习记忆功能减退中起重要作用     

超声处理对羊瘙痒病脑组织中PrPSc聚集状态的影响

目的 研究超声处理对感染羊瘙痒症仓鼠脑组织中PrP^Sc聚集状态的影响，寻找产生PrP^Sc低聚体的条件。方法 裂解液制备脑组织提取物，用各种超声条件处理不同阶段的脑组织提取物；以蛋白酶消化后的Western blot方法和图象分析系统观测PrP^Sc蛋白的分布和聚集状态。结果 适当的超声处理(15s共30次)可增加脑组织匀浆上清中PrP^Sc含量1．29～1．58倍；同样条件下超声处理可明显增加羊瘙痒因子263K感染仓鼠脑组织匀浆上清中PrP蛋白总量，而对正常对照仓鼠脑组织匀浆上清中PrP蛋白总量影响不大；对经常规高速离心获得的PrP^Sc的超声处理显示约90％的PrP^Sc存在于离心上清液中。结论 对感染动物脑组织进行超声处理可增加PrP^Sc的提取量，利于实验室检测。适当的超声处理可破碎大的相对分子质量的PrP^Sc聚集物，产生小的相对分子质量的PrP^Sc产物。     

苯丙胺对大鼠学习能力和海马结构GFAP阳性细胞结构的影响

目的观察苯丙胺对大鼠学习能力、GFAP免疫阳性细胞结构和亚细胞结构的影响。方法苯丙胺腹腔注射SD大鼠,用水迷宫检测其空间辨别性学习能力,用免疫组织化学方法观察海马结构胶质纤维酸性蛋白(GFAP)免疫阳性细胞的形态变化;用电镜方法检测GFAP免疫阳性细胞超微结构变化。结果学习能力检测发现,在14d以前,苯丙胺组大鼠较生理盐水组的平均运行时间和平均潜伏期缩短(P0.05);在15～28d,苯丙胺组大鼠较生理盐水组正确率降低(P0.05);在29～42d,苯丙胺组大鼠较生理盐水组正确率降低、平均运行时间延长(P0.05)。GFAP阳性细胞形态学观察发现,正常对照组大鼠海马结构星形胶质细胞主要分布在海马以及齿状回的多形层和分子层;生理盐水组大鼠海马结构星形胶质细胞分布与游水组相似,但细胞突起变长及增粗,分支增多,且各亚区比正常对照组相应亚区的星形胶质细胞数量增多(P0.05);苯丙胺组大鼠海马星形胶质细胞分布与正常对照组相似,但细胞突起变长、增粗和分支增多更明显,各亚区的星形胶质细胞比正常对照组和生理盐水组相应亚区增多(P0.05);电镜观察发现苯丙胺组大鼠脑内星形胶质细胞增生肥大,亦可见退变星形胶质细胞。结论在本实验条件下,苯丙胺导致大鼠空间辨别性学习记忆能力下降,引起大鼠海马结构星形胶质细胞增生和损害。     

一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

大鼠癫痫敏感性增强过程中c-jun与GFAP基因表达的关系

本文利用免疫细胞化学技术研究了海人酸(KA)致大鼠癫痫敏感性长期增强过程中,原癌基因c-jun的表达规律及其与星形胶质细胞增生、星形胶质细胞的标志物-神经胶质原纤维酸性蛋白(GFAP)基因表达的关系。一次性给予惊厥剂量(10mg/kg,i.p.)的KA后,海马CA1区出现c-jun的一过性高表达,之后呈持续表达状态直至给予KA后6个月。c-jun的表达明显地早于给予KA 2 d后方开始出现的海马内CA1区锥体细胞层神经元的死亡和C-JUN免疫反应阳性的星形胶质细胞的增生。星形胶质细胞GFAP基因的表达与其细胞内的c-jun表达的时间和部位一致。结果提示,c-jun在癫痫敏感性长期增强过程中持续表达,并可能参与介导神经元死亡和维持星形胶质细胞增生以及调控其细胞内GFAP基因的表达。     

Experimental scrapie in golden Syrian hamsters: temporal comparison of in vitro cell-fusing activity with brain infectivity and histopathological changes.

Golden Syrain hamsters were inoculated intracerebrally with the hamster-adapted 263K strain of scrapie virus, and the evolution of in vitro cell fusing activity induced by brain suspensions was compared with brain infectivity titers and histological changes. Cell-fusing activity abruptly appeared 4 weeks after inoculation, 1 week before the earliest detectable histopathological changes, at an infectivity level of 7.6 log 50% lethal doses per g of brain. Cell-fusing activity was sustained throughout the remaining 4 weeks of the incubation period and the subsequent 1- to 3-week stage of clinical illness but did not increase with the logarithmic progression of infectivity, which reached a level of 11 log 50% lethal doses per g in the agonal stage of disease. Gliosis was most sensitively detected by a monoclonal antibody reacting with astrocyte intermediate filaments in an indirect immunofluorescence test, anticipating histological recognition of gliosis and spongiform change by 1 to 2 weeks. In vitro cell-fusing activity is thus one of the earliest known biological markers (apart from infectivity itself) of experimental scrapie infection.     

Absence of Spiroplasma or other bacterial 16s rRNA genes in brain tissue of hamsters with scrapie

Spiroplasma spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained > or =10(10) infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control Spiroplasma mirum genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.     

Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture.

A previous paper (Kimberlin & Walker, 1977) described an experimental model of scrapie in hamsters in which the incubation period decreased progressively over the first 4 passages before becoming stable at the 5th and subsequent passes. Studies have been made of some of the agent strains present in brains taken from the 2nd, 3rd, 4th and 6th hamster passes. The results indicate the presence of at least two strains of agent at the 3rd passage level. One of these (431K) is highly pathogenic for mice and the other (263K) has an extremely low pathogenicity for mice. However only one of these strains (263K) is present in hamster brain after the 6th serial passage. It is suggested that the 'adaptation' of scrapie to hamsters may involve the selection, from a mixture, of a single strain which is highly pathogenic for hamsters. The possibility of modification of the properties of agent strains on passage discussed.     

The ultrastructural diversity of scrapie-associated fibrils isolated from experimental scrapie and Creutzfeldt-Jakob disease.

Several different samples of scrapie-associated fibrils (SAF) were extracted in identical fashion from the brains of golden Syrian hamsters infected with the 263K strain of scrapie agent and NIH Swiss mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) agent. Based on a total of over 500 measurements in individual fibrils in different extracts, hamster fibrils were more abundant, thicker and had better defined substructure than mouse fibrils. Hamster protofibrils were usually either twisted helically or in parallel arrays, whereas mouse protofibrils were often twisted, occasionally parallel, or could not be morphologically defined. Thus, SAF preparations from scrapie-affected hamsters can be ultrastructurally distinguished from those of CJD-affected mice, an observation that presumably reflects differences in their respective host-encoded amyloid protein subunits.     

Purification of non-infectious ganglioside preparations from scrapie-infected brain tissue

Summary The extraction and purification of gangliosides from brains of animals infected with the scrapie agent was evaluated by scaling-down a large-scale procedure currently used with bovine brains. Inactivation experiments employed hamster brains infected with the 263 K strain of scrapie. Residual infectivity was determined at different points of the procedure and in the final preparation by an in vivo animal bioassay. The efficacy of single steps, which included chemicals or physical techniques known to affect the viability of the scrapie agent, was verified by spiking experiments where known amounts of infectivity were added just before each single step. Infectivity was significantly diminished at early stages of the purification, and no infectivity was detected in the final preparation containing purified gangliosides. The significant reduction of infectivity at intermediate steps and the total absence of detectable infectivity in the final product confirmed that a combination of heat and exposure to sodium hydroxide, even in the presence of organic solvents, completely inactivated the scrapie agent.     

Reduction of protein kinase MARK4 in the brains of experimental scrapie rodents and human prion disease correlates with deposits of PrP(Sc)

Microtubule affinity-regulating kinase 4 (MARK4) belongs to a family of kinases that are able to actively phosphorylate the neuronal microtubule-associate proteins (MAPs), such as tau, MAP2 and the ubiquitous MAP4. Abnormal changes in tubulin and the profiles of tau have been previously reported in the human brain and animal transmissible spongiform encephalopathies (TSEs), which may be associated with abnormal alterations of various cellular kinases. To elucidate the possible role of MARK4 in TSE pathogenesis, the MARK4 levels in the brain tissues of scrapie-infected rodents and human prion diseases were evaluated using western blotting and immunohistochemical assays. The results revealed that at terminal stages of the diseases, MARK4 levels in the brain tissues of the scrapie 263K-infected hamsters, 139A-infected mice and a case of Creutzfeldt-Jakob disease (CJD, G114V gCJD) correlated with amounts of PrP(Sc) deposits that were almost undetectable. On the other hand MARK4 signals were noticeable in the brain tissues of a fatal familial insomnia (FFI) patient without PrP(Sc). The reduction of MARK4 was closely related to the prolonged incubation times. These results could be reproduced in SK-N-SH and PC12 cell lines after being exposed to the synthetic peptide PrP106-126. Accordingly, the levels of phosphorylated tau at Ser262 (p-tau262) in cultured cells exposed to PrP106-126, or the ratios of p-tau262/total tau in the brain tissues of 263K-infected hamsters were also significantly decreased. According to our data there is a correlation between a TSE pathological-associated decline of MARK4 in the brain tissues with the deposits of PrP(Sc). Reduction of MARK4 will result in abnormalities of tau phosphorylation, and possibly induce further detachment of microtubules and hinder microtubule transportation.     

Serial ultrastructural studies of scrapie in hamsters

We report sequential studies of the ultrastructural neuropathology of the subcortical grey matter of hamsters infected with the 263K strain of scrapie virus. Vacuolation in this model develops relatively late in the incubation period, while tubulovesicular structures appear early. Furthermore, neuroaxonal dystrophy, accumulations of branching tubules, and neuronal change consisting of intracytoplasmic "whorls" of proliferating membranes constitute prominent features of scrapie-related neurodegeneration.