Shapshak P, Tourtellotte W W, Wolman M, Verity N, Verity M A, Schmid P, Syndulko K, Bedows E, Boostanfar R, Darvish M
J Neurosci Res. 1986;16(1):281-301. doi: 10.1002/jnr.490160124.
In order to obtain a useful and readily applicable in situ hybridization (ISH) protocol for progressive central nervous system (CNS) diseases of unknown etiology that are possibly due to persistent viral infection, known and well described diseases were studied, namely, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE). The procedures described were validated by confirming results obtained by other investigators using histology, immunocytochemistry, electron microscopy, and ISH. A number of frequently encountered problems of tissue preparation are addressed as well as techniques to reduce autoradiography exposure times. A multi-staged specific, sensitive, reliable, and valid procedure for detection of viral genomes, mRNA and proteins is approached. Formalin-fixed and paraffin-embedded (FFPE) brain material from six patients who died with PML and one patient who died from SSPE were studied using ISH with a tritium-labeled cloned JC virus DNA probe and a measles-cloned nucleocapsid (NC) gene cDNA probe, respectively. This report constitutes a methodological framework as well as a detailed neuropathological analysis of identified brain cell populations within which in situ hybridization was detected. In early PML lesions, swollen nuclei or oligodendrocytes were the predominant cells labeled, whereas older lesions revealed increased numbers of reactive and bizarre hypertrophic astrocytes hybridized at the outer periphery of the demyelinated lesions. The hybridization varied greatly in intensity in different cells. Intense hybridization was noted very rarely in microglial cells, including rod cells and rarely in venular pericytes, intravascular mononuclear cells, or in vascular endothelial cells. These results, considered together with previous findings, indicate that in PML the viral infection runs different courses in the various cells: in astrocytes the viral genome persists for a long time inducing pathological changes in some cells. In oligodendrocytes the infection rapidly lyses the cells. There was a good correlation between chromatic changes observable in routinely stained sections and virus presence. In addition, in situ hybridization using a measles-NP-cloned probe in white matter from FFPE SSPE brain is presented confirming earlier results in SSPE cryopreserved brain.
为了获得一种适用于病因不明的进行性中枢神经系统（CNS）疾病（可能由持续性病毒感染引起）的实用且易于应用的原位杂交（ISH）方案，我们研究了已知且描述详尽的疾病，即进行性多灶性白质脑病（PML）和亚急性硬化性全脑炎（SSPE）。通过确认其他研究者使用组织学、免疫细胞化学、电子显微镜和ISH所获得的结果，验证了所描述的程序。文中讨论了一些组织制备过程中经常遇到的问题以及减少放射自显影曝光时间的技术。我们提出了一种用于检测病毒基因组、mRNA和蛋白质的多阶段特异性、灵敏性、可靠性和有效性的程序。分别使用氚标记的克隆JC病毒DNA探针和麻疹克隆核衣壳（NC）基因cDNA探针，对6例死于PML的患者和1例死于SSPE的患者的福尔马林固定石蜡包埋（FFPE）脑材料进行ISH研究。本报告构成了一个方法框架以及对检测到原位杂交的已识别脑细胞群体的详细神经病理学分析。在早期PML病变中，肿胀的细胞核或少突胶质细胞是主要的标记细胞，而较老的病变显示在脱髓鞘病变外周有更多反应性和怪异的肥大星形胶质细胞杂交。不同细胞中的杂交强度差异很大。在小胶质细胞（包括杆状细胞）中很少观察到强烈杂交，在小静脉周细胞、血管内单核细胞或血管内皮细胞中也很少见。这些结果与先前的发现一起表明，在PML中病毒感染在不同细胞中呈现不同进程：在星形胶质细胞中病毒基因组长期存在，在一些细胞中诱导病理变化。在少突胶质细胞中感染迅速裂解细胞。在常规染色切片中观察到的染色变化与病毒存在之间存在良好的相关性。此外，还展示了使用麻疹NP克隆探针在FFPE SSPE脑白质中进行原位杂交的结果，证实了在SSPE冷冻保存脑中的早期结果。
