Barth Martin, Oulmi Yasmina, Ehrenreich Hannelore, Schilling Lothar
Department of Neurosurgery, Division of Neurosurgical Research, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany.
Acta Neuropathol. 2002 Dec;104(6):621-36. doi: 10.1007/s00401-002-0595-8. Epub 2002 Aug 1.
In the brain apoptosis may occur as a physiological phenomenon during periods of programmed cell death as well as under pathological conditions such as ischemia, trauma, tumor, and degenerative diseases. While the definition of apoptotic cell death was originally based on ultrastructural alterations, the detection of DNA double-strand breaks has become an important feature in studies of apoptosis. Currently, the terminal transferase-mediated dUTP nick-end labeling (TUNEL) procedure is widely used for detection of apoptotic cell death. However, there is a growing body of evidence to suggest that the TUNEL staining does not label apoptotic alterations exclusively. Therefore, a new staining procedure was developed combining TUNEL methodology with pre-embedding nanogold labeling to detect DNA double-strand breaks in individual cells by electron microscopy and assess the accompanying ultrastructural alterations. In vitro DNAse-treated vibratome sections (thickness, 20 micro m) from normal adult rat brains were used to develop the staining procedure consisting of the following steps: (i) TUNEL staining of free-floating vibratome sections using fluorescein isothiocyanate (FITC)-labeled UTP, (ii) conversion of the fluorescence signal into an electron-dense signal using an anti-FITC antibody coupled with ultrasmall (diameter, 0.8 nm) gold particles followed by silver enhancement, and (iii) osmification, embedding in Spurr resin and cutting of ultrathin sections. Early postnatal brain tissue was used to study physiologically occurring apoptotic cell death. Under these conditions different patterns of gold staining were observed probably representing different states of cellular decay along the apoptotic avenue. Severe focal brain ischemia was studied as a pathological situation in which intense TUNEL staining occurs. Under these conditions TUNEL labeling of cells was regularly observed in conjunction with ultrastructural alterations indicative of necrosis. These results suggest that under pathological conditions apoptosis and necrosis are not mutually exclusive mechanisms but rather may occur concurrently along a continuum in which cell death occurs.
在大脑中，细胞凋亡可能作为一种生理现象发生在程序性细胞死亡期间，以及诸如缺血、创伤、肿瘤和退行性疾病等病理状况下。虽然凋亡性细胞死亡的定义最初基于超微结构改变，但DNA双链断裂的检测已成为凋亡研究中的一个重要特征。目前，末端转移酶介导的dUTP缺口末端标记（TUNEL）方法被广泛用于检测凋亡性细胞死亡。然而，越来越多的证据表明，TUNEL染色并非专门标记凋亡改变。因此，开发了一种新的染色方法，将TUNEL方法与包埋前纳米金标记相结合，通过电子显微镜检测单个细胞中的DNA双链断裂，并评估伴随的超微结构改变。使用来自正常成年大鼠大脑的体外经DNA酶处理的振动切片（厚度为20微米）来开发该染色方法，其包括以下步骤：（i）使用异硫氰酸荧光素（FITC）标记的UTP对游离的振动切片进行TUNEL染色，（ii）使用与超小（直径0.8纳米）金颗粒偶联的抗FITC抗体将荧光信号转化为电子致密信号，随后进行银增强，以及（iii）进行锇化、包埋在Spurr树脂中并切割超薄切片。产后早期脑组织用于研究生理性发生的凋亡性细胞死亡。在这些条件下，观察到不同模式的金染色，可能代表沿凋亡途径细胞衰变的不同状态。严重局灶性脑缺血作为一种出现强烈TUNEL染色的病理情况进行了研究。在这些条件下，经常观察到细胞的TUNEL标记与指示坏死的超微结构改变同时出现。这些结果表明，在病理条件下，凋亡和坏死并非相互排斥的机制，而是可能沿着细胞死亡发生的连续统一体同时发生。
