Soriano E, Del Río J A, Auladell C
Unidad de Biología Celular, Facultad de Biología, Universidad de Barcelona, Spain.
J Histochem Cytochem. 1993 Jun;41(6):819-27. doi: 10.1177/41.6.8315274.
Cells displaying highly condensed pyknotic nuclei, the most characteristic feature of apoptosis, are considered as dead cells in neural tissue. The present study aimed to devise methods that could allow the neurogenetic and phenotypic characterization of dying pyknotic cells. In the first set of experiments, pregnant mice were labeled at embryonic days E10-E16 with pulses of 5'-bromodeoxyuridine visualization of BrdU after an immunoperoxidase reaction. In addition to normal, healthy immunopositive nuclei, these preparations displayed a number of pyknotic nuclei that were immunoreactive for BrdU. Both the regional and the temporal distribution of BrdU-positive pyknotic cells were coincidental with the peaks of dead cells in neural tissue. For example, pulses of BrdU at E10-E11 resulted in the visualization of immunoreactive pyknotic cells in the subplate and white matter of the cerebral cortex in early postnatal (P) animals. Thus, the times of generation (birthdates) of cells subjected to degenerative processes can be unequivocally identified. In the second set of experiments, brain sections from unlabeled littermates were immunostained for a variety of neural and glial markers and counterstained with bisbenzimide, to find antigens which, by being present in degenerate pyknotic cells, could indicate the phenotype of such cells. Although no pyknotic cells were positively immunostained for neurofilaments, neuropeptide Y, somatostatin, vasoactive intestinal polypeptide, or vimentin, a number of pyknotic cells were found to be immunoreactive for microtubule-associated protein 2, gamma-aminobutyric acid, calbindin 28KD, and glial fibrillary acidic protein. The percentage of pyknotic cells labeled with neural antigens accounted for more than 20% of the total number of pyknotic cells in a given brain region. In contrast, GFAP-positive pyknotic cells represented up to 50% of the total pyknotic cell population. The method shown here has enabled us to determine that both neurons and glial cells undergo degeneration during normal development.
细胞核高度浓缩呈固缩状是细胞凋亡最典型的特征，在神经组织中，呈现这种特征的细胞被视为死亡细胞。本研究旨在设计一些方法，以便对濒死的固缩细胞进行神经遗传学和表型特征分析。在第一组实验中，在胚胎期第10 - 16天给怀孕小鼠注射5'-溴脱氧尿苷脉冲标记，免疫过氧化物酶反应后进行BrdU可视化检测。除了正常、健康的免疫阳性细胞核外，这些标本还显示出一些对BrdU呈免疫反应的固缩细胞核。BrdU阳性固缩细胞的区域和时间分布与神经组织中死亡细胞的峰值一致。例如，在胚胎期第10 - 11天注射BrdU脉冲，可在出生后早期（P）动物的大脑皮质板下区和白质中观察到免疫反应性固缩细胞。因此，可以明确鉴定出经历退化过程的细胞的生成时间（出生日期）。在第二组实验中，对未标记的同窝幼崽的脑切片进行多种神经和神经胶质标志物的免疫染色，并用双苯甲酰亚胺复染，以寻找那些存在于退化固缩细胞中、能够指示此类细胞表型的抗原。尽管没有固缩细胞对神经丝、神经肽Y、生长抑素、血管活性肠肽或波形蛋白呈阳性免疫染色，但发现一些固缩细胞对微管相关蛋白2、γ-氨基丁酸、钙结合蛋白28KD和胶质纤维酸性蛋白呈免疫反应。在给定脑区，用神经抗原标记的固缩细胞百分比占固缩细胞总数的20%以上。相比之下，GFAP阳性固缩细胞占固缩细胞总数的比例高达50%。本文展示的方法使我们能够确定，在正常发育过程中神经元和神经胶质细胞都会发生退化。
