Lucassen P J, Chung W C, Vermeulen J P, Van Lookeren Campagne M, Van Dierendonck J H, Swaab D F
Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, The Netherlands.
J Histochem Cytochem. 1995 Nov;43(11):1163-71. doi: 10.1177/43.11.7560899.
In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.
原位末端标记（ISEL）可识别组织切片中凋亡或坏死细胞核内的DNA片段化。然而，将ISEL应用于人类大脑需要在死后延迟（PMD）期间保持DNA完整性，并且在（长时间）组织固定后，片段化DNA具有良好的可及性。因此，我们研究了ISEL与大鼠和人类大脑中PMD及固定的关系。在灌注大鼠脑的单侧损伤模型上的应用表明，延长后固定时间会显著降低ISEL结果。然而，微波预处理可以抵消这些掩盖效应，且不会在对侧诱导非特异性标记。另一方面，在短暂后固定的灌注脑或浸泡固定的大鼠和人类PMD脑标本中，微波预处理是有害的，并会诱导强烈的非特异性标记。在幼鼠大脑中，直到PMD达24小时出现大量核标记之前，PMD对少量凋亡细胞核并无影响。在人类皮质中，DNA片段化模式与固定时间或PMD持续时间无关，并且从PMD 4.25小时起就已存在。我们的数据表明，人类大脑上的ISEL代表生前DNA损伤而非PMD假象。此外，微波预处理似乎仅在特定的固定条件下才有益。
