EXAMINATION OF MECHANISMS OF EARLY PROGRAMMED MOTONEURON DEATH IN THE DEVELOPING CHICK CERVICAL SPINAL CORD

在鸡胚胎脊髓中，运动神经元（MNs）的程序性细胞死亡（PCD）有两种类型，一种发生在相对较晚的分化阶段，另一种发生在分化较早的阶段。在第二种类型中，MNs的PCD仅发生在E4和E5之间的非肢体支配颈脊髓。为了阐明半胱天冬酶在颈脊髓MNs早期PCD中的作用，我们在体内检测了半胱天冬酶的激活和半胱天冬酶抑制剂的作用。在24 （E4.5）时，颈节段腹侧含MN区Caspase-3样活性升高，此时观察到许多收缩性死亡神经元。免疫组化观察显示，caspase-3的激活先于DNA断裂和宫颈MNs PCD的形态学表现。为了进一步研究caspase-3样活性的作用，我们用Ac-DEVD-CHO处理胚胎，抑制caspase-3样活性。当计数缩缩细胞时，包括这些中度缩缩细胞，对照组和Ac-DEVD-CHO处理的胚胎之间没有显著差异。TUNEL染色显示，在这些适度凝聚的细胞核中没有发生DNA断裂，尽管电镜观察清楚地显示细胞质中发生了退行性变化。这些结果提示caspase-3样活性在凋亡细胞核改变中起作用。为了研究其他负责细胞质凋亡变化的半胱天冬酶的参与，我们用抑制广谱半胱天冬酶的Boc-Asp-FMK （BAF）处理胚胎。经BAF处理12小时后，固缩细胞数量明显少于对照组。即使在Ac-DEVD-CHO治疗后也很少观察到中度固缩细胞。相反，有许多细胞核较小且细胞质嗜酸性稍强的细胞。这些细胞缺乏DNA断裂，并保留了MN特异性标记物。电镜观察显示有许多小细胞，细胞核不规则，细胞质微电子致密，细胞器明显正常。BAF处理24小时后，固缩细胞的数量增加到与Ac-DEVD-CHO处理的胚胎相当的水平，尽管TUNEL阳性细胞的数量仍然少于对照组。电镜观察显示有许多异常变性细胞。存活的MNs总数不多于对照和Ac-DEVD-CHO处理的胚胎。这些结果表明，其他可被BAF抑制而不能被Ac-DEVD-CHO抑制的caspase也参与了死亡MNs的凋亡变化。尽管抑制这些caspase可显著延缓PCD的速率，但MNs不能通过抑制caspase而最终获救。少

In the chick embryo spinal cord, two types of programmed cell death (PCD) of motoneurons (MNs) are known to occur, one at a relatively late stage and the other at an earlier stage of differentiation. In the second type, the PCD of MNs occurs only in the non-limb innervating cervical spinal cord between E4 and E5. To elucidate the roles for caspases in this early PCD of MNs in the cervical spinal cord, we examined the activation of caspases and the effects of inhibitors of caspases in vivo.Caspase-3 like activity was increased in ventral MN containing region of the cervical segments at st 24 (E4.5) when many pyknotic dying neurons are observed. Immunohistochemical observations with an antibody against activated form of caspase-3 suggested that activation of caspase-3 precedes both DNA fragmentation and the morphological manifestation of PCD in cervical MNs. To further investigate roles for casapase-3 like activity, we treated embryos with Ac-DEVD-CHO that inhibits caspase-3 like activit … More y. In Ac-DEVD-CHO treated embryos, the nuclei of pyknotic cells showed only moderate condensation. When pyknotic cells were counted, including these moderately pyknotic cells, there was no significant difference between controls and Ac-DEVD-CHO treated embryos. TUNEL staining revealed that DNA fragmentation did not occur in these moderately condensed nuclei, even though electron microscopic observations clearly revealed that degenerative changes were occurring in the cytoplasm. These results suggest that caspase-3 like activity plays a role in the apoptotic nuclear changes.To investigate involvement of other caspases that are responsible for apoptotic changes of cytoplasm, we treated embryos with Boc-Asp-FMK (BAF) that inhibits broad spectrum of caspases. After treatment with BAF for 12 hours the number of pyknotic cells remarkably fewer than controls. Even moderately pyknotic cell that were observed after treatment with Ac-DEVD-CHO were rarely observed. Instead, there were many cells whose nuclei are smaller and whose cytoplasm was slightly more eosinophilic. These cells lacked DNA fragmentation and retained MN specific marker. Electron microscopic observation revealed that there are many smaller cells that have irregular shaped nuclei, slightly electron dense cytoplasm and apparently normal organelles. Following treatment with BAF for 24 hours, the number of pyknotic cells increased to the level comparable to embryos treated with Ac-DEVD-CHO, although the number of TUNEL positive cells was still fewer than controls. Electron microscopic observation revealed that there are many aberrantly degenerating cells. The total number of surviving MNs was not more than controls and Ac-DEVD-CHO treated embryos. These results suggest that other caspases that can be inhibited by BAF but not by Ac-DEVD-CHO play roles in execution of apoptotic changes in dying MNs. Although inhibition of such caspases considerably delayed the rate of PCD, MNs can not be finally rescued by inhibition of caspases. Less