van der Zee D C, de Heer E, Piersma J, Vermeij-Keers C
Department of Pediatric Surgery, Wilhelmina Children's Hospital, University of Utrecht, The Netherlands.
Teratology. 1995 Aug;52(2):57-70. doi: 10.1002/tera.1420520202.
The effects of intracardiac injection of 0.5 microliter allospecific hemolyzing rat-antirat antibodies, directed against the blood group antigens, on the endothelium of the dorsal aortae were studied in 9-14 somite-staged Wistar and RIV:Tax rat embryos, using both transmission electron microscopy (TEM) and immunoelectron microscopy (IEM). In a TEM study it was further investigated if either apoptosis or cell necrosis occurred as a result of the forementioned intracardiac injection. The results were compared to ultrastructural findings of the dorsal aortae in sham- and noninjected rat embryos of the same gestational age. In the control rat embryos, the aortic vascular wall consisted of a single continuous layer of endothelial cells. No clear basal lamina was present in TEM. Furthermore, no immunoreactivity against the endothelium or the intravascular blood cells was noted. Embryos injected with hemolyzing rat-antirat antibodies displayed clefts or pores, and diaphragmatic fenestrations of the endothelial lining of the dorsal aortae after 2 hr. Alterations resembled those induced by vasoactive mediators such as histamine, serotonin, bradykinin, and prostaglandins. The above changes had disappeared 4 and 6 hr after injection with complete restoration of the endothelial lining. Immunogold staining demonstrated Ig depositions along the luminal side of the endothelium, in the vicinity of the intercellular spaces, and in the subendothelial space of the dorsal aortae. Numerous particles were seen located inside intracytoplasmatic vesicles, indicating involvement of transcytoplasmatic transport as well as intracytoplasmatic phagocytosis. Similar depositions were observed in and around intravascular embryonic blood cells. Apoptosis, or programmed cell death, an important component in immunological reactions, occurred in rat embryos injected with hemolyzing rat-antirat antibodies. The excessive amount of apoptosis seen in this study is in accordance with the pathogenetic cell degeneration found in our earlier studies. Cell necrosis was not observed. The results from this study indicate that the endothelium of the dorsal aortae and intravascular blood cells only display a transient reaction following injection with hemolyzing rat-antirat (RAR) antibodies. The temporary reaction is presumably due to the release of vasoactive mediators. The smaller vessels and capillaries are still in an earlier stage of development, displaying fenestration, making them more susceptible for injury after immunological interaction. The results are indicative that the pathogenetic effect of the immunological reaction after intracardiac injection takes place at the level of the microcirculation by "switching on" apoptosis. Programmed cell death is essential in embryogenesis and development. Therefore excessive apoptosis, i.e., inappropriate apoptosis, will eventually induce congenital malformations.
在9至14体节期的Wistar和RIV:Tax大鼠胚胎中，使用透射电子显微镜（TEM）和免疫电子显微镜（IEM），研究了向心脏内注射0.5微升针对血型抗原的同种异体溶血大鼠抗大鼠抗体对背主动脉内皮的影响。在TEM研究中，进一步调查了上述心脏内注射是否导致细胞凋亡或细胞坏死。将结果与相同胎龄的假手术和未注射大鼠胚胎背主动脉的超微结构结果进行比较。在对照大鼠胚胎中，主动脉血管壁由单一连续的内皮细胞层组成。TEM中未发现明显的基膜。此外，未观察到针对内皮或血管内血细胞的免疫反应性。注射溶血大鼠抗大鼠抗体的胚胎在2小时后，背主动脉内皮衬里出现裂隙或孔隙以及隔膜窗孔。这些改变类似于组胺、5-羟色胺、缓激肽和前列腺素等血管活性介质诱导的改变。上述变化在注射后4小时和6小时消失，内皮衬里完全恢复。免疫金染色显示Ig沉积沿内皮的管腔侧、细胞间隙附近以及背主动脉的内皮下间隙。在细胞质内小泡内可见大量颗粒，表明涉及跨细胞质转运以及细胞质内吞噬作用。在血管内胚胎血细胞内及其周围也观察到类似的沉积。细胞凋亡，即程序性细胞死亡，是免疫反应的重要组成部分，发生在注射溶血大鼠抗大鼠抗体的大鼠胚胎中。本研究中观察到的过量细胞凋亡与我们早期研究中发现的致病细胞变性一致。未观察到细胞坏死。本研究结果表明，背主动脉内皮和血管内血细胞在注射溶血大鼠抗大鼠（RAR）抗体后仅表现出短暂反应。这种暂时反应可能是由于血管活性介质的释放。较小的血管和毛细血管仍处于发育的早期阶段，表现为有窗孔，使其在免疫相互作用后更容易受到损伤。结果表明，心脏内注射后免疫反应的致病作用是通过“开启”细胞凋亡在微循环水平发生的。程序性细胞死亡在胚胎发生和发育中至关重要。因此，过量的细胞凋亡，即不适当的细胞凋亡，最终将导致先天性畸形。
