Ultrastructural alterations caused by immunological reactions after intracardiac injection of allogeneic antibodies against blood group antigens: an experimental study using the in vitro whole-rat embryo culture.

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.