Apoptosis induced in cultured rat embryos by intraamniotically microinjected sodium nitroprusside

Previously, we reported that massive cell death was induced in the mesencephalic area of cultured rat embryos after embryos of gestational day 10.5 were intra-amniotically microinjected with sodium nitroprusside (SNP, 800 μM) and cultured for 24 hr at 37°C. The massive cell death apparently was the result of NO-mediated embryotoxicity. Damage was concentration dependent and tissue specific. In follow-up studies, we now report evidence that NO generated from SNP induces apoptosis in organogenesis stage cultured rat embryos. Nile blue sulfate (NBS) staining suggested that microinjections of 400 μM SNP induced apoptosis in the mesencephalic area. Since we observed no massive cell death (“white caps”) at this concentration, it appeared that early stages of apoptosis preceded “white cap” formation. At 800 μM SNP, total disintegration of cell bodies was evident and may have resulted from later stages of aoptosis or necrosis, or both. The “white caps” per se, an accumulation of disintegrated cell bodies, did not stain with NBS, probably due to total loss of cell integrity and resultant coagulation. The majority of the coagulated dead cells in the “white caps” were heavily stained with 3,3′-diaminobenzidine via in situ 3′ end-labeling with terminal transferase. However, it is now known that NO can damage DNA directly and that in situ 3′ end-labeling by terminal transferase detects not only apoptosis but also random DNA breakage. Increased 3′ end-labeling and a “DNA ladder” were detectable within 5–10 hr after exposure of day 10.5 embryos to 400 or 800 μM of microinjected SNP. Some smear background was also observed in the “ladder.” Rostral aspects of embryos exhibited more prominent indices of apoptosis than caudal regions. The results suggested that microinjections of SNP into the amniotic fluid of day 10.5 cultured rat embryos induces NO-mediated cell death in the mesencephalic and rhombencephalic regions by the process of apoptosis or of both apoptosis and necrosis, depending on the timing, concentration, and stage of gestation.restrictio