The in vitro fate of rabbit fetal brain cells after acute in vivo hypoxia.
In the investigation of ischemia-induced brain damage, traditional methods using histopathology estimate brain cell death at a time remote from ischemic insult. These observations fail to take into account endogenous repair processes or ongoing injury cascades like apoptosis. The cells that are injured but not killed initially are the population most amenable to rescue. The hypothesis was that in vivo uterine ischemia-reperfusion would result in more cell death and apoptosis in fetal brain cells cultured in vitro. Near-term, 29 d gestation, pregnant New Zealand White rabbits were subjected to repetitive uterine ischemia for a cumulative time of 40 min ischemia and 20 min reperfusion. Immediately after uterine ischemia, the fetal brains were removed and dissociated into a cell suspension. The ischemic group had more cell death than non-ischemic controls as assessed by Trypan Blue exclusion and propidium iodide (PI) uptake on a flow cytometer. Aliquots of cells were plated and cultured for 24 and 48 hr. The ischemic group had significantly more cell death (propidium iodide) than non-ischemic controls at 24 hr and significantly more apoptosis, as assessed by annexin-V binding in cells at 24 hr and caspase-3 activity at 48 hr. Fewer cells attached to the culture plates at 48 hr in the ischemia group. After uterine ischemia, certain fetal brain cells die immediately, and other cells undergo ongoing damage resulting in necrosis and apoptosis that is manifest later. This method offers insight into the fate of those cells and provides a tool for assessing interventions to decrease cell injury.
Derrick
M
M
Department of Pediatrics, Evanston Hospital, Northwestern University, Evanston, Illinois 60201, USA. m-derrick@northwestern.edu
He
J
J
Brady
E
E
Tan
S
S
D000208:Acute Disease; D000818:Animals; D001921:Brain / Q000473:pathology*; D001926:Brain Death / Q000473:pathology*; D002478:Cells, Cultured; D005260:Female; D005333:Fetus / Q000473:pathology; D000860:Hypoxia / Q000473:pathology*; D066298:In Vitro Techniques; D007511:Ischemia / Q000473:pathology*; D011817:Rabbits; D014599:Uterus / Q000098:blood supply*