The Journal of Neuroscience, 2001, 21:RC138:1-5
RAPID COMMUNICATION
The In Vitro Fate of Rabbit Fetal Brain Cells after
Acute In Vivo Hypoxia
Matthew
Derrick,
Jie
He,
Elizabeth
Brady, and
Sidhartha
Tan
Department of Pediatrics, Evanston Hospital, Northwestern
University, Evanston, Illinois 60201
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.
Key words:
apoptosis; cell culture; cell death; fluorescence; flow
cytometry; neurons; propidium iodide; mitochondria
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