Mild hypothermia mitigates post-ischemic neuronal death following focal cerebral ischemia in rat brain: Immunohistochemical study of Fas, caspase-3 and TUNEL

Mild hypothermia is considered to have a protective effect during ischemic neuronal cell death. The present study provides experimental evidence for this beneficial role of mild hypothermia using reversible middle cerebral artery occlusion (MCAo) in a Sprague-Dawley (SD) rat model. MCAo was induced in rats for 1 h followed by reperfusion at different periods. Hematoxylin-eosin (HE) staining in normothermic (NT) 37 degreesC and hypothermic (HT) 33 degreesC groups of rats confirmed cerebral infarcts. The mean per cent infarct area was significantly reduced in the HT group of rats. Immunohistochemical analysis was done using anti-Fas and caspase-3 antibodies. The immunohistochemical expression of Fas and caspase-3 was demonstrable as early as 5 h after reperfusion, but the expression pattern maximized at 24 h after reperfusion. The expression of Fas and caspase-3 proteins showed a clear decrease in the HT group over the NT group. In situ detection of DNA fragmentation was done using the terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end- labeling method (TUNEL). TUNEL-positive cells were first observed at 5 h after reperfusion and progressively increased by 24 h. A higher number of TUNEL-positive cells was found in the NT group, but they were significantly decreased in the HT group. Further, DNA fragmentation was confirmed by size fractionation in agarose gel. These findings demonstrate a positive relation between the expression of Fas, caspase-3 and TUNEL-positive cells. Mild expression of Fas and caspase-3 proteins and a reduced number of TUNEL-positive cells in the HT group is clear evidence for the protective role of hypothermia in ischemia-induced cell death.