Introduction
Mild hypothermia is protective, and is associated with less apoptosis. Prior work has established that this protective effect is associated with better mitochondrial preservation and thus, suppress of the intrinsic apoptotic pathway as evidenced by reduced cytosolic cytochrome c redistribution 3 , decreased caspase-3 processing 5 , and, in some reports, increased Bcl-22, 4 and decreased Bax 1 expression. Only recently has it become recognized that brain tissue may undergo apoptotic death via the extrinsic, or receptor mediated pathways. One of the most studied of these pathways is that triggered by Fas/FasL, leading to caspase-8 activation.
Methods
Male Sprague Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) with 2 h intraischemic mild hypothermia (brain temperature=33C). Brain tissue was collected 2 (n=9), 6 (n=8) and 24 h (n=18) for histology, immunostaining and Western blot analysis in a manner previously published by our group (3), and assessed for Fas, FasL and caspase-8 expression. Double immunofluorescent labeling was also performed to identify Fas and caspase-8 levels in neurons (NeuN), astrocytes (GFAP) and phagocytes (ED1). To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, 11 rats were treated with 400 mg of anti-FasL antibody (Pharmingen) (or vehicle) i.p. immediately upon reperfusion and 3 days later, then assessed at 7 d.
Results
Mild hypothermia significantly reduced infarct size and reduced numbers of TUNEL positive cells (P<0. 05). Double immunofluorescent labeling at 24 h indicated that Fas was expressed largely in neurons and phagocytes as well as some astrocytes. Interestingly, caspase-8 was observed in phagocytes, rare astrocytes but no neurons. There was no significant change in the staining pattern among hypothermic animals. Mild hypothermia significantly reduced expression of Fas (P<0.05) and processed (but not pro-) caspase-8 (P<0.05). Interestingly, FasL levels were increased by mild hypothermia. Among rats treated FasL antibody, infarct size was significantly reduced by 87% compared to vehicle treatment (P<0.01). Similarly, numbers of TUNEL positive cells were also reduced in FasL antibody treated brains (P<0.05).
Conclusions
The Fas/FasL apoptotic pathway is involved in cell death following experimental stroke, as FasL antibody treatment is neuroprotective. Mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Even though mild hypothermia increases FasL expression, the reduction in Fas receptor may explain the reduction in apoptosis by this pathway. Alternatively, some reports have proposed that proteases such as the matrix metalloproteinases (MMPs) trigger the Fas pathway by cleaving FasL, liberating it to stimulate its receptors. Since mild hypothermia also reduces MMPs6, 7, it is possible that mild hypothermia may prevent FasL cleavage and thus inhibit Fas-mediated apoptosis by inhibiting upstream MMPs.