Study of the activation of the RISK and SAFE signaling pathways by cyclosporin A pre- and post-conditioning in a mouse model of cerebral infarction

Abstract

Cyclosporin A (CsA) has been shown to be useful in preventing ischemia-reperfusion injury in basic models of myocardial or cerebral infarction. CsA limits the opening of the mitochondrial transition pore (mPTP) through its direct interaction with cyclophilin D. However, recent studies in cardioprotection and neuroprotection suggest a role of CsA on the classic RISK and SAFE pathways, upstream of mitochondria. We aim to study the effect of CsA in ischemic pre- and post-conditioning on the RISK and SAFE pathways in a mouse model of stroke. Mice underwent 60 min ischemia and 30 min or 24 h reperfusion and received either no treatment (IR), a bolus of CsA (10 mg/kg) 10 min before the ischemia (pre-CsA) or 10 min after the reperfusion (post-CsA). Infarction of the middle cerebral artery was performed with a microfilament placed intra-arterially for the desired duration. Surgery was performed on Sham mice, but the filament was not occlusive. mPTP opening was evaluated by calcium retention capacity (CRC) on isolated mitochondria from ischemic right hemisphere and non-ischemic left hemisphere. Activation of the RISK and SAFE pathways was evaluated by determining the phosphorylation state of key proteins by Western blots: STAT3, GSK3-β, Akt and Erk. As expected, CRC was significantly reduced in the mitochondria from the right hemisphere in the IR group (Sham, 103 ± 21 vs. IR, 57 ± 18 nmol Ca2+/mg, n = 12/gp, P < 0.05). There was a slight increase in PTP resistance in the ischemic mitochondria from the pre-CsA and post-CsA groups. Experiments are ongoing to measure infarct size after 24 h of reperfusion. There was no significant change in protein phosphorylation in the left hemisphere. In the right ischemic hemisphere, there was a trend of activation of GSK3-β in post-CsA mice and STAT3 in pre-CsA mice. Our results suggest an effect of CsA upstream of the mPTP during stroke, which could correspond to a positive feedback loop.