Introduction: In recent studies conducted by our lab, acute administration of chlorpromazine and promethazine (C+P) in ischemic stroke has been shown, through the induction of a metabolically-slowed, hibernation-like state, to reduce infarct size and neurological deficit, and thus to exert a neuroprotective effect on infracted subjects. Although its mechanism has not been fully elucidated, this effect is associated with alterations in the expression of protein kinase C-δ (PKC-δ) and Akt. Because PKC-δ and Akt are also known to be involved in the regulation of apoptotic cell death after ischemic stroke, we hypothesized that treatment with C+P would result in reductions in post-ischemic apoptosis. Additionally, because the neuroprotective effect of C+P is associated with reductions in NADPH oxidase (NOX) expression and activity, and because of the involvement of NOX in the PKC-Akt apoptotic pathway, we hypothesized that adding a NOX inhibitor would potentiate the anti-apoptotic effect of C+P.
Methods: 8mg/kg C+P with or without a NOX inhibitor was administered intraperitoneally to Sprague-Dawley rats subjected to 2h MCAO, followed by 6 or 24h reperfusion. The amount of apoptotic cell death in control, non-treatment ischemic, and all treatment ischemic groups was quantified using ELISA for cytoplasmic histone-associated DNA fragments. Levels of pro-apoptotic (caspase-3, BAX, and AIF) and anti-apoptotic (Bcl-2 and Bcl-xL) proteins were determined by Western blot.
Results: Rats treated with C+P followed by 6h and 24h reperfusion demonstrated significant reductions in apoptotic cell death, increased expression of anti-apoptotic proteins, and decreased expression of pro-apoptotic proteins, compared with counterparts in the non-treatment ischemic group. Rats treated with C+P together with a NOX inhibitor exhibited further reductions in apoptotic cell death.
Conclusion: These results demonstrate that C+P administration causes significant reductions in apoptotic cell death following ischemic stroke, when measured both at 6 and 24h reperfusion, and that addition of a NOX inhibitor potentiates this reduction. Therefore, the neuroprotective function observed upon C+P administration appears to involve the prevention of post-stroke apoptosis.