Purpose and background
Mild hypothermia may reduce apoptosis after stroke, and the PI3/AKT kinase pathway may be involved in ischemic apoptosis, but the effects of hypothermia on the AKT pathway in ischemia is not known. AKT kinase may block apoptosis by phosphorylating the substrates FKHR and GSK3β, and AKT is activated by growth factors via a pathway that requires the kinases PI3 Kinase and PDK1. PTEN, a lipid phosphotase, downregulates PI3 K activity. We studied the effect of moderate hypothermia on phosphorylation (P) of PTEN (Ser380), PDK1 (Ser241), AKT(Ser473), FKHR(Ser256), and GSK3β(Ser9) in a model of focal ischemia. AKT activity in vitro was analyzed, and we tested whether inhibition of the AKT pathway reverses the protective effect of hypothermia.
Methods
Focal ischemia was induced by occluding the left MCA permanently and both CCAs for 1 h in rats maintained at 37°C or 30°C for 1 h. For another two groups, 10 μl of a specific PI3 kinase inhibitor, LY 294002, dissolved in DMSO and ethanol to 10 mM, or its vehicle, was injected intraventricularly 1 h before and 24 h after hypothermic ischemia onset. Infarct size was measured 2 days later. Other groups were sacrificed at 30 min after MCA occlusion and 30 min, 4, 8, 24 and 48 h after reperfusion. Whole cell homogenates from the penumbra of the ischemic cortex were prepared for Western blots. An AKT kinase assay was performed for tissues harvested at 4 and 24 h. Behavior was assessed up to 2 months.
Results
Hypothermia reduced infarct size by 84% (P<0. 001) and improved neurological function (P<0.05). At 37°C P-AKT decreased during ischemia, increased at 30 min and 4 h (n=3-5/group; P<0.05) after reperfusion, and decreased after 8 h. AKT kinase assay indicated that AKT activity decreased at 4 h, so phosphorylation levels of P-AKT may not represent AKT activity. P-FKHR, P-GSK3β and P-PDK1 decreased after ischemia at all time points (P<0.001). P-PTEN decreased from 30 min after occlusion to 8 h post-reperfusion, then recovered from 24 to 48 h. Hypothermia did not increase P-AKT at 30 min or 4 h after reperfusion compared to sham, but attenuated its decrease during ischemia and 8 h after reperfusion. Nevertheless, AKT kinase assay showed that AKT activity at 4 and 24 h was maintained by hypothermia. Although P-PDK1 decreased at 4 and 8 h after reperfusion in hypothermic animals, decreases in overall levels of P-PDK1 were smaller compared to normothermia (P<0.05). P-FKHR transiently increased at 30 min after reperfusion and then decreased in hypothermic rats. The most striking effect of hypothermia was to block the decrease in P-PTEN at all time points (P<0.05). Surprisingly, hypothermia did not attenuate dephosphorylation of P-GSK3β, suggesting that dephosphorylation of GSK3β may not contribute to brain damage. In hypothermic animals the PI3 kinase inhibitor worsened infarct size by 53% (n=5) compared to vehicle (n=6) (p<0.05).
Conclusion
The AKT pathway plays a critical role in preventing ischemic damage. Hypothermia protects in part by preserving AKT activity and attenuating the apoptotic effects of PTEN, PDK1 and FKHR.