Postconditioning provided long-term protection
Postconditioning reduced lesion size approximately 40% in rats subjected to ischemia when measured 30 days after ischemia, suggesting long-term protection (). Neurological functions were assayed by the vibrissae test. The percentage of contralateral forelimb placements dropped from 100% before ischemia to approximately 25–60% after ischemia, indicating an asymmetrical deficit in forelimb use. Postconditioning attenuated the overall deficit from 2 to 30 days after ischemia ().
Fig. 2 Long-term protective effect of postconditioning. (a). Post-conditioning reduced ischemic damage up to 1 month after focal ischemia. Representative whole brains (top) and the cresyl violet staining of four coronal sections (second panel) from rats that (more ...)
Increased Akt activity contributed to the protective effect postconditioning
Western blot analysis was used to evaluate the effect of postconditioning on Akt phosphorylation (P-Akt). Levels of P-Akt increased at 1 and 5 h and returned to normal levels at 24 h postischemia in the penumbra of control rats; they did not change significantly in the ischemic core (). Postconditioning significantly increased P-Akt levels compared with control ischemia both in the ischemic penumbra and in the core at 5 and 24 h after stroke (). However, levels of total Akt decreased after stroke in the ischemic penumbra and the core, and this was not prevented by postconditioning (densitometry quantitation not shown). Thus, postconditioning increased P-Akt without affecting total (decreased) Akt in the ischemic penumbra and core.
Fig. 3 Akt activity contributed to the protective effect of postconditioning. (a) Changes in phosphorylated-Akt (P-Akt) and total Akt after stroke in rats with and without postconditioning. Representative protein bands for P-Akt (Ser473), total Akt and β-actin (more ...)
An in vitro Akt kinase assay was used to assay Akt activity. Despite the robust increase in P-Akt, 5 or 24 h after control ischemia (), Akt activity decreased to approximately 70% of sham (). Postconditioning significantly increased Akt kinase activity to approximately 110% at 5 h post-stroke (p < 0.05 vs. control ischemia), but had no effect at 24 h (). Injection of the PI3K inhibitor, LY294002, reduced Akt activity in rat brains that received postconditioning (). Injection of LY294002 enlarged infarct by 42% in rats with postconditioning (). However, it did not completely reverse the protective effect of postconditioning, as the infarct in rats that received postconditioning was still smaller than that in rats receiving vehicle. Notably, LY294002 did not change the infarction volume in rats treated with control ischemia. These data indicate that postconditioning increases Akt activity, and that inhibition of upstream Akt-activating PI3K activity does not completely abolish the protective effects of postconditioning on infarct size.
The levels of phosphorylated PDK1, which activates Akt and of phosphorylated PTEN (P-PTEN), which cannot inhibit Akt as well as their total protein levels were examined next (). Levels of phosphorylated PDK1and P-PTEN decreased in both the ischemic penumbra and the core after ischemia (). Postconditoning did not significantly affect their levels compared with control ischemia, though postconditioning slightly enhanced levels of P-PTEN at 1, 5, and 24 h in the penumbra. Total protein levels of PDK1 and PTEN decreased after ischemia; postconditioning did not affect their levels (densitometry quantitation not shown). Thus, postconditioning had no effect on the decrease of phosphorylated or total PDK1 or total PTEN, though it slightly increased P-PTEN (inactive PTEN).
Fig. 4 The effect of postconditioning on phosphorylated and total protein levels of phosphoinositide-dependent protein kinase-1 (PDK1), phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and glycogen synthase kinase 3β (GSK 3β) (more ...)
The levels of Akt substrate GSK3β and its inactive phosphorylated form, P-GSK3β were examined in the context of postconditioning. The P-GSK3β level was transiently increased at 1 h and then decreased at 5 and 24 h in the penumbra after stroke (); postconditioning blocked the increase at 1 h, but had no effect on its levels at 5 and 24 h. In the ischemic core, P-GSK3β levels were decreased at 5 h in brains subjected to control ischemia; with postconditioning, it was decreased as early as 1 h. Total protein levels of GSK3β did not change significantly in ischemic brains with or without postconditioning (densitometry quantitation not shown). Postconditioning therefore had no effect on total GSK3β levels; its effect of lowering P-GSK3β levels was limited to the 1 h time point.
The last Akt signaling cascade molecule we examined was β-catenin. Levels of phosphorylated β-catenin were increased as early as 1 h, peaked at 5 h and then returned to normal levels in the penumbra of control ischemic brains; postconditioning blocked the increase at 1 h, but had no effect on phosphorylated β-catenin levels at other time points (). In the ischemic core, the level of phosphorylated β-catenin was slightly increased at 5 h, but did not reach a significant difference compared with sham; postconditioning reduced phosphorylated β-catenin at 1 h, whereas it increased phosphorylated β-catenin at 5 h. Protein levels of total β-catenin were decreased from 1 through 24 h postischemia; postconditioning had no effect on them (). We also measured the level of active β-catenin, i.e., non-phosphorylated () and obtained similar results with total β-catenin. Postconditioning thus decreased phosphorylated β-catenin levels at 1 h in the ischemic penumbra and core, while increasing phosphorylated β-catenin at 5 h in the core, without affecting total phosphorylated β-catenin levels.
Fig. 5 Effects of postconditioning on levels of phosphorylated, total, and active β-catenin after stroke. Western blot protein bands and corresponding mean optical densities (bar graphs) are shown. (a) Phosphorylated-β-catenin levels postischemia (more ...)
Inhibition of MAPK signaling molecules JNK and ERK1/2 correlates with postconditioning’s protection
We determined the effect of postconditioning on levels of phosphorylated-JNK (P-JNK) and total JNK protein in both the ischemic penumbra and the core (). P-JNK transiently increased at 1 h in the ischemic penumbra, but decreased to approximately 50% of sham from 1 through 24 h in the ischemic core. Postconditioning blocked the increase of P-JNK at 1 h in the penumbra, but did not affect P-JNK levels in the ischemic core. Total protein levels of JNK did not decrease until 24 h after ischemia and they were unaffected by postconditioning (densitometry quantitation not shown). Thus, postconditioning’s effect on P-JNK levels were limited to blocking the increase of P-JNK at 1 h in the ischemic penumbra.
Fig. 6 The effects of postconditoning on the mitogen-activated protein kinase pathway. Representative western blot protein bands (top panels) and corresponding mean optical densities (bar graphs) are shown. (a) Postconditioning reduced overall increases in phosphorylated-c-jun (more ...)
We then measured protein levels of phosphorylated-ERK1/2 (P-ERK1/2) and ERK1/2 (). P-ERK1/2 transiently increased after reperfusion in both the ischemic penumbra and core of control ischemic rats. Postconditioning reduced overall levels of P-ERK1/2 from 1 to 24 h in the penumbra, and reduced P-ERK1/2 levels at 5 h but not at 1 and 24 h in the ischemic core. Total protein levels of ERK1/2 did not change significantly after ischemia with or without postconditioning (densitometry quantitation not shown).
The effect of postconditioning on the PKC pathway
We assayed for changes in total and cleaved δPKC, phosphorylated δPKC (Thr 505; P-δPKC), and phosphorylated εPKC (P-εPKC). Total δPKC decreased in the ischemic core of both control and postconditioning groups at 5 and 24 h (p < 0.05) and in the penumbra of control at 5 h (p < 0.05); postconditioning had no significant effect on protein levels of total δPKC (bar graphs for densitometry quantitation of total δPKC are not shown). However, levels of the cleaved form of δPKC, indicative of δPKC activity were transiently increased 1 h after stroke in the penumbra; postconditoning attenuated this increase (). P-δPKC levels were decreased by 24 h to less than 70% of sham in the penumbra and to less than 40% in the core in ischemic brains (); postconditioning did not significantly change P-δPKC levels compared with control. P-εPKC decreased to approximately 60–20% of sham at 1 and 24 h after stroke, respectively; postconditioning attenuated such decreases in the penumbra but not in the core (). Taken together, these data suggest postconditioning blocked the increase of cleaved δPKC at 1 h and attenuated the decrease in P-εPKC at 1 h; these effects were limited to the ischemic penumbra.
Fig. 7 The effect of postconditioning on the protein kinase C (PKC) pathway. Representative western blot protein bands (top panels) and corresponding mean optical densities, normalized to same protein in sham rats (bar graphs) are shown. (a) Postconditioning (more ...)