Tissue corresponding to the ischemic penumbra and core. The gray region (I), including the black region (II) represents ischemic injury in controls with ischemia alone; the black region (II) represents infarction in ischemia plus postconditioning. The region spared by postconditioning is defined as the penumbra (region I) and the black area is defined as the ischemic core (region II). These regions were dissected for western blotting. The corresponding non-ischemic cortex from sham animal without ischemia was dissected for comparison.

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 received sham surgery, postconditioning, or control ischemia are presented. Arrows indicate injury sites. A bar graph (third panel) presents average lesion sizes measured 1 month after ischemia. Postconditioning reduced infarction from 36 ± 4% (n = 6) to 22 ± 3% (n = 7) in control ischemic animals (*p = 0.006, postconditioning vs. control). Post, ischemia plus postconditioning; Con, control ischemia without postconditioning. (b). Postconditioning attenuates the asymmetrical behavioral abnormalities measured up to 1 month after stroke. In the vibrissae test, the number of contralateral forelimb placements was compared with that of the ipsilateral forelimb, n = 5–7/group. Postconditioning attenuates the overall deficit from 2 to 30 days after stroke (two-way ANOVA, p < 0.001). *p < 0.05 and **p < 0.01 versus sham (one-way repeated measures ANOVA); #p < 0.05 versus postconditioning at 21 days (two-way ANOVA, Student—Newman—Keuls).

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 in both the ischemic penumbra and the core are shown. Relative optical densities of protein bands in ischemic rats were normalized to those in sham rats and calibrated with β-actin. Postconditioning up-regulated the overall protein level of P-Akt across all time points compared with control ischemia (two-way ANOVA: p = 0.005 in the penumbra, p = 0.027 in the ischemic core). *p < 0.05 and **p < 0.01 versus sham, respectively; #p < 0.01 and ##p < 0.05 versus control at a corresponding time point, respectively. Total Akt decreased after ischemia in both the ischemic core and penumbra; postconditioning had no statistically significant effect. *p < 0.01 and *p < 0.05 versus sham, n = 5–7/group. c, control; p, postconditioning. (b) Postconditioning increased in vitro Akt activity at 5 h. Akt activity as assessed by protein levels of phosphophorylated Akt substrate, a fusion protein of glycogen synthase kinase (GSK3β) is expressed as percentages of phosphorylated GSK3β (P-GSK3) from sham animals. *p < 0.05 versus sham; #p < 0.05 versus 5-h control, n = 5/group. (c) Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 inhibited Akt activity at 5 h in postconditioned animals. Antibody directed against P-GSK3β was used for detection. *p < 0.05 versus sham; #p < 0.05 versus PI3K inhibitor. (d) The PI3K inhibitor LY294002, partially blocked the protective effect of postconditioning. Infarct size was detected by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining 2 days after stroke. The left columns show representative TTC staining from rat brains that received postconditioning or control ischemia treated with vehicle [dimethylsulfoxide (DMSO)] or with the PI3K inhibitor LY294002. Bar graph (right panel) shows quantitation of the data. *p < 0.001 versus other groups and #p < 0.05 versus DMSO/con and inhib/con. Post, postconditioning; con, control ischemia; inhib, PI3K inhibitor.

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) after stroke. Representative protein bands of phosphorylated and total PDK1, PTEN, and GSK 3β are shown along with their corresponding mean optical densities (bar graphs). For brevity, β-actin bands were omitted. Levels of phosphorylated proteins are shown in a, b, and c for PDK1, PTEN, and GSK 3β, respectively. Protein levels of phosphorylated-PDK1 (P-PDK1), phosphorylated-PTEN (P-PTEN), and phosphorylated glycogen synthase kinase 3β (P-GSK3β) decreased after ischemia. (a) The P-PDK1 levels were reduced at 24 h in postconditioned control ischemic brains in the penumbra; they were reduced at 24 h postischemia with or without postconditioning in the ischemic core.*p < 0.05, **p < 0.01 versus sham. (b) Although postconditioning slightly attenuated the reduction in P-PTEN levels at 1, 5, and 24 h in the penumbra, no significant difference was detected between postconditioning and control ischemia. *p < 0.05, **p < 0.01, and ***p < 0.001 versus sham. (c) P-GSK3β levels were transiently increased at 1 h in only the penumbra, and then reduced at 5 and 24 h in both the penumbra and core in rats with control ischemia; they were reduced in rats with postconditioning in both the penumbra and core postischemia. *p < 0.05, **p < 0.01,***p < 0.001 versus sham; #p < 0.05 versus 1 h/con in the penumbra; ##p < 0.01 versus 1 h/con in the core, n = 4–5/group.

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 with and without postconditoning. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; and #p < 0.05, versus 1 h/con. (b) Decreased levels of β-catenin postischemia. An antibody that recognizes total β-catenin was used for detection.†p < 0.05 versus other time points, n = 3–5/group. (c) Decreased levels of active β-catenin postischemia. An antibody recognizing the active, non-phosphorylated form of β-catenin was used for detection. †p < 0.05 versus other time points, n = 3–5/group.

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 N terminal kinase from 1 to 24 h in the penumbra (two-way ANOVA, p = 0.023) but not in the core. *p < 0.05 versus sham; ##p < 0.01 versus control/1 h; ††p < 0.01 versus other time points. (b) Postconditioning reduced overall levels of phosphorylated-ERK (P-ERK) from 1 to 24 h in the penumbra but not in the core (p = 0.015, two-way ANOVA). *p < 0.05 versus sham; #p < 0.05 versus control at a corresponding time point, n = 5–9/group.

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 reduced δPKC cleavage in the ischemic penumbra. The levels of cleaved δPKC were increased in the ischemic penumbra after stroke (see bar graph). The increase at 1 h was significantly inhibited by postconditioning. *p < 0.05, ***p < 0.001 versus sham; ###p < 0.001 versus 1 h/control, n = 5–10/group. (b) Phosphorylated-δPKC level decreased postischemia. No significant difference was found between control ischemia and postconditioning. *p < 0.05 versus sham. †††p < 0.001 versus the rest of the groups in the core, n = 3–5/group. (c) Postconditioning increased P-εPKC levels at 1 h compared with control ischemia. *p < 0.05, **p < 0.01 versus sham; #p < 0.05 versus 1 h/con; ††p < 0.01 versus the rest of the groups in the core, n = 3– 10/group.