Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats

Abstract

Activation of the Akt/protein kinase B (PKB) kinase pathway can be neuroprotective after stroke. Akt is activated by growth factors via a phosphorylation-dependent pathway involving the kinases phosphoinositide 3 (PI3) kinase and phosphoinositide-dependent protein kinase-1 (PDK1) and is negatively regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Akt kinase blocks apoptosis by phosphorylating the substrates forkhead transcription factor (FKHR) and glycogen synthase kinase 3beta (GSK3beta). We found that intra-ischemic hypothermia (30 degrees C) reduced infarct size and improved functional outcomes up to 2 months. Changes in phosphorylation levels of Akt, as measured by Western blots and immunostaining, differed from levels of Akt activity measured in an in vitro assay in normothermic animals. Hypothermia blocked most of these changes and maintained Akt activity. Inhibition of PI3/Akt enlarged infarct size in hypothermic animals. Hypothermia improved phosphorylation of PDK1, PTEN, and FKHR. Hypothermia did not improve GSK3beta (Ser9) phosphorylation but blocked the nuclear translocation of phosphorylated beta-catenin (Ser33/37/Thr41) downstream of GSK3beta. Phosphorylation levels of PTEN, Akt, and Akt substrate decreased before apoptotic cytochrome c release and degradation of microtubule-associated protein-2, a marker of neuronal survival. Hypothermia may protect from ischemic damage in part by preserving Akt activity and attenuating the apoptotic effects of PTEN, PDK1, and FKHR.

Figure 1.

A diagram of Akt/PKB survival signal pathways. Fas L, Fas ligand; Cyto C, cytochrome c; Cas-3, caspase-3; Cas-8, caspase-8.

Figure 2.

Tissue corresponding to the ischemic penumbra was dissected for Western blot. Ischemic lesion in a normothermic rat is represented as black plus gray, and black alone represents ischemic infarct in a hypothermic rat. The region spared by hypothermia is defined as the penumbra (gray), and the black area is the ischemic core. The region of the penumbra (within the solid lines, I) was dissected for Western blotting. The corresponding contralateral non-ischemic cortex (II) or similar cortex from sham animal without ischemia was dissected as a control.

Figure 3.

Hypothermia reduces infarct size after permanent dMCAo. A, Representative infarcts stained with cresyl violet from level 3 in rats killed 2 d after stroke. The pale area with asterisks represents the infarct region. Normothermic ischemia damaged the cortex ipsilateral to the occluded MCA, whereas hypothermia spared all or most of the injured cortex. Only a small lesion was observed in the presented section from a hypothermic rat. B, Statistical analysis of infarct size 2 d after stroke. Two-way ANOVA (two factors, temperature and brain section level) was used to compare the effect of temperature on the infarct size at each level (data not shown) and on the mean of all four levels. Hypothermia (n = 7) reduced the mean infarct size by >80% compared with normothermia (n = 7; p < 0.001). C, Representative sections stained with cresyl violet from animals surviving 2 months after stroke. Most of the cortex in the infarcted hemisphere was lost in normothermic but not hypothermic rats. D, Infarct size 60 d after stroke. Hypothermia (n = 9) reduced infarct size 60 d after stroke compared with normothermia (n = 8; p < 0.001).

Figure 4.

Hypothermia attenuates behavioral deficits up to 2 months after ischemia. A, Vibrissa test. All sham animals (n = 6) showed normal forelimb placing. There was unsuccessful placing of the contralateral forelimb at 2, 3, and 7 d after stroke in normothermic animals (n = 8) but gradual recovery to normal levels at 1 month. Hypothermia (n = 9) attenuated the deficit from 2 to 7 d after stroke. ^*p < 0.05 and ^**p < 0.001 versus sham; ^#p < 0.05 and ^##p < 0.001 versus 30°C; ⁺p < 0.05 versus sham.B, Postural reflex test. Scores significantly increased at all time points in normothermic animals. Hypothermia decreased the scores at 2, 3, 7, 14, and 60 d after stroke. ^*p < 0.01 and ^**p < 0.001 versus sham; ^#p < 0.01 and ^##p < 0.001 versus 30°C; ⁺p < 0.05 versus sham. C, Tail hang test. The number of large right turns increased in normothermic animals, an effect blocked by hypothermia. ^#p < 0.01 versus sham and 30°C; ^*p = 0.005 and ^**p < 0.001 versus 37°C. D, Home cage test. The percentage of the ipsilateral forelimb placing increased 2, 3, 7, 14, 30, and 60 d after ischemia in normothermic animals. Hypothermia decreased this biased usage of the forelimb at all time points. ^#p < 0.05 and ^##p < 0.001 versus sham; ^*p < 0.05 and ^**p < 0.001 versus 30°C.

Figure 5.

The effect of hypothermia on Akt activity after stroke. A, Representative protein bands from Western blots for P-Akt (Ser473), P-Akt (Thr308), β-actin, and total Akt. Biphasic changes in P-Akt (Ser473) were observed in normothermic rats, whereas hypothermia blocked all such changes except at 24 h. No changes in P-Akt (Thr308) were detected in normothermic animals until 48 h or hypothermic animals at all time points. Total Akt did not decrease after ischemia until 48 h, an effect blocked by hypothermia.β-Actin was used to show equal protein loading of each lane. The same samples were used for the subsequent Western blot for detecting GSK3β, PDK1, and PTEN. For the sake of brevity, bands of β-actin confirming equal loading of each lane are not shown. B, Relative optical densities for P-Akt (Ser473). Optical densities indicate that P-Akt (Ser473) decreased 30 min into dMCAo, increased from 1.5 to 5 h after ischemia onset, decreased at 9 and 24 h, and recovered at 48 h. Hypothermia maintained P-Akt (Ser473) levels at most time points, with a transient decrease observed at 24 h. n = 3-5 per group. ^*p < 0.05 versus control (con); ^**p < 0.01 versus control and other time points at 30°C; ^#p < 0.05 and ^##p < 0.01 versus 30°C at 1.5 and 5 h, respectively. Black bars, 37°C; gray bars, 30°C. n = 3-5 per group. C, An experiment was performed to verify that a pure Akt kinase extraction was obtained for the Akt kinase assay. Western blots indicate that β-actin was not seen in solution II containing Akt extraction, suggesting that the Akt kinase assay was not contaminated by the supernatant (solution I) and potential unwanted precipitation from the whole-cell extraction itself (a control solution from sham animal was used). In addition, no GSK3 fusion protein band was observed in the whole-cell extraction, suggesting that it did not contain the endogenous GSK3 protein to confound the Akt kinase assay. D, Representative protein bands from an in vitro Akt kinase assay. The solution used for Akt immunoprecipitation was subjected to β-actin measurement to confirm that equal amounts of protein were used for this assay. E, Akt kinase activity was expressed as percentages of P-GSK3 band compared with that from sham animals. Akt kinase activity decreased at 5 and 24 h after ischemia onset at 37°C; this effect was inhibited by hypothermia. ^*p < 0.001 versus control and 30°C, 24 h; ^**p < 0.001 versus control; ^#p < 0.05 versus 37°C, 24 h; ^##p < 0.001 versus 37°C, 5 h; ⁺p < 0.001 versus control. Black bars, 37°C; gray bars, 30°C.

Figure 6.

Triple staining of P-Akt (Ser473), MAP-2, and 4′, 6′-diamidino-2-phenylindole (DAPI). Overview pictures were taken at the border between the ischemic core and the penumbra defined by MAP-2 staining (dashed line in MAP staining). Both P-Akt (Ser473) and MAP-2 expressed in non-ischemic cortex (Sham). P-Akt (Ser473) colocalized with MAP-2 as shown in the high-magnification inset. MAP-2 expression decreased in the ischemic core at 5 h in normothermic animals, was slightly decreased in the penumbra, and disappeared at 24 h. P-Akt (Ser473) expression decreased in the core at 5 h but was maintained in the penumbra and decreased 24 h after normothermic stroke. Both MAP-2 and P-Akt (Ser473) expression decreased in the core even in the hypothermic animals, but MAP-2 still expressed in the penumbra at 24 h whereas P-Akt (Ser473) decreased. Scale bars: low magnification, 100 μm; inset, 10 μm.

Figure 7.

Triple staining of P-Akt substrate (P-Akt SUB), MAP-2, and DAPI. P-Akt substrate strongly expressed in the non-ischemic contralateral cortex of ischemic animals and cortex of sham animals and colocalized with MAP-2 (arrows). In normothermic animals, immunoreactivity of P-Akt substrate decreased at 5 h (arrowheads) and 24 h (data not shown) after stroke. Hypothermia improved its expression both at 5 and 24 h (see also Fig. 12). Scale bar, 20 μm.

Figure 8.

Hypothermia attenuated decreases in P-PDK1 and P-PTEN after stroke. A, Representative protein bands from Western blots of P-PDK1 and P-PTEN. B, Optical densities of P-PDK1. P-PDK1 protein significantly decreased at all time points after normothermic ischemia (black bars). Although P-PDK1 decreased at 5 and 9 h in hypothermic animals (gray bars), it recovered from 24 h, and total levels of P-PDK1 across all time points were substantially higher under hypothermia. ⁺p < 0.001 versus all groups, 37°C; ^*p < 0.05 and ^**p < 0.01 versus control (con); ^#p < 0.05 and ^##p<0.001 versus 37°C at the same time point. n = 3-5 per group. C, Mean optical densities demonstrate that P-PTEN decreased from 0.5 to 9 h after ischemia and started to recover at 24 h. No decreases in P-PTEN were observed in hypothermic animals after ischemia, and total level of P-PTEN in hypothermic rats were higher than in normothermic animals (p < 0.05), suggesting that hypothermia maintained protein levels of P-PTEN after stroke. ^*p < 0.05 versus control. n = 3-5 per group. Black bars, 37°C; gray bars, 30°C.

Figure 9.

A, Triple staining of P-PTEN, MAP-2, and DAPI confirms that hypothermia maintained P-PTEN expression after ischemia. The photomicrographis of the ischemic penumbra. P-PTEN ubiquitously expressed in cortical neurons and decreased at 5 and 24 h in neurons both in the ischemic core (data not shown) and penumbra of normothermic brains. However, P-PTEN overexpressed in capillaries at 24 h (arrow). Hypothermia attenuated the decrease in immunoreactivity of P-PTEN both at 5 and 24 h after stroke. Scale bar, 20 μm. B, Triple staining of P-PTEN, CD-31 (an endothelial cell marker), and DAPI, indicating that P-PTEN expressed in capillaries and some larger vessels 24 h after stroke but not in blood vessels of the non-ischemic cortex. Scale bar, 20 μm.

Figure 10.

Phosphorylation levels of FKHR and GSK3β after cerebral ischemia. A, Representative protein bands from Western blots for P-FKHR, P-GSK3β (Tyr216), and P-GSK3β (Ser9). P-GSK3β (Tyr216) did not change after cerebral ischemia in either normothermic or hypothermic animals. B, Optical densities indicate that P-FKHR significantly decreased at all time points after stroke, whereas hypothermia transiently preserved P-FKHR at 0.5 h and enhanced P-FKHR at 1.5 h. Although P-FKHR decreased to low levels at 5 and 9 h with hypothermia, it began to recover earlier than under normothermia. ⁺p < 0.001 versus all groups of 37°C; ^*p < 0.01 versus control (con); ^#p < 0.001 and ^##p < 0.01 versus 37°C at the same time point. n = 3-5 per group. Black bars, 37°C; gray bars, 30°C. C, Optical densities of P-GSK3β (Ser9) bands shows that P-GSK3β (Ser9) gradually decreased after ischemia, and hypothermia did not block this. In fact, hypothermia seemed to magnify the decrease in P-GSK3β, suggesting that dephosphorylation of GSK3β may not play a role in ischemic cell damage. ⁺p < 0.001 versus all groups, 30°C; ^*p < 0.05 versus control; ^**p < 0.001 versus all groups except 0.5 h, 37°C; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus 30°C. n = 3-5 per group. Black bars, 37°C; gray bars, 30°C.

Figure 11.

Triple staining of P-β-catenin, MAP-2, and DAPI. Pictures were taken from sham, normothermic, and hypothermic animals at 5 and 24 h after ischemia, and some merged higher-magnification images are presented. Immunostainings in both ischemic penumbra (IP) and ischemic core (IC) were also compared. P-β-Catenin normally expresses in the cytosol (arrows) in non-ischemic brain but translocated into the nuclei (asterisks) of neurons in the penumbra of normothermic brains at 5 h, although few positive cells in the core were detected. Twenty-four hours later, nuclear-positive staining (arrowheads) was observed in most cells. However, nuclear translocation of P-β-catenin was detected only in the ischemic core (arrowheads) in the hypothermic brain both at 5 and 24 h. A picture showing the transitional pattern of subcellular distribution of P-β-catenin from ischemic penumbra to core was taken in a hypothermic rat at 24 h after ischemia, indicating that P-β-catenin mainly expressed in the cytosol in the penumbra but in the nuclei in the ischemic core. A dotted line indicates the border between the ischemic core, with less MAP-2 staining, and the penumbra, with more MAP-2 staining. Scale bars: low magnification, 20 μm; higher magnification, 10 μm. con, Control.

Figure 12.

Cytochrome c (Cyto C) releases only after P-Akt, P-Akt substrate (P-Akt SUB), and P-PTEN decrease in normothermic but not in hypothermic animals. Triple staining of P-Akt (Ser473) (red), P-Akt substrate (red), and P-PTEN (red) with cytochrome c (green) and DAPI (blue) was performed. Cytochrome c immunostaining was not observed in the cortex of sham animals and was detected in a few cells at 5 h after stroke (data not shown). Strong immunostaining of cytochrome c (arrows) was detected in the ischemic cortex penumbra 24 h after normothermic ischemia when P-Akt (Ser473), P-Akt substrate, and P-PTEN substantially decreased. Hypothermic blocked the decreases in P-Akt substrate and P-PTEN but not the Akt dephosphorylation at 24 h in the penumbra. Scale bar, 20 μm.