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(Stroke. 2007;38:375.)
© 2007 American Heart Association, Inc.

Original Contributions

PKC May Contribute to the Protective Effect of Hypothermia in a Rat Focal Cerebral Ischemia Model

Takayoshi Shimohata, MD, PhD; Heng Zhao, PhD Gary K. Steinberg, MD, PhD

From the Department of Neurosurgery (T.S., H.Z., G.K.S.), Department of Neurology and Neurological Science (H.Z., G.K.S.), and Stanford Stroke Center (G.K.S.), Stanford University, Stanford, Calif.

Correspondence to Gary K. Steinberg, MD, PhD, Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Dr R200, Stanford, CA 94305-5327. E-mail cerebral{at}stanford.edu

	Abstract

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Background and Purpose— Protein kinase C epsilon (PKC) has been implicated as a neuroprotectant in vitro. We studied PKC activation (by its localization and proteolysis) in a rodent stroke model and correlated the effects of hypothermia with PKC activity after cerebral ischemia.

Methods— Rats were subjected to permanent distal middle cerebral artery occlusion plus 1 hour of bilateral common carotid artery occlusion. Body temperatures were maintained at 37°C or 30°C during common carotid artery occlusion. Brains were harvested at 10 minutes, 4 hours, and 24 hours after common carotid artery release, and the cortex corresponding to the ischemic core and penumbra was dissected. PKC localization after stroke was assessed by Western blot and immunofluorescence microscopy. A caspase-3 inhibitor was used to test whether PKC cleavage is caspase dependent.

Results— PKC in the membrane fraction and whole-protein homogenates decreased moderately in the penumbra but decreased markedly in the ischemic core. Hypothermia blocked this decrease in both the ischemic core and penumbra. Confocal microscopy confirmed that neuronal PKC expression decreased in the ischemic core at 4 hours after reperfusion, and this loss was prevented by hypothermia. Two carboxyl-terminal cleavage products of PKC with molecular masses of 43 and 35 kDa were detected. Although the protein band of 43 kDa decreased after stroke, the 35-kDa band increased. Such changes were not dependent on caspase-3. However, hypothermia blocked changes in the cleavage form of 35 kDa but not 43 kDa after stroke.

Conclusions— Moderate hypothermia preserves PKC activity after stroke.

Key Words: PKC • focal cerebral ischemia • hypothermia

	Introduction

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Protein kinase C epsilon (PKC) belongs to the novel subgroup of the PKC family and has been associated with antiapoptotic functions in a variety of cellular systems.^1–3 Several studies have shown that PKC is activated via subcellular translocation from the cytosol to the particulate membrane fraction.^4–6 In addition, generation of a 43-kDa carboxyl-terminal catalytic fragment by caspase activity also results in the activation of PKC^7–9 (Figure 1A).

View larger version (44K): [in this window] [in a new window]   Figure 1. Hypothermia attenuates PKC proteolytic cleavage. A, Proteolytic cleavage sites of PKC by caspases. Arrows indicate cleavage sites, and an arrowhead indicates the recognition site of the PKC (C-15) antibody. B, Experimental protocol. See details in the text. C, Tissue corresponding to the ischemic core (region II) and penumbra (region I) that was dissected for Western blots. D and E, Levels of PKC and its cleavage products in the ischemic core (D) and the penumbra (E) from normothermic or hypothermic rats. Short exposure=3 seconds; long exposure=30 seconds (43 kDa=black arrowhead; 35 kDa=white arrowhead). F to H, Optical densitometric analyses for PKC isoforms between normothermic and hypothermic rat brains in both the ischemic core and penumbra. Black bars indicate 37°C; gray bars, 30°C. *P<0.05 vs sham; #P<0.05 vs 30°C, 4 hours; P<0.05 vs 30°C, 4 hours; n=5 per group.

PKC is an important component of the signal transduction pathways in ischemic preconditioning–induced neuroprotection.¹⁰ A recent study demonstrated that ischemic preconditioning neuroprotection could be emulated in vitro with an agonist of PKC or blocked with its antagonist.⁶ Despite its importance, the spatial kinetics of PKC activation after focal cerebral ischemia are unknown.

The neuroprotective effects of hypothermia are not understood completely. Recently, we have shown that proapoptotic PKC activation via subcellular translocation and proteolytic cleavage is suppressed by hypothermia in a rat focal cerebral ischemia model.¹¹ In this report we study PKC activation in response to stroke and the effects of hypothermia on the kinetics of its activity.

	Methods

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Focal Cerebral Ischemia and Hypothermia
Experimental protocols were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Focal ischemia was generated as described¹² in male Sprague-Dawley rats (weight, 350 to 450 g). The distal middle cerebral artery was permanently cauterized, and the bilateral common carotid arteries (CCAs) were transiently occluded for 1 hour. Core temperature was maintained at 37°C throughout the surgery for normothermic animals; for hypothermia, the temperature was maintained at 30°C during ischemia by spraying 70% methanol onto the rat’s body (Figure 1B).¹²

Whole-Cell Homogenization, Subcellular Fractionation, and Western Blots
At 10 minutes, 4 hours, and 24 hours after CCA release, rat brains were harvested, and tissue corresponding to the ischemic core and penumbra was dissected as described¹² (Figure 1C). Samples from sham-operated rats were also prepared. For whole-cell homogenates, tissues were homogenized in cell lysis buffer (Cell Signaling Technology) and centrifuged, and the supernatant was collected.¹² Subcellular cytosolic (soluble) and membrane (particulate) fractions were also prepared as described.¹² Proteins (10 µg) were loaded and separated by SDS-PAGE.¹² A primary PKC antibody (C-15, 1:10 000, Santa Cruz Biotechnologies) was probed, and signals were detected by enhanced chemiluminescence (Amersham). Densities of protein bands were analyzed with the use of ImageJ (NIH).

Caspase-3 Inhibitor Injection and PKC Cleavage Assay
A cell-permeable caspase-3–specific inhibitor, Z-DQMD-FMK (Calbiochem), was dissolved in dimethyl sulfoxide and PBS (Z-DQMD-FMK, 0.3 µg/µL in 1% dimethyl sulfoxide in PBS; vehicle, 1% dimethyl sulfoxide in PBS). Rats were anesthetized and placed in stereotactic frames. The drug solution (1.5 µg) or the vehicle was injected into the ventricular space ipsilateral to the ischemia (5 µL; from bregma: 0.9 mm posterior, 1.5 mm lateral, 3.5 mm deep) at 2 hours after CCA release. At 24 hours after CCA release, brains were removed, and whole-cell extracts were prepared for full-length or cleaved PKC detection by Western blots.

Immunofluorescence Staining
Rat brains were collected and fixed in 4% paraformaldehyde as described,¹² then cut on a vibratome into slices of 50 µm. Sections were stained with anti-PKC (1:200) and anti-MAP2 (1:500, Sigma) antibodies 4°C overnight, then incubated with FITC or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories) for 2 hours. The slices were mounted with medium containing DAPI (Vector Laboratories) and examined under a laser-scanning confocal microscope (LSM510, Carl Zeiss).

Statistical Analysis
All data are presented as mean±SEM. All statistical analyses were performed with the use of ANOVA followed by the Tukey post hoc test.

	Results

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In normothermic brains, Western blots indicated that PKC in whole-cell homogenates decreased significantly at 4 hours after CCA release in the penumbra, and greater decreases were detected in the ischemic core at 4 hours and 24 hours (Figure 1D to 1F). Such decreases in PKC in both the ischemic core and penumbra at 4 hours (Figure 1D to 1F) were blocked by hypothermia. Carboxyl-terminal cleavage products of PKC molecular masses of 43 and 35 kDa were detected (Figure 1D and 1E). The 35-kDa fragment increased at 10 minutes and 4 hours, returned to baseline at 24 hours after CCA release in the ischemic core (Figure 1D and 1H), and increased at 4 hours in the penumbra (Figure 1E and 1H). Hypothermia blocked all accumulation of the 35-kDa fragment. In contrast, the larger fragment (43 kDa) decreased in the ischemic core from 4 to 24 hours after stroke but did not change in the penumbra (Figure 1D and 1G). Hypothermia did not block such changes (Figure 1G).

To investigate whether PKC cleavage after stroke is caused by caspase-3 activation, we examined the effect of a cell-permeable caspase-3–specific inhibitor, Z-DQMD-FMK, on generation of cleaved PKC fragments (Figure 2). Caspase-3 inhibition did not suppress the decrease in full-length PKC and the 43-kDa fragment in the ischemic core and penumbra after stroke.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of a caspase-3–specific inhibitor on PKC cleavage. Representative protein bands of PKC in the ischemic core and penumbra from rat brains treated with Z-DQMD-FMK or vehicle are shown (n=3 per group). Short exposure=3 seconds; long exposure=30 seconds (43 kDa=black arrowhead; 35 kDa=white arrowhead).

Confocal microscopy indicated that PKC is distributed in the cytoplasm and axons of neurons in nonischemic brains. Consistent with our results from Western blots, PKC immunofluorescence decreased in the ischemic core at 4 hours after CCA release, and this decrease was suppressed by hypothermia (Figure 3).

View larger version (60K): [in this window] [in a new window]   Figure 3. Brain tissues of sham-operated rats or operated rats (4 hours after CCA release) were stained for PKC (green), MAP2 (red), and DNA (blue). PKC decreased in the ischemic core at 4 hours after CCA release. Bar=10 µm.

Subcellular distribution of PKC between the cytosolic and membrane fractions was also studied. In the ischemic core from normothermic brains, PKC decreased at 4 hours after CCA release in both the cytosolic and membrane fractions compared with controls (Figure 4A and 4C). This decrease in both fractions was blocked by hypothermia. In the penumbra, cytosolic PKC levels did not change after stroke, and hypothermia did not affect cytosolic PKC levels. However, membrane-associated PKC significantly decreased at 4 hours after stroke, and this decrease was blocked by hypothermia (Figure 4B and 4D).

View larger version (56K): [in this window] [in a new window]   Figure 4. Assessment of membrane translocation and levels of PKC. A, PKC from the cytosolic (C) and membrane (M) fractions of the ischemic core and penumbra of normothermic rats. B, PKC from the cytosolic and membrane fractions of the ischemic core and penumbra at 37°C or 30°C. C, Levels of PKC in the cytosolic and membrane fractions from the ischemic core. D, PKC partitioning in the penumbra. Black bars indicate 37°C; gray bars, 30°C. *P<0.05 vs sham; #P<0.05 vs 30°C at 4 hours; n=3 per group.

	Discussion

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We report here that levels of full-length PKC in cell lysates and membrane fractions decreased after stroke. This reduction was blocked by hypothermia. The manner by which hypothermia prevents a decrease in PKC levels after stroke is not understood. It has been demonstrated that PKC is cleaved by caspases including caspase-3, -7, and -9.⁹ Our previous study has detected caspase-3 activity in this ischemic model.¹⁶ However, caspase-3 inhibition did not blocked decreases in the full-length and 43-kDa PKC, despite the fact that we have previously shown that PKC cleavage was blocked via inhibiting caspase-3 activity.¹¹ This suggests that PKC cleavage is not mediated by caspase-3. Hypothermia may block PKC cleavage through inhibiting other proteases.

Our characterization of PKC activation and proteolysis in the normothermic and hypothermic stroke brain is consistent with functional studies showing that PKC activity can be neuroprotective in an in vitro model.⁶ The fact that 2 disparate neuroprotective treatments, mild hypothermia and adenosine administration,¹³ both enhance PKC activity indicates that this kinase lies at an important node of a neuroprotective cascade.

The present study has some limitations. First, because hypothermia protects ischemic brain through its multiple effects on various detrimental signals, we cannot exclude the possibility that preservation of PKC levels is secondary to hypothermic protection. However, hypothermia also specifically modifies some gene expression or protein levels to reduce ischemic damage. For example, hypothermia inhibits Bax overexpression 4 hours after 30 minutes of incomplete cerebral ischemia but does not affect Bcl-2, p53, and Mdm-2 expression.¹⁴ Yenari et al¹⁵ demonstrated that hypothermia transiently attenuated cytochrome c release but did not alter Bcl-2 and Bax expression in a focal ischemia model. Most recently, we found that hypothermia generally maintains the Akt pathway signals but does not attenuate dephosphorylation of GSK-3ß.¹² In our present study, we found that hypothermia reduces PKC cleavage and its subcellular translocation, suggesting, but not unequivocally proving, that preservation of PKC protein level is an important component contributing to the protective effect of hypothermia. Another limitation of this study is that intraischemic hypothermia was employed. The manner by which postischemic hypothermia, a paradigm more relevant to clinical application, blocks PKC cleavage should be pursued in the future.

	Acknowledgments

 
The authors thank Dr Bruce Schaar for editing the manuscript and David Kunis for technical assistance.

Sources of Funding

This study was supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke grant R01 NS27292 (Dr Steinberg).

Disclosures

None.

Received September 12, 2006; accepted October 2, 2006.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 38/2/375    most recent 01.STR.0000254616.78387.eev1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shimohata, T. Articles by Steinberg, G. K. Search for Related Content PubMed PubMed Citation Articles by Shimohata, T. Articles by Steinberg, G. K. Related Collections Apoptosis Neuroprotectors Related Article