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. Author manuscript; available in PMC: 2025 Jun 8.
Published in final edited form as: J Neurosci Res. 2018 Nov 29;97(4):444–455. doi: 10.1002/jnr.24362

Genetic inhibition of PKCε attenuates neurodegeneration after global cerebral ischemia in male mice

Varun Kumar 1, Yi-Chinn Weng 2, Yu-Chieh Wu 2, Yu-Ting Huang 2, Tung-Hsia Liu 2, Tibor Kristian 3, Yu-Li Liu 2, Hsiao-Hui Tsou 4,5, Wen-Hai Chou 1,2
PMCID: PMC12147233  NIHMSID: NIHMS2085927  PMID: 30488977

Abstract

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC sub-family and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.

Keywords: cardiac arrest, global cerebral ischemia, mitochondrial translocation, neurodegeneration, PKC

1 |. INTRODUCTION

Cardiac arrest is an important public health issue worldwide; there are approximately 400,000 cases in the United States and 8,000 cases in Taiwan annually (Mozaffarian et al., 2015; Wang, Wang et al., 2015; Zipes & Wellens, 1998). Cardiac arrest is an unexpected loss of heart function, resulting in severe impairment of cerebral blood flow and ischemic damage to brain cells (Stub, Bernard, Duffy, & Kaye, 2011). Therapeutic hypothermia was introduced a decade ago and remains the standard for post-resuscitation care (Varon, Marik, & Einav, 2012). Despite the expanded use of therapeutic intervention, less than 25% of the patients with cardiac arrest ultimately survive after hospital discharge (Go et al., 2013). Brain injury is a major cause of morbidity and mortality, responsible for two thirds of deaths after cardiac arrest (Laver, Farrow, Turner, & Nolan, 2004).

Protein kinase C (PKC) is a family of 10 serine-threonine kinases that regulate a broad spectrum of cellular functions in both healthy and diseased (Newton, 2010; Steinberg, 2008) conditions. Based on their amino-terminal structures and sensitivities to Ca2+ and diacylglycerol (DAG), the PKCs are classified into conventional PKCs (α, βI, βII, and γ), novel PKCs (δ, ε, η, and θ), and atypical PKCs (ζ and λ/ι). Of these, Protein Kinase C epsilon (PKCε) has been demonstrated to play a vital role in ischemic preconditioning (Perez-Pinzon, Stetler, & Fiskum, 2012). Translocation of PKC isozymes to the subcellular compartments mediated by the binding with receptors for activated C kinase (RACKs) has been characterized as a unique feature of PKC activation (Murriel & Mochly-Rosen, 2003).

Treatment with PKCε peptide activator (ψεRACK) before cerebral ischemia confers neuroprotection by increasing the phosphorylation of the mitochondrial respiratory chain proteins and the respiration of synaptic mitochondria (Bright, Sun, Yenari, Steinberg, & Mochly-Rosen, 2008; Dave et al., 2008; Raval, Dave, DeFazio, & Perez-Pinzon, 2007; Raval, Dave, Mochly-Rosen, Sick, & Perez-Pinzon, 2003). These data suggest that the pharmacological activation of the PKCε pathway can have a protective effect against cerebral ischemia. The physiological role of endogenous PKCε has not been reported.

PKCε-null mice were used to assess the effects of endogenous PKCε after transient global cerebral ischemia (tGCI). Global cerebral ischemia causes extensive neurodegeneration in the hippocampus (Kristian & Hu, 2013; Onken, Berger, & Kristian, 2012; Owens, Park, Gourley, Jones, & Kristian, 2014; Sheng, Laskowitz, Pearlstein, & Warner, 1999). Neurodegeneration can be detected in the hippocampus as early as one day after ischemia (Kristian & Hu, 2013; Onken et al., 2012; Owens et al., 2014; Sheng et al., 1999). The number of degenerating neurons continues to rise between 2 and 3 days but thereafter is stable up to 5 days, suggesting that neuronal cell death matures by 3 days after ischemia.

We found that tGCI-induced Fluoro-Jade C labeling, cytochrome C release, and translocation of PKCδ in the hippocampus were significantly reduced, while spatial learning was improved in PKCε-null mice. Our data demonstrate that the reduction in endogenous PKCε mitigates ischemic brain injury. As brief episodes of sublethal stress protects cells from a subsequent episode of lethal ischemia through preconditioning (Perez-Pinzon et al., 2012), our data may also support the preconditioning role of PKCε in stroke.

2 |. MATERIALS AND METHODS

2.1 |. Mice

Prkce−/− mice were generated by homologous recombination (Chou et al., 2010; Khasar et al., 1999). Prkce+/− mice were maintained on inbred 129S4/SvJaeJ (RRID: IMSR_-JAX:009104) and C57BL/6J (RRID: IMSR_-JAX:000664) backgrounds and crossed to produce F1 hybrid mice for breeding. The F1 mice were intercrossed to generate F2 hybrid mice. Genotyping was performed by PCR of mouse tail DNA. Male co-housed Prkce+/+ and Prkce−/− littermates between 2 and 4 months of age were used for the experiments. The decision to use male mice was based on the evidence that sexual dimorphism occurs for cerebral ischemia (Hanamsagar & Bilbo, 2016; Spychala, Honarpisheh, & McCullough, 2017; Thagard et al., 2017). Female mice exhibit attenuated ischemic brain injury as compared to male mice. All procedures were performed in accordance with Institutional Animal Care and Use Committee policies at Kent State University and NHRI. Mice were housed at 23 ± 1°C with a controlled 12:12 hr light/dark cycle (lights on 0700–1,900), and fed standard lab chow and water ad libitum.

2.2 |. Transient global cerebral ischemia

tGCI was induced by a combination of hypotension and bilateral common carotid artery occlusion (BCCAO) following a previously described procedure with minor modifications (Onken et al., 2012). The major difference between these two procedures is the ventilator used. A mouse ventilator (Model 687, Harvard Apparatus, Holliston, MA, USA) was used in a previous publication (Onken et al., 2012). MouseVent G500 Automatic Ventilator (Kent Scientific, Torrington, CT, USA) was used in this study.

Before inducing tGCI, mice were fasted overnight with free access to water. Blood collected from the tail vein was assessed for glucose levels using the ACCU-CHEK® Aviva system (Roche, Indianapolis, IN, USA). Mice with a blood glucose level between 150 and 200 mg/dl were chosen for surgery. Mice were then anesthetized with 5% isoflurane in a mixture of N2O:O2 (70:30), intubated using BioLite Small Animal Intubation System (Braintree Scientific, MA, USA), and mechanically ventilated with 1.5% isoflurane using a pressure-controlled MouseVent G500 Automatic Ventilator (Kent Scientific, Torrington, CT, USA). The common carotid arteries were isolated via a neck incision and encircled with loose-fitting sutures for later clamping. To induce tGCI, the isoflurane level was increased to 5%, and 3 min later the bilateral common carotid arteries were occluded using microclips. After 9 min of 5% isoflurane, the isoflurane level was reduced to 0%. After 4 min, the microclips were removed from the carotid arteries. During anesthesia, body and pericranial temperature were monitored and maintained at 37.0 ± 0.5°C using a Homeothermic Blanket System (Fine Scientific Tools, Foster City, CA, USA), with a needle Thermistor probe (ThermoWorks, American Fork, UT, USA) and a heating lamp to avoid hypothermia-induced neuroprotection. After surgery, the mice were allowed to recover for 2 hr in a V1200 chamber set at 36–37°C (Peco Services Ltd., Cumbria, UK) to prevent post-ischemic hypothermia (Wellons et al., 2000; Zhen & Dore, 2007). A total of 107 mice were subjected to 10 min of tGCI and randomly allocated into different groups of analysis. Mice developing seizures after tGCI were excluded from further studies. The exclusion rates were about 16.4% and 11.5% for Prkce+/+ and Prkce−/− mice, respectively.

2.3 |. Barnes maze

The Barnes maze was used to assess spatial memory before and after tGCI (Patil, Sunyer, Hoger, & Lubec, 2009; Sun et al., 2013). The Barnes maze is a circular platform (92 cm diameter) with 20 evenly spaced holes (5 cm diameter) along the perimeter. The maze was positioned at 105 cm above the floor in the center of the testing room with visual cues hanging on the wall. A black acrylic box (27 × 9 × 6 cm) with a stepped-down ramp was placed under one hole as the escape target. Blowing fans and bright lights positioned above the platform were used as mildly aversive stimuli to increase the motivation to find the escape box (Sun et al., 2013). Mice were trained in the Barnes maze to learn to locate the escape box. Four training sessions (3 min each) were performed per day. The training sessions were continued for four consecutive days starting from day 4 after tGCI. Latencies to locate the escape hole were recorded and analyzed.

2.4 |. Open field

Mice were placed in a 40 × 40 cm open field chamber with a video camera mounted above the experimental arena for 15 min to assess their locomotor activities (Lesscher et al., 2008). EthoVision software v. 7.0 (Noldus Information Technology, Leesburg, VA, USA; RRID:SCR_004074) was used for analysis of distance traveled in the open field.

2.5 |. Fluoro-Jade C staining

Three days after tGCI, mice were perfused intracardially with 4% PFA and coronal brain sections (50 μm) were mounted on slides (Wang, Weng et al., 2015). Degenerating neurons were detected using a Fluoro-Jade C staining kit following the manufacturer’s protocol (Histo-Chem, Jefferson, AR, USA) (Wang et al., 2010). To quantify the areas with degenerating neurons, an investigator blinded to the treatment conditions outlined the hippocampus and measured the total number of Fluoro-Jade C-stained pixels using a densitometric thresholding technique with NIH ImageJ (RRID:SCR_003070) (Belousov et al., 2012). The threshold was set at a level just above that which would have counted the background and nonspecific staining in areas outside the outlined region. For each mouse, three sections between 1.67 and 1.91 mm posterior to bregma were stained with Fluoro-Jade C and used to quantify the intensity of Fluoro-Jade C labeling.

2.6 |. Cresyl violet staining

Neuronal cell death after ischemia was assessed using cresyl violet staining. Hippocampal neuronal damage was evaluated by an investigator blinded to the studies on the basis of a scoring system (Kawase et al., 1999). Grade 0, no injury to any area in the hippocampus; grade 1, scattered ischemic injury in the CA1 area; grade 2, moderate ischemic injury in the CA1 area (less than half of pyramidal neurons damaged); grade 3, severe damage to pyramidal cells in the CA1 area (more than half of pyramidal neurons damaged); grade 4, extensive injury in all hippocampal areas. The hippocampal areas of the brain sections showing pyknotic, shrunken nuclei were outlined, measured, and quantified using NIH ImageJ (Wang et al., 2012). For each mouse, three sections between 1.67 and 1.91 mm posterior to bregma were stained with Fluoro-Jade C and used to assess neuronal cell death after tGCI.

2.7 |. Cerebrovascular anatomy and regional cerebral blood flow

Cerebrovascular anatomy was assessed after injection of Higgins Black Magic waterproof ink (200–250 μl; Sanford Corp.) into the left ventricle (Wang, Weng et al., 2015). The regional cerebral blood flow (rCBF) was recorded 3 min before ischemia and continued until 5 min after reperfusion by Laser Doppler Flowmetry (Perimed, Ardmore, PA, USA) (Wang, Weng et al., 2015).

2.8 |. Brain homogenate and subcellular fractionation

Several studies showed that the cytosolic PKCδ translocates to the mitochondria and mediates neuronal cell death after cerebral ischemia (Dave et al., 2011; Shimohata, Zhao, Sung et al., 2007). Hippocampal tissues were dissected from mouse brains at 24 and 48 hr after tGCI, and homogenized in 1× RIPA buffer with protease inhibitors (Cell Signaling Technology, Beverly, MA, USA) using a Teflon-glass homogenizer (Chou et al., 2010). A mitochondrial isolation kit (Thermo Scientific Pierce, Rockford, IL, USA) was used to separate the mitochondrial and cytosolic fractions. The mitochondrial fractions were sonicated six times (10 s each time) at 70% amplitude in an ice bath using a Sonifier 450 (Branson Sonic Power, Danbury, CT, USA) and centrifuged at 12,000 × g for 10 min at 4°C. The mitochondrial and cytosolic fractionations were resuspended in the RIPA buffer. The protein concentrations were determined by Bradford assays (Thermo Scientific Pierce, Rockford, IL, USA).

2.9 |. Western blotting

The whole cell lysates and the subcellular fractions from the hippocampus were separated by NuPAGE 4%–12% Bis-Tris gels using the NuPAGE MOPS SDS Running Buffer (Thermo Scientific Invitrogen, Carlsbad, CA, USA), transferred to PVDF membranes, and analyzed by the western blot technique using rabbit anti-Cytochrome C (1:1,000, Cell Signaling Technology, Cat# 11940; RRID:AB_2637071), mouse anti-PKCε (1:1,000, BD, Cat# 610086; RRID:AB_397493), rabbit anti-PKCδ (1:1,000, Cell Signaling, Cat# 2058; RRID:AB_10694655), mouse anti-β-Actin (1:1,000, Sigma-Aldrich, Cat# A4700; RRID:AB_476730), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000, GeneTex, Cat #GTX100118; RRID:AB_1080976), and rabbit anti-COXIV (1:1,000, Sigma Aldrich, Cat# AV42784; RRID:AB_1847177) antibodies. Western blotting for PKC isozymes was assessed using the PKC Sampler Kit (BD Transduction Laboratories, Cat# 611421). Immunoreactive bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA), acquired by Luminescent Image Analyzer LAS-3000 (Fujifilm, Edison, NJ, USA), and analyzed by NIH ImageJ.

2.10 |. Statistics

Quantitative data were expressed as means ± SD and analyzed using Prism 5.0 (GraphPad, La Jolla, CA, USA) (RRID:SCR_002798). All data were tested for normality using the Kolmogorov–Smirnov normality tests and met the criteria for normality. t-test and Newman–Keuls post hoc tests were used for statistical analyses. Two-way repeated measures ANOVA was performed using the MIXED procedure in the mixed model of SAS 9.4 program (SAS Institute, Inc., Cary, NC) (RRID:SCR_008567). A statistically significant difference was defined as p < 0.05.

3 |. RESULTS

3.1 |. Neurodegeneration in the hippocampus after tGCI

To assess the effect of PKCε deficiency on ischemia-induced neurodegeneration, male Prkce+/+ and Prkce−/− mice were subjected to 10 min of tGCI followed by 3 days of reperfusion. Ischemia-induced neurodegeneration revealed by Fluoro-Jade C staining was significantly reduced by 59.3% in the hippocampus of Prkce−/− mice compared to Prkce+/+ mice after tGCI (p = 0.0413; Figure 1). Since Fluoro-Jade C detects both degenerating neurons and stressed neurons that may not ultimately die (Larsson, Lindvall, & Kokaia, 2001; Schmued & Hopkins, 2000; Schmued, Stowers, Scallet, & Xu, 2005; Wang et al., 2010), we used cresyl violet staining to confirm cell death and neurodegeneration after ischemia (Figure 2) (Onken et al., 2012). Histological assessment of hippocampal injury after tGCI showed reduced neuronal damage in Prkce−/− mice (Figure 2a,b). The area of neuronal cell death in the hippocampus was significantly reduced by 43.4% in Prkce−/− mice (2.2 ± 0.4 mm2) compared to Prkce+/+ mice (3.8 ± 0.5 mm2; p = 0.0188; Figure 2c).

FIGURE 1.

FIGURE 1

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Neurodegeneration after tGCI was reduced in the hippocampus of Prkce−/− mice. (a) Representative images of Fluoro-Jade C-stained brain slices from Prkce+/+ and Prkce−/− mice subjected to 10 min of tGCI and 3 days of recovery. (b) Quantification of hippocampal Fluoro-Jade C labeling using a densitometric thresholding technique with NIH ImageJ in Prkce+/+ (n = 5) and Prkce−/− mice (n = 5) following3 days of post-ischemic recovery. *p < 0.05 compared with Prkce+/+ mice (t-test). (c) Enlarged views of the CA1–CA2 border. There are relatively fewer cells stained with Fluoro-Jade C in the CA1–CA2 border of Prkce−/− than Prkce+/+ mice. White arrowheads indicate degenerating neurons with a shrunken nucleus. Scale bars are 500 μm (a) and 50 μm (c) [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2.

FIGURE 2

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Neural cell death after tGCI was attenuated in the hippocampus of Prkce−/− mice. (a) Representative images of cresyl violet-stained brain slices from Prkce+/+ and Prkce−/− mice 3 days after tGCI. Arrowheads indicate degenerative neurons with pyknotic and shrunken nuclei in the enlarged CA1 region of a Prkce+/+ brain slice (lower panels). Scale bars are 500 and 50 μm in the upper and lower panels, respectively. (b) Qualitative analysis of neuronal damage in the hippocampus of Prkce+/+ and Prkce−/− mice 3 days after tGCI. Scattered dots show scores of the hippocampal injury of Prkce+/+ (n = 5) and Prkce−/− mice (n = 5). (c) Quantification of areas showing neural cell death in the hippocampus of Prkce+/+ (n = 5) and Prkce−/− mice (n = 5) after 3 days of post-ischemic recovery. *p < 0.05 compared with Prkce+/+ mice (t-test). (d, e) Cytochrome C release after tGCI was reduced in the hippocampus of Prkce−/− mice. (d) The hippocampus was isolated from Prkce+/+ and Prkce−/− mice at 48 hr after tGCI. The cytosolic fraction of the hippocampus was analyzed by the western blotting technique using antibodies against cytochrome C (Cyto C). β-Actin was used as a loading control. (e) Cyto C and β-Actin protein bands were quantified by densitometry (n = 3). Cyto C immunoreactivity normalized to β-Actin (Cyto C/Actin) was compared between means using a two-tailed t test (*p < 0.05) [Color figure can be viewed at wileyonlinelibrary.com]

Cytochrome C is released from the mitochondria into the cytosol after cerebral ischemia (Sugawara, Fujimura, Morita-Fujimura, Kawase, & Chan, 1999). To determine the level of cytochrome C in the cytosol, we isolated the subcellular fractions of the hippocampus at 48 hr after tGCI. The cytosolic cytochrome C expression was significantly reduced in the hippocampus of Prkce−/− mice as compared to Prkce+/+ mice (p = 0.0312; Figure 2d,e), corresponding to the attenuated neurodegeneration and cell death observed in the hippocampus of Prkce−/− mice.

3.2 |. Spatial learning and memory in the Barnes maze

To determine whether the deletion of PKCε alters spatial learning and memory, experimentally naive Prkce+/+ (n = 8) and Prkce−/− mice (n = 8) were assessed using the Barnes maze (Figure 3a). Two-way repeated measures ANOVA showed that both genotypes learned the task as demonstrated by spending progressively less time to locate the escape box across the four training days [Fday (3,42) = 18.85, p < 0.0001]. No significant difference was found between naive Prkce+/+ and Prkce−/− mice in their performance [Fgenotype (1,14) = 0.35, p = 0.57], suggesting that PKCε deficiency did not alter basic spatial learning and memory function. No interaction was found between genotype and time (training day) [Finteraction (3,42) = 0.08, p = 0.97].

FIGURE 3.

FIGURE 3

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Prkce−/− mice showed improved spatial learning and memory after tGCI. (a) The latency (sec) to locate the target hole across the four training days during the Barnes maze was recorded for naive Prkce+/+ (n = 8, blue open circle) and Prkce−/− (n = 8, red open square) mice, as well as Prkce+/+ (n = 7, blue filled circle) and Prkce−/− (n = 7, red filled square) mice 3 days after tGCI. (b) The total distance traveled in an open field chamber for 15 min was similar between naive Prkce+/+ (n = 8, blue open circle) and Prkce−/− (n = 8, red open square) mice, as well as Prkce+/+ (n = 7, blue filled circle) and Prkce−/− (n = 7, red filled square) mice 3 days after tGCI [Color figure can be viewed at wileyonlinelibrary.com]

To assess the effect of cerebral ischemia on spatial learning and memory, Prkce+/+ and Prkce−/− mice were subjected to tGCI followed by 3 days of recovery (Figure 3a). Both genotypes learned the task after tGCI. Two-way repeated measures ANOVA showed that the latencies to locate the escape box were significantly decreased across the four training days [Fday (3,36) = 23.21, p < 0.0001]. However, both Prkce+/+ and Prkce−/− mice after tGCI demonstrate longer latency in locating the escape box than the naive mice. The main effect of ischemia was statistically significant for Prkce+/+ [Fischemia (1,13) = 17.55, p = 0.0011] and Prkce−/− mice [Fischemia (1,13) = 11.52, p = 0.0048]. Interestingly after tGCI, Prkce−/− mice spent significantly less time to locate the escape hole than Prkce+/+ mice. The main effect of genotype was statistically significant [Fgenotype (1,12) = 4.81, p = 0.0487). No interaction was found between genotype and time (training day) [Finteraction (3,36) = 0.08, p = 0.9729]. These data suggest attenuated neurodegeneration in Prkce−/− mice. Moreover, the total distance traveled in the open field was similar between Prkce+/+ and Prkce−/− mice before and after tGCI (Figure 3b), suggesting that the observed difference in the Barnes maze is related to cognitive function and not due to difference in general locomotion function.

3.3 |. Physiological parameters, cerebrovascular anatomy, and function

Prkce−/− mice display normal motor and sensory functions at baseline and showed no significant differences in their body weight (Table 1) (Chou et al., 2010; Hodge et al., 1999; Qi et al., 2007). Hyperglycemia is known to exacerbate brain damage and induce seizures after cerebral ischemia (Shukla, Shakya, Perez-Pinzon, & Dave, 2017). Therefore, blood glucose levels were measured before tGCI (Table 1). There was no significant difference in the blood glucose levels measured before tGCI in Prkce+/+ and Prkce−/− mice.

TABLE 1.

Physiological parameters

PKCε WT (n = 5) PKCε KO (n = 5)
Body weight (g) 26.74 ± 0.58 26.72 ± 0.23
Blood glucose (mg/dL) 181 ± 8.46 180 ± 17.99
Pre-ischemic head temperature (°C) 36.58 ± 0.16 36.62 ± 0.19
Intra-ischemic head temperature (°C) 36.68 ± 0.08 36.62 ± 0.11
Post-ischemic head temperature (°C) 37.22 ± 0.16 37.1 ± 0.10
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Note. Values represents mean ± SD.

To investigate whether the attenuated neurodegeneration in Prkce−/− mice was due to developmental defects in the cerebrovascular anatomy, we examined the distribution of cerebral arteries by black ink perfusion. There was no significant difference in the origins of the middle cerebral artery (MCA) or distribution of other major blood vessels of the circle of Willis (Upper panels in Figure 4a). The patency of the posterior communicating arteries (PcomA) is considered a key factor for the development of neuronal injury in the rodent models of tGCI. The degree of PcomA patency (Lower panels in Figure 4a,b) was similar between Prkce+/+ and Prkce−/− mice. The size of MCA territory (Figure 4c,d) was also comparable between these two genotypes. To assess the dynamic changes in cerebral blood flow during tGCI, we used laser Doppler flowmetry and found that cerebral blood flow before and during ischemia, and after reperfusion, was similar for the two genotypes (Figure 4e). These results indicate that the deletion of PKCε did not affect cerebrovascular anatomy and function, and suggest that the attenuated ischemia-induced neurodegeneration in Prkce−/− mice was not due to altered physiological parameters, cerebral vasculature, or blood flow.

FIGURE 4.

FIGURE 4

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Cerebrovascular anatomy and cerebral blood flow were comparable between Prkce+/+ and Prkce−/− mice. (a) Representative images of the brains from Prkce+/+ and Prkce−/− mice perfused with black ink. The major arteries in the circle of Willis (upper panels) and PcomA (indicated by arrowheads in lower panels) were identified. (b) Scores of PcomA plasticity in Prkce+/+ (n = 3) and Prkce−/− mice (n = 3). (c) Dorsal images of the brains from Prkce+/+ and Prkce−/− mice perfused with black ink. The points of anastomoses were circled and connected to form the line of anastomoses. (d) Distances from the line of anastomoses to the midline in Prkce+/+ (n = 3) and Prkce−/− (n = 3) mice were measured at coronal planes2, 4, and 6 mm from the frontal pole. (e) rCBF of Prkce+/+ (blue line, n = 3) and Prkce−/− mice (red line, n = 3) was monitored continuously during tGCI. The percentage of rCBF (%) was calculated as the percentage relative to the baseline. Isoflurane was increased to 5% for 9 min. At the 3rd min, bilateral common carotid arteries’ occlusion (BCCAO) was induced using microclips. The combination of the high isoflurane level and BCCAO caused the reduction of rCBF to less than 20% of the baseline. At 10 min, anesthesia was discontinued (0% isoflurane). After 10 min of BCCAO, the microclips were removed which resulted in a rapid recovery of rCBF [Color figure can be viewed at wileyonlinelibrary.com]

3.4 |. Expression of PKC isozymes in hippocampal total cell lysates

To assess the effect of PKCε deficiency on the expression of other PKC isozymes, we performed a western blotting analysis of the total cell lysates prepared from the hippocampus of Prkce+/+ and Prkce−/− mice before and at 24 hr after tGCI. The abundance of PKCα, PKCβ, PKCδ, PKCε, PKCι, and PKCθ in the hippocampus was similar between Prkce+/+ and Prkce−/− mice (Figure 5a–f). The expression of PKCδ was significantly increased in Prkce−/− mice after tGCI (Figure 5c, p = 0.0173).

FIGURE 5.

FIGURE 5

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PKC isozyme immunoreactivity in the hippocampus of Prkce+/+ and Prkce−/− mice. Hippocampi were isolated from Prkce+/+ and Prkce−/− mice 24 hr after tGCI. Hippocampi isolated from mice without tGCI were used as normal controls (con). PKC isozyme and β-Actin protein bands were quantified by densitometry (n = 3 in each group). (a–f) The expression of PKCα, PKCβ, PKCδ, PKCε, PKCι, and PKCθ was similar between Prkce+/+ and Prkce−/− mice in control mice. (c) There was a significant induction of PKCδ/Actin in Prkce−/− mice at 24 hr after tGCI compared with Prkce−/− control mice (*p < 0.05, t-test)

3.5 |. Mitochondrial translocation of PKCδ

To assess the effect of PKCε deficiency on the mitochondrial translocation of PKCδ, we fractionated the hippocampus of Prkce+/+ and Prkce−/− mice isolated before and at 24 hr after tGCI (Figure 6). Verification of the purity of the cytoplasmic and mitochondrial fractions from mouse hippocampus was determined using specific markers (GAPDH and COXIV) (Figure 6a). The expression of PKCδ was significantly reduced in the cytosolic fractions after tGCI in both Prkce+/+ (p = 0.0292) and Prkce−/− mice (p = 0.0041) (Figure 6b). The mitochondrial level of PKCδ was significantly increased after tGCI in Prkce+/+ mice (Figure 6c, p = 0.0378). Interestingly, in Prkce−/− mice, the mitochondrial level of PKCδ was elevated before tGCI and the elevated level was maintained after tGCI, suggesting that the accumulation of PKCδ in the mitochondria before tGCI may prevent the mitochondrial translocation of PKCδ after tGCI in Prkce−/− mice.

FIGURE 6.

FIGURE 6

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PKCδ immunoreactivity in the cytosolic and mitochondrial fractions of hippocampus (a) Verification of fraction purification from the mouse hippocampus. Representative western blots showing the enrichment of COXIV in the mitochondrial (Mito) fraction and GAPDH in the cytosolic fraction (Cyto). (b, c) Hippocampi were isolated from Prkce+/+ and Prkce−/− mice 24 hr after tGCI. Hippocampi isolated from mice without tGCI were used as normal controls (con). The cytosolic (b) and mitochondrial (c) fractions of the hippocampus were analyzed by western blotting using antibodies against PKCδ, β-Actin, and COXIV. PKCδ, β-Actin, and COXIV protein bands were quantified by densitometry (n = 3 in each group). (B) There was a significant reduction in PKCδ/Actin in the cytosol of both Prkce+/+ and Prkce−/− hippocampi after tGCI compared with the controls (*p < 0.05, t-test). (c) There was a significant induction of PKCδ/COXIV in the mitochondria of Prkce+/+ hippocampus after tGCI compared with the Prkce+/+ control (*p < 0.05, t-test)

4 |. DISCUSSION

We found that ischemia-induced Fluoro-Jade C labeling, cytochrome C release, and PKCδ mitochondrial translocation in the hippocampus were significantly reduced, while spatial learning and memory were improved in PKCε-null mice. Cerebrovascular anatomy and function were comparable between wild-type and PKCε-null mice, suggesting that they are not the major contributors to attenuated neurodegeneration in PKCε-null mice. Our data suggest that reduction in endogenous PKCε mitigates ischemic brain injury. To our knowledge, this study is the first to characterize the role of endogenous PKCε after global cerebral ischemia using PKCε-null mice.

Our finding is rather unexpected because previous reports have demonstrated that the activation of PKCε by pharmacological activators before cerebral ischemia provides neuroprotection (Dave et al., 2008; Raval et al., 2007). Given that both pharmacological activation of PKCε before cerebral ischemia and genetic deletion of PKCε showed neuroprotective effects, it demonstrates the complex nature of PKCε actions in the process of cerebral ischemia, and suggests that whether PKCε participates in detrimental or neuroprotective pathways depends on the timing and the intensity of the ischemic stimuli. Preconditioning is a sublethal dose of ischemia lasting only for 2 min, while global cerebral ischemia is a more severe insult lasting for at least 10 min (Perez-Pinzon et al., 2012). Several genes with opposite roles in preconditioning and cerebral ischemia have been reported. A brief activation of toll-like receptors (i.e., TLR2, TLR4), hypoxia-inducible factor 1-alpha (HIF1-α), and NF-κB before ischemia induces preconditioning (Gesuete, Kohama, & Stenzel-Poore, 2014; Ran, Xu, Lu, Bernaudin, & Sharp, 2005; Ridder & Schwaninger, 2009), while mice lacking these genes exhibit reduced brain injury after cerebral ischemia (Gesuete et al., 2014; Helton et al., 2005).

Neurodegeneration induced by global cerebral ischemia continues over days and months even after successful resuscitation from cardiac arrest (Kiryk et al., 2011). More than 50% of cardiac arrest survivors exhibit cognitive dysfunction, manifested as learning and memory impairment. Morris water maze and Barnes maze have been used to assess spatial learning and memory in rodent models of cerebral ischemia (Iivonen, Nurminen, Harri, Tanila, & Puolivali, 2003; Patil et al., 2009; Vorhees & Williams, 2006). Both mazes involve learning and remembering the relationship between extra-maze cues and escape localization. However, the Barnes maze is less physically demanding and avoids potential hypothermia in water, which might interfere with neurodegeneration after ischemia (Iivonen et al., 2003; Vorhees & Williams, 2006). Also, while rats are natural swimmers, mice are not (Whishaw, 1995). After surgery, mice might sink or drown, and have a higher risk of infection in the water maze.

We used the Barnes maze, and found that mice spent longer times to locate the target hole after global cerebral ischemia, suggesting that their spatial learning and memory were impaired. The impairment could have been caused by damage to the circuitry connecting the hippocampus and ventromedial prefrontal cortex after global cerebral ischemia (Nieuwenhuis & Takashima, 2011). We also found that memory formation after global cerebral ischemia was impaired, but not totally inhibited. This phenomenon has been reported in other rodent models of cerebral ischemia (Samson, Kajitani, & Robertson, 2010; Sun et al., 2013) and cardiac arrest survivors (Horstmann et al., 2010).

In this study, we found that genetic deletion of PKCε did not affect the expression of other PKC isozymes in the hippocampus under physiological conditions, which is consistent with previous studies using dorsal root ganglia (Khasar et al., 1999), forebrain (Hodge et al., 1999), and heart tissues (Gray et al., 2004) of the PKCε-null mice. Both PKCε and PKCδ are members of the novel PKC subfamily. These isozymes, however, have different and sometimes opposing roles in the same signaling pathways (Steinberg, 2008). In signaling after cerebral ischemia, PKCε is anti-apoptotic according to studies using PKCε peptide activator (ψεRACK) and inhibitor (εV1–2) (Perez-Pinzon et al., 2012). In contrast, PKCδ is pro-apoptotic based on studies using the PKCδ peptide inhibitor δV1–1 (Bright & Mochly-Rosen, 2005) and PKCδ-null mice (Chou & Messing, 2005). In response to ischemic preconditioning, PKCε translocates to the mitochondria and confers neuroprotection by increasing the phosphorylation of Kir6.2, a specific subunit of the mitochondrial K+ATP channel (Dave et al., 2008; Raval et al., 2007). In response to apoptotic signals, PKCδ translocates to the mitochondria and increases the phosphorylation of phospolipid scramblase 3 (PLSCR3) and mediates the release of cytochrome C from the mitochondria to the cytosol (Dave et al., 2011).

Therapeutic hypothermia has been used in post-resuscitation care to improve the survival rates for cardiac arrest patients (Varon et al., 2012). Degradation of PKCε (Shimohata, Zhao, Sung et al., 2007) and mitochondrial translocation of PKCδ (Shimohata, Zhao, & Steinberg, 2007) in experimental models of cerebral ischemia were reduced after therapeutic hypothermia. In this study, we confirmed the mitochondrial translocation of PKCδ after cerebral ischemia in wild-type mice (Figure 6b) (Dave et al., 2011; Shimohata, Zhao, Sung et al., 2007). Interestingly, we found that PKCδ accumulates in hippocampal mitochondria of PKCε-null mice before ischemia (Figure 6c). The accumulation may prevent the mitochondrial translocation of PKCδ from the cytosol after ischemia, resulting in a reduced release of mitochondrial cytochrome C and neurodegeneration in PKCε-null mice. The continuous elevation of PKCδ in the mitochondria before and after ischemia may also signify a chronic activation of PKCδ, resulting in phosphorylation changes of PKCδ protein substrates in the mitochondria and protection against ischemic attack. Moreover, the level of cytosolic PKCδ was reduced in PKCε-null mice after ischemia (Figure 6b) and the total amount of PKCδ was increased after ischemia (Figure 5c), suggesting that cytosolic PKCδ may have translocated to other subcellular compartments (e.g., plasma membrane, nucleus).

In summary, we established genetic approaches to assess the in vivo functions of PKCε after global cerebral ischemia. Our results demonstrate that PKCε expression was not required for the development of cerebral vasculature, blood flow, and the formation of spatial memory under physiological conditions. Deletion of PKCε protected mice against global cerebral ischemia, and reduced ischemia-induced neurodegeneration and spatial memory deficits. This study also reveals for the first time that chronic activation of PKCδ is a contributing factor toward the attenuated neurodegeneration after global cerebral ischemia in PKCε-null mice.

Significance.

Pharmacological activation of PKCε by specific peptide activator induces a state of tolerance to subsequent brain injury. Here we demonstrate that the genetic inhibition of PKCε mitigates ischemic brain injury. Ischemia-induced neurodegeneration and learning deficits were significantly attenuated in male mice lacking PKCε-null mice after global cerebral ischemia. Thus, PKCε appears to exert a dualistic effect on the progression of ischemic brain damage. Whether PKCε is neuroprotective or detrimental depends on the timing and the intensity of ischemic insults.

ACKNOWLEDGMENTS

This study was supported by Ministry of Science and Technology, Taiwan (MOST-105-2320-B-400-024 and MOST-106-2320-B-400-017), the National Health Research Institutes, Taiwan (NP-107-PP-09), and a start-up fund from Kent State University, USA to WHC. The funding agencies were not involved in the study design, collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the article for publication. We appreciate Dr. Robert Messing of the University of Texas Austin for providing us the PKCε-null mice, and comments and suggestions from Dr. Yun Wang at NHRI and Dr. William J. Freed.

Funding information

Ministry of Science and Technology in Taiwan, National Health Research Institutes in Taiwan, Grant/Award Number: (MOST-105-2320-B-400-024 and MOST-106-2320-B-400-017)

Footnotes

CONFLICT OF INTEREST

The authors have declared that no competing interests exist.

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