Transient global cerebral ischemia (tGCI)
PKCε null mice generated by homologous recombination were generous gifts from Dr. Robert Messing at the University of Texas Austin [16, 17]. Male and female PKCε heterozygous mice maintained on C57BL/6J and 129S4 backgrounds were crossed to produce F1 hybrid mice. The F1 hybrid mice were intercrossed to generate F2 hybrid littermates for experiments. Mice were genotyped via PCR of DNA extracted from mouse tails. Male F2 hybrid wild-type (WT) and PKCε null mice between 2 and 4 months of age were used for all experiments [16, 17].
Total 105 mice were subjected to tGCI and randomly assigned into different groups of analysis (3 WT and 3 PKCε null mice for PKCε phosphorylation of ATF2 at T52, 29 WT mice for PKCε expression in the hippocampus after tGCI, 43 WT mice for temporal expression of ATF2 in the hippocampal mitochondria after tGCI, 12 WT mice for histological analysis of the CA1 region of the hippocampus after tGCI, 9 WT mice for mitochondrial translocation of ATF2 after tGCI, 6 WT mice for expression of ATF2 in degenerating neurons labeled with Fluoro-Jade C).
Before tGCI, mice were fasted overnight but allowed free access to water. Mice were anesthetized with 5% isoflurane in a mixture of N2O/O2 (70%/30%) and maintained on 1.5% isoflurane using the MouseVent G500 Automatic Ventilator (Kent Scientific, Torrington, CT, USA). TGCI was induced by hypotension and occlusion of bilateral common carotid arteries using microclips for 10 min, following a previously-described procedure . Control mice were sham-operated under the same surgical conditions.
All mouse-related experiments were conducted in compliance with the guidelines of the Office of Research Compliance at Kent State University and the Laboratory Animal Center of National Health Research Institutes. The animal protocols were approved by Research Involving Animal (Institutional Animal Care and Use Committee, IACUC) under Office of Research Compliance at Kent State University (Protocol# 359 CW 14-8 rev 3 Sub 27 August 2013) and the Laboratory Animal Center at National Health Research Institutes (Protocol# NHRI-IACUC-105087-A), according to the Guide for the Care and Use of Laboratory Animals (NRC 2011). Management of animal experiment and animal care and use of Kent State University and National Health Research Institutes have accredited by the AAALAC. All efforts were made to minimize the number of animals used.
Western blot analysis
At different time points after tGCI, mice were deeply anesthetized with 4% isoflurane in N2O/O2 (70%/30%) and euthanized by cervical dislocation. Mouse hippocampi were isolated at different time points after tGCI and homogenized using a Teflon-glass homogenizer as previously described . Mitochondrial and cytosolic fractions were prepared using a mitochondrial isolation kit (Thermo Scientific Pierce, Rockford, lL, USA). The whole cell lysates, mitochondrial and cytosolic fractions were analyzed by Western blot using rabbit anti-Thr-52 ATF2 (1:200, Phosphosolution, Aurora, CO, USA, Cat# p115-52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9226), mouse anti-Actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA, Cat# A4700), mouse anti-PKCε (1:1000, BD, Franklin Lakes, NJ, USA, Cat# 610086) and rabbit anti-COXIV (1:1000, Sigma-Aldrich, Cat# AV42784). Immunoreactive bands were detected by enhanced chemiluminescence (ECL) (Thermo Scientific Pierce, Rockford, lL, USA), imaged by Luminescent Image Analyzer LAS-3000 (Fujifilm, Edison, NJ, USA) and quantified by scanning densitometry using the NIH ImageJ program.
Hippocampal neuronal culture and oxygen–glucose–deprivation
Primary neuronal cultures were prepared from the hippocampus of postnatal day 1 (P1) to P3 mice as previously described [17, 18]. The cultures were subjected to 1 h of oxygen–glucose–deprivation and 4 h of re-oxygenation to simulate transient ischemia . To label mitochondria, cultures were incubated with 100 nM MitoTracker for one hour (Thermo Scientific Invitrogen, Carlsbad, CA, USA), fixed with 4% paraformaldehyde (PFA), and immunostained with an antibody against ATF2 (1:200, Sigma-Aldrich, Cat#SAB4300315) and DAPI as previously described . The images were obtained using an Olympus (Center Valley, PA, USA) FV500/IX81 confocal microscope.
Mice were deeply anesthetized with 4% isoflurane in N2O/O2 (70%/30%) at different time points after tGCI and perfused transcardially with 4% PFA in 0.15 M phosphate buffer (pH 7.3) . Brains were isolated and postfixed in 4% PFA. Coronal Sects. (1 mm) were obtained using a brain matrix (Braintree Scientific, Braintree, MA, USA) and processed for paraffin embedding. Coronal Sects. (5 μm) from the hippocampus (-3.5 mm caudal to bregma) were obtained using a microtome. Paraffin sections mounted on slides were stained with hematoxylin and eosin (H&E).
Primary neurons and paraffin brain Sects. (5 μm) were fixed with 4% PFA and incubated overnight at 4 °C with rabbit anti-ATF2 (1:200, Cell Signaling Technology, Cat# 9226) and mouse anti-COXIV (1:200, Cell Signaling Technology, Cat#11967) [17, 18]. Degenerating neurons were detected using a Fluoro-Jade C staining kit (Histo-Chem, Jefferson, AR, USA) . After washing, the cultured neurons and brain sections were stained with Alexa Fluor conjugated secondary antibodies (1:200, Jackson ImmunoResearch, West Grove, PA, USA) and mounted in media containing 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The images were acquired using an Olympus FV500/IX81 confocal microscope or a Leica TCS SP5 II confocal laser scanning microscope.
Quantitative data are mean ± SEM t test, one-way ANOVA, and Newman-Keuls post hoc tests were used for statistical analysis. Values of P< 0.05 in all tests were considered to be statistically significant.