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Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia Heng Zhao1,2, Midori A Yenari1,2,3, Danye Cheng1,2, Robert M Sapolsky4,2 and Gary K Steinberg1,2 1

Department of Neurosurgery, Stanford University, Stanford, California, USA; 2Stanford Stroke Center, Stanford University, Stanford, California, USA; 3Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, USA; 4Department of Biological Sciences, Stanford University, Stanford, California, USA Hypothermia is effective in preventing ischemic damage. A caspase-dependent apoptotic pathway is involved in ischemic damage, but how hypothermia inhibits this pathway after global cerebral ischemia has not been well explored. It was determined whether hypothermia protects the brain by altering cytochrome c release and caspase activity. Cerebral ischemia was produced by two-vessel occlusion plus hypotension for 10 mins. Body temperature in hypothermic animals was reduced to 331C before ischemia onset and maintained for 3 h after reperfusion. Western blots of subcellular fractions revealed biphasic cytosolic cytochrome c release, with an initial peak at about 5 h after ischemia, which decreased at 12 to 24 h, and a second, larger peak at 48 h. Caspase-3 and -9 activity increased at 12 and 24 h. A caspase inhibitor, Z-DEVD-FMK, administered 5 and 24 h after ischemia onset, protected hippocampal CA1 neurons from injury and blocked the second cytochrome c peak, suggesting that caspases mediate this second phase. Hypothermia (331C), which prevented CA1 injury, did not inhibit cytochrome c release at 5 h, but reduced cytochrome c release at 48 h. Caspase-3 and -9 activity was markedly attenuated by hypothermia at 12 and 24 h. Thus, biphasic cytochrome c release occurs after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase of cytochrome c release, possibly by blocking caspase activity. Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129. doi:10.1038/sj.jcbfm.9600111; published online 23 March 2005 Keywords: apoptosis; caspases; cerebral ischemia; cytochrome c; global ischemia; hypothermia

Introduction Hypothermia has profound protective effects in animal models of experimental cerebral ischemia (Busto et al, 1989a; Colbourne et al, 1997; Corbett and Thornhill, 2000; Ginsberg and Busto, 1998; Miyazawa et al, 2003; Yenari et al, 2003). Understanding the protective mechanisms of hypothermia may both enhance its therapeutic potential and reveal targets for treatment by other means. Apoptosis has recently been shown to play critical roles in ischemic damage after transient global and focal ischemia (Fujimura et al, 1998; Graham and Chen, 2001; Namura et al, 1998). During apoptosis, Correspondence: Dr GK Steinberg, Department of Neurosurgery, Stanford University School of Medicine, 300 Pasteur Drive R200, Stanford, CA 94305-5327, USA. E-mail: [email protected] This study was supported by NINDS Grants R01 NS27292 (GKS), R01 NS 40516 (MAY), and P01 NS37520 (GKS and RMS). Received 6 October 2004; revised 18 November 2004; accepted 22 November 2004; published online 23 March 2005

cytochrome c is released from the mitochondria into the cytosol and causes the subsequent caspase-9 and -3 activity (Budihardjo et al, 1999; Zou et al, 1999). This mitochondrial apoptotic pathway is mediated by pro- and anti-apoptotic proteins of the Bcl-2 family (Adams and Cory, 1998). Caspase-8 also contributes to apoptosis, either by mediating Bid/ Bax activity or directly activating caspase-3 (Li et al, 1998; Yuan and Horvitz, 2004). Additionally, apoptosis can occur independently of caspase-3 activation. Translocation of apoptosis-inducing factor (AIF) or endogenous nuclease G from the mitochondria into the nuclei produces large-scale DNA fragmentation and chromatin condensation (Cande et al, 2004; Li et al, 2001). While many reports agree that both global and focal ischemia can cause cytochrome c release and caspase activation (Cao et al, 2003; Fujimura et al, 1998; Schulz et al, 1999) and AIF nuclear translocation (Cao et al, 2003; Ferrer and Planas, 2003; Zhao et al, 2004a, 2003; Zhu et al, 2003), whether and how apoptosis is involved in cerebral ischemia is

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not yet clear. For example, caspase activity is not always observed after stroke, and may vary depending on ischemic severity or animal age (Gill et al, 2002; Hu et al, 2000; Yenari et al, 2002). In addition, cytochrome c release has been observed as early as 2 h after stroke (Sugawara et al, 1999), or as late as 36 h after global ischemia (Ouyang et al, 1999), and after permanent focal ischemia it may not be observed at all (Gill et al, 2002). The mechanisms underlying the neuroprotective effects of mild hypothermia have been studied extensively (Busto et al, 1989b, 1994; Cardell et al, 1991; Chopp et al, 1992; Hamann et al, 2004; Harada et al, 2002; Maier et al, 2002; Yenari et al, 2002; Zhang et al, 2001). It has been suggested that mild hypothermia improves energy levels (Erecinska et al, 2003), delays anoxic depolarization (Kaminogo et al, 1999; Takeda et al, 2003), attenuates or completely suppresses glutamate release (Busto et al, 1989b; Mitani and Kataoka, 1991), and suppresses production of reactive oxygen species (ROS) (Maier et al, 2002). It has also been shown that hypothermia blocks the opening of the blood–brain barrier (BBB) (Dietrich et al, 1990; Huang et al, 1999; Kawanishi 2003), inhibits inflammation (Han et al, 2003, 2002; Huang et al, 1999; Kawanishi, 2003; Wang et al, 2002), and regulates the expression of various genes (Akaji et al, 2003; Boris-Moller et al, 1998; Friedman et al, 2001; Kumar et al, 1996; Wu et al, 1995). However, only a few studies have investigated the effects of hypothermia on apoptotic pathways after stroke. One report showed that hypothermia inhibits Fas and caspase-3 expression after focal ischemia (Phanithi et al, 2000). Our laboratories recently showed that mild hypothermia transiently blocks cytochrome c release after focal ischemia, but did not alter Bcl-2 expression (Yenari et al, 2002). In contrast, mild hypothermia increased Bcl-2 expression and decreased apoptosis after global ischemia (Zhang et al, 2001). To our knowledge, there are no prior studies examining the effects of hypothermia on the cytochrome c/caspase-dependent apoptotic pathway after transient global ischemia. In this study, we explored these apoptotic events after transient global ischemia, and investigated the effects of hypothermia.

Materials and methods Global Cerebral Ischemia All experimental protocols performed on animals were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Male Sprague–Dawley rats (350 to 450 g) were used. Core body temperatures were monitored with a rectal probe throughout the surgical procedure. Anesthesia was induced by 5% isoflurane and maintained with 2% to 3% isoflurane during surgery. Ten minutes of global ischemia was induced by bilateral occlusion of the common carotid arteries (CCAs) combined with hypotension (30 mm Hg) as described by Smith Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

et al (1984), with minor changes as previously noted (Kelly et al, 2002). Briefly, the left femoral artery was cannulated with PE-50 tubing to monitor mean arterial blood pressure (MABP) and arterial blood gases. A midline incision was made in the neck and the CCAs were exposed and encircled by a suture. An external jugular vein was cannulated to allow blood withdrawal. pCO2 and pO2 of the arterial blood were adjusted to a normal range before ischemia onset. Common carotid arteries were occluded by tightening the sutures around them when MABP was reduced to 30 mm Hg by blood withdrawal. After 10 mins, the sutures were removed and the blood was returned to the animal, cannulae were removed and the wound sites were closed. After surgery, all animals were allowed to regain consciousness and were monitored throughout their recovery periods. Animals were divided into normothermic or hypothermic groups according to body temperature (Zhang et al, 2001). For rats in the hypothermic group, core temperature was decreased to 331C by spraying 100% alcohol onto the rat’s body, and was brought back to normal with a light and a heating pad. Hypothermia was induced 10 mins before ischemia and was maintained for 3 h after reperfusion. Core temperature of rats in the normothermic group was maintained at 371C throughout the experiment. Zhao et al (1997) have shown that brain temperature spontaneously drops from 371C to 331C to 341C at the end of 10 mins of global ischemia in the same model, and recovers to normothermia within 20 mins after reperfusion (Zhao et al, 1997), which is consistent with a previous study (Busto et al, 1987). A high correlation between rectal temperature and brain temperature in hypothermic rats was observed (Zhao et al, 2004b). All rats were maintained under anesthesia for 3 h after reperfusion, except for those killed at 1 h after reperfusion. Sham animals were prepared for control.

Caspase Assays Caspase activity was quantified using a fluorometric assay based on cleavage of caspase substrates as described previously (Yenari et al, 2002). Rats were killed at different time points relative to ischemia with an overdose of halothane and perfused with ice-cold saline. For normothermic rats, fresh slices of CA1 were dissected on ice 1, 4, and 12 h, and 1, 2, and 3 days after the onset of ischemia. For hypothermic rats, CA1 samples were dissected 12 h and 1, 2, 3, 4, 5, and 7 days after ischemia. Samples were sonicated in 10 mL lysis buffer per gram of tissue. Positive controls were similarly prepared from whole-brain lysates of neonatal rat brains. Samples were centrifuged for 10 mins at 10,000 r.p.m., and the supernatants were collected. Fifty microliters of each sample and doses of standards (free 7-amino-4-trifluoromethyl coumarin; #T-7, Enzyme Systems Products, Livermore, CA, USA) ranging from 2 to 32 mL of 80 mmol/L were loaded into 96-well, black polystyrene plates with clear flat bottoms (#07200656, Fisher). Two microliters of 10 mmol/L caspase-3 substrates (acetyl-A sp-Glut-ValAsp-7-amido-4-trifluoromethyl coumarin; #AFC-138,

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Enzyme Systems Products) or caspase-9 substrates (acetylLeu-Glut-His-Asp-7-amido-4-trifluoromethyl coumarin; #AFC-154, Enzyme Systems Products) were added to the wells. Baseline readings were obtained using a fluorometric plate reader (F-max, Molecular Devices; excitation ¼ 355 nm, emission ¼ 538 nm), and were taken again after 60 mins incubation at 371C. Increased values were corrected with the standard curve and normalized to protein concentrations. Activity was expressed as the foldchange over sham control (not subjected to ischemia). Three to five animals were studied for each time point.

Subcellular Fractionation and Western Blots Cytochrome c release into the cytosol and mitochondria was detected using Western blots as described previously (Fujimura et al, 2000; Yenari et al, 2002; Zhao et al, 2003). Brains of normothermic rats were harvested 5, 12, 24, and 48 h after the onset of ischemia. Brains from hypothermic rats were harvested 5 and 48 h after ischemia. Animals were perfused transcardially with normal saline, the brain was quickly removed and the hippocampal CA1 region was rapidly dissected. For mitochondrial and cytosolic subcellular fractionation, samples were prepared as described previously using a multiple centrifugation method (Yenari et al, 2002). Twenty micrograms protein/ lane was subjected to 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for 1.5 h. Protein bands were transferred from the gel to polyvinylidinene fluoride (Millipore, Bedford, MA, USA) membranes for 1 h. The primary antibody of cytochrome c (BD-Pharmingen, San Jose, CA, USA, Cat# 556433, 1:2,000) was incubated for 1 h and followed by a horseradish peroxidase-conjugated secondary anti-mouse antibody (1:2,000, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) for 1 h. To verify accurate separation of the mitochondria and cytosol fractions, separate mitochondria and cytosol samples were subjected to 15% SDS-PAGE and membranes were probed for cytochrome c oxidase (1 g/mL; 20E8-C12, Molecular Probes, Eugene, OR, USA). To confirm even loading of cytosolic protein, separate samples were subjected to 15% SDS-PAGE and membranes were probed for b-actin (1:5,000, A-5441, Sigma Chemical, St. Louis, MO, USA). Protein bands were detected using an enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA) and exposed to Hyperfilm (Amersham Pharmacia Biotech UK Limited, England). Films were scanned by an optical densitometer and analyzed using Multi-Analyst software (Bio-Rad).

Caspase Inhibition To determine if the second phase of cytochrome c release after global ischemia is caspase mediated, a caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-ValAsp-(OMe)-fluoro-methylketone (Z-DEVD-FMK, ICN Biomedicals, Inc., Irvine, CA, USA, Cat# FK010) was injected intraventricularly as described previously (Chen et al, 1998; Sugawara et al, 2002) The drug was diluted in dimethylsulfoxide (DMSO) to 30 mg/mL and diluted

further with sterilized phosphate-buffered saline (PBS) to 3 mg/10 mL. It was injected in a volume of 5.0 mL (1.5 mg) into the right lateral ventricle (1.5 mm lateral and 0.8 mm anterior from bregma, and 3.5 mm deep) at 5 and 24 h after the onset of ischemia, using a 10 mL Hamilton syringe (Hamilton, Reno, NV, USA) connected to a microinfusion pump (World Precision Instruments, Sarasota, FL, USA). The infusion rate was 1.0 mL/min. The dose used effectively blocks caspase-3 activity after transient global ischemia, according to previous reports (Chen et al, 1998; Sugawara et al, 2002). Five microliters solution containing 1% DMSO served as vehicle. Rat brains were harvested 2 days after ischemia onset for subcellular fractionation and cytochrome c detection by Western blot.

Immunofluorescence Staining Immunofluorescence staining of cytochrome c was performed as described in prior reports (Fujimura et al, 1998; Sugawara et al, 2002; Yenari et al, 2002; Zhao et al, 2003, 2004b). Animals were perfused transcardially with saline followed with 3% paraformaldehyde 5, 12, 24, 48, and 72 h after ischemia, and brains were postfixed in 3% paraformaldehyde plus 20% sucrose for 24 h. Thirtymicrometer sections were cut onto glass slides in the coronal plane using a cryostat. Sections were blocked in PBS containing 5% donkey serum (Sigma), 1% bovine serum albumin (BSA), and 0.3% Triton X-100 for 1 h at room temperature and then incubated in the primary antibody of purified mouse anti-cytochrome c antibody (1:500, Cat. No 556432, PharMingen) diluted in blocking solution at 41C overnight. Sections were incubated for 2 h at room temperature in the secondary antibody of fluorescein (FITC)-conjugated donkey anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) diluted in blocking solution. Sections were washed with PBS, one drop of propidium iodide (PI) mounting media was added, and sections were coverslipped and examined by confocal microscopy (Carl Zeiss). Negative controls, in which the primary antibodies were omitted, were run in parallel.

Histopathology/Assessment of Ischemic Injury For histopathologic study, animals subjected to normothermic ischemia plus Z-DEVD-FMK, normothermic ischemia plus vehicle, or hypothermic ischemia were then allowed to survive 3 days, 3 days, and 7 days, respectively. Animals not subjected to ischemia were prepared as controls. Animals were euthanized with an overdose of isoflurane and perfused with saline followed by 10% formalin. Brains were postfixed in 10% formalin for 3 days and then embedded in paraffin. Sections (8-mm thick) were cut on a microtome and mounted on Superfrost-Plus glass slides (Fisher Scientific), then stained with hematoxylin–eosin (H&E). The extent of ischemic injury in hippocampi was determined from H&E-stained sections. Morphologically intact neurons in the CA1 were counted by investigators masked to the experimental Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

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conditions (  400, four fields each side) and compared across groups (Sugawara et al, 2002; Zhang et al, 2001).

Statistics For comparing caspase-9 or -3 activities between normothermic and hypothermic groups, statistical analyses were performed using SAS v8.2 (SAS Institute, Cary, NC, USA). Standard analysis of variance (ANOVA) methods and Friedman’s nonparametric two-way ANOVA for ranked data were used to evaluate the association of caspase levels with time and temperature. Pair-wise comparisons of the data were performed only for variables found to be statistically significant in the overall ANOVA model. In the parametric models, pair-wise comparisons were performed using the Tukey–Kramer test for least-squares means. In the nonparametric model pair-wise comparisons of statistically significant factors were performed using Wilcoxon’s test. All statistical tests were two-sided and considered statistically significant for P-values o0.05. The remaining statistical analyses used one-way ANOVA followed by Fisher’s LSD post hoc test. Data are presented as means7s.d.

Results Biphasic Cytochrome c Release and Caspase Activity After Global Ischemia

Biphasic cytochrome c release was observed after transient global ischemia in normothermic rats. Western blots indicated that cytosolic cytochrome c increased at 5 h, but decreased at 12 and 24 h after global ischemia (Figure 1A). A second phase of cytochrome c release appeared at 48 h after ischemia, and was significantly larger than the first phase at 5 h (Figure 1B). Consistent with Western blots, confocal immunostaining confirmed that cytosolic cytochrome c staining was observed in some CA1 neurons at 5 h and strikingly increased at 48 and 72 h (Figure 2A, 371C). Cytochrome c immunostaining in the sham animals appeared in a punctate pattern and the signal was weak, which may reflect its mitochondrial localization. This suggests that cytochrome c was released from the mitochondria into the cytosol after ischemia. Finally, both caspase-9 (Figure 3) and caspase-3 activity (Figure 4) increased after ischemia/reperfusion under normothermic conditions. Caspase Inhibition Attenuates the Second Phase of Cytochrome c Release

Because the second phase of cytochrome c release under normothermia occurred after the peaks of caspase activity, we tested the hypothesis that the second phase of cytochrome c release is caspasedependent. Western blots showed that the amount of Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

Figure 1 Biphasic cytochrome c release after global ischemia. (A) Western blots of subcellular fractions isolated from ischemic CA1 show that levels of cytochrome c changed in cytosol and mitochondria 5, 12, 24, and 48 h after ischemia onset when core temperature was maintained at 371C. Western blots of cytochrome oxidase (COX) show bands only within the mitochondrial fractions, confirming successful separation of mitochondria and cytosol fractions. Bands of b-actin show equal loading of protein. Each band represents three to four animals. Western blots were repeated three times. (B) Optical densities of Western blots show a bimodal pattern of cytochrome c release. An increase in cytochrome c was detected 5 h after ischemia, followed by a decrease at 12 and 24 h. A second peak of cytochrome c release was observed at 48 h. Data represent optical densities of brain samples of separate animals run on the same gel for each time and condition. * versus sham, 12, 24 h (ANOVA, Po0.01), ** versus sham, 5, 12 or 24 h (ANOVA, Po0.001, n ¼ 3 to 5/group).

cytochrome c released was significantly reduced in animals treated with the caspase inhibitor administered at 5 and 24 h after ischemia onset (Figure 5), suggesting that the second phase of cytochrome c release is because of caspase activity. Mild Hypothermia Attenuated the Second Phase of Cytochrome c Release

The amount of cytochrome c released in hypothermic and normothermic rats at 5 and 48 h after ischemia was compared using Western blotting. Mild hypothermia did not inhibit the first phase

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Figure 2 Double immunofluorescence staining of cytochrome c (green) and propidium iodide (PI) (red) after global ischemia. Sections of ischemic CA1 regions from normothermic rats (top panels, 371C) and hypothermic rats (bottom panels, 331C) are shown using confocal microscopy. A representative section from a rat not subjected to ischemia (sham) is also shown. In the normal rat brain (sham), weak punctate cytochrome c stain was observed. A diffuse pattern of cytochrome c staining was observed 5 h after ischemia in normothermic rats (arrow). Some punctate staining was observed in neurons at 12 and 24 h after ischemia (arrowhead). At 48 h, an intense, diffuse pattern of cytosolic cytochrome c was observed (arrow). Fewer nuclei remained 72 h after ischemia, which indicates that CA1 neurons were damaged or lost; intense cytochrome c staining was observed in the remaining cell debris (arrow). Hypothermia did not affect the diffuse pattern of cytochrome c staining at 5 h (arrow), but in contrast to normothermic brains, only a punctate pattern of cytochrome c stain was observed at 12, 24, 48, and 72 h after ischemia (arrowheads).

Figure 3 Caspase-9-like activity after global ischemia. Caspase-9 activity increased slightly from 1 h to 2 days after reperfusion in normothermic rats (371C) compared to sham. In contrast, slight increases in caspase-9 activity were not observed until 3 days in hypothermic rats (331C). Although there was no significant difference between various time points across temperatures, there was a main effect of temperature (i.e., overall levels of caspase-9 activity were significantly higher under normothermia than hypothermia; Po0.05, n ¼ 3 to 5/group). NR—neonatal rat brain, which served as positive control.

Figure 4 Caspase-3-like activity after global ischemia. Caspase-3-like activity increased 1 h after ischemia and reached a peak at 12 h and 1 day, then decreased to normal levels at 2 and 3 days. With hypothermia, caspase-3-like activity did not increase until 3 days after ischemia. Caspase-3-like activity under normothermia was significantly higher than under hypothermia at 12 h and 1 day, and lower at 3 days (Po0.05) (n ¼ 3 to 5/group). *, **, *** (Po0.01, 0.001, 0.0001, respectively, versus sham); # versus 371C at 12 h, 1 and 3 days (Po0.05).

of cytochrome c release at 5 h, but markedly attenuated the large second phase of cytochrome c release (Figure 6). Consistent with this, confocal imaging showed neurons at 5 h stained with a diffuse pattern of cytochrome c, but neurons at other time points showed weak, punctate staining (Figure 2B, 331C).

Hypothermia Attenuated Caspase-3 and -9 Activity

Overall levels of caspase-3 and -9 activity were decreased by mild hypothermia (Figures 3 and 4). Although there was a delayed increase in caspase-3 activity at 72 h, mild hypothermia markedly reduced caspase-3 activity at 12 and 24 h (Figure 4). Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

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Figure 5 Caspase inhibition blocks the second phase of cytochrome c release at 48 h after global ischemia in normothermic rats. (A) Western blots showing cytochrome c levels in the cytosol 48 h after global ischemia in animals treated with a caspase inhibitor (CI) or its vehicle, and in sham rats. Bands of b-actin show even loading of protein. (B) Optical densities of Western blots show cytochrome c release in normothermic, ischemic rats treated with vehicle (vehicle) or a CI, and in shams. Caspase inhibition significantly blocked cytochrome c release 48 h after global ischemia (n ¼ 3 in each group). * versus sham or CI (ANOVA, Po0.05).

Mild Hypothermia Completely Blocked and Caspase Inhibition Significantly Attenuated CA1 Neuronal Damage After Transient Global Ischemia

In normothermic animals, most CA1 neurons were damaged, with shrunken, condensed nuclei and vacuolar morphology at 3 days after ischemia (Figure 7). However, many normal neurons were observed in animals that received the caspase inhibitor, suggesting it attenuated CA1 neuronal damage. Almost no damage was observed in hypothermic animals at 7 days after ischemia. Cell counts showed that caspase inhibition significantly reduced the number of damaged neurons in the hippocampal CA1. Hypothermia nearly completely spared CA1 neuronal damage.

Figure 6 Hypothermia suppresses cytochrome c release at 2 days but not 5 h. (A) Western blots showing cytochrome c levels in the cytosol at 5 and 48 h after global ischemia with or without hypothermia. Bands of b-actin revealed equal loading of protein. (B) Optical densities of Western blots show cytochrome c release in normothermic (371C) or hypothermic (331C) ischemic rats. Hypothermia did not inhibit cytosolic cytochrome c accumulation at 5 h (upper panel), but significantly attenuated cytochrome c release at 2 days after global ischemia (lower panel). * versus 5 h at 331C or 5 h at 371C; # versus sham or 2 days at 331C (ANOVA, Po0.05, n ¼ 3/group).

Discussion Several novel phenomena were shown in the current study. First, biphasic cytochrome c release was observed after transient global ischemia in normothermic animals. Because increased caspase Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

activity occurred before the second phase of cytochrome c release and delayed administration of the caspase inhibitor Z-DEVD-FMK blocked the second phase of cytochrome c release, caspases may be

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Figure 7 Representative H & E stains of CA1 hippocampus (A) and counts of intact neurons in CA1 region (B) At 3 days after ischemia, most CA1 neurons were damaged, with shrunken, triangular condensed nuclei in normothermic animals. Caspase inhibition attenuated CA1 neuronal damage. Because some caspase activity was observed at 3 days after ischemia in hypothermic animals, it is possible that such caspase activity may cause further delayed neuronal damage. Therefore, hypothermic animals were allowed to survive for 7 days for histopathologic assessment. Almost no damage was observed in hypothermic animals at 7 days after ischemia. Counts of intact neurons showed that there was no significant neuronal damage in hypothermic rats, and caspase inhibition significantly attenuated neuronal damage (n ¼ 6/group). * versus sham, 331C, or 371C/CI (ANOVA, Po0.001), # versus sham or 331C (Po0.001).

responsible for this larger, delayed cytochrome c release. Second, and unexpectedly, mild hypothermia did not block the first phase of cytochrome c release, but inhibited the increase in caspase activity and the second phase of cytochrome c release. This result suggests that the first phase of cytochrome c release may not contribute to eventual ischemic damage. Furthermore, delayed caspase activity was found 3 days after hypothermic global ischemia, which apparently was sublethal because little obvious CA1 neuronal damage was observed at 7 days after ischemia.

Biphasic Release of Cytochrome c and Caspase Activity After Transient Global Ischemia

How cytochrome c is released after cerebral ischemia and whether it mediates the activity of caspases

is controversial (Fujimura et al, 1998; Gill et al, 2002; Hu et al, 2000; Ouyang et al, 1999; Sugawara et al, 1999; Yenari et al, 2002). One report indicated that it was released as early as 2 h after global ischemia (Sugawara et al, 1999) while another study showed no cytochrome c release until 36 h (Ouyang et al, 1999). A recent study showed no definitive evidence for cytochrome c release or caspase activity after permanent focal ischemia (Gill et al, 2002), and another report revealed no caspase activity despite cytochrome c release after transient focal ischemia (Gill et al, 2002; Yenari et al, 2002). In contrast, the current study showed a clear biphasic pattern of cytochrome c release after global ischemia, and caspase activity was observed before the second phase of cytochrome c release. The mechanisms of cytochrome c release after global ischemia are not clear. It is well known that ATP depletion after global ischemia triggers anoxic depolarization (Kaminogo et al, 1998), glutamate release (Benveniste et al, 1989; Globus et al, 1991) and calcium dysregulation (Kristian and Siesjo, 1996). In addition, mitochondria are injured early after ischemia (Hillered et al, 1984; Siesjo et al, 1999). It has been shown that overloading of mitochondrial Ca2 þ induces the mitochondrial permeability transition (mPT) (Budd et al, 2000; Kroemer and Reed, 2000), which leads to cytochrome c release and caspase activity. Mitochondrial permeability transition has also been observed in in vitro ischemia, and cytochrome c release associated with mPT was noted after ischemia in vivo (Kroemer and Reed, 2000). It may be that the initial first cytochrome c release in the current study was caused in part by glutamate release and NMDAreceptor-mediated intracellular Ca2 þ increase and early mitochondrial dysfunction. Furthermore, excessive free radicals are produced immediately after reperfusion (Chan, 2001). It has been shown that overexpression of the antioxidant protein superoxide dismutase (SOD) attenuates early cytochrome c release after global ischemia (Sugawara et al, 2002) and glutathione peroxidase (GPX) blocks cytochrome c release 2 days after focal ischemia (Hoehn et al, 2003), which suggests that ROS are involved in cytochrome c release. Production of ROS after reperfusion may lead to mitochondrial injury and cytochrome c release. Cytochrome c release has been shown to play a critical role in apoptosis (Yuan and Horvitz, 2004; Zou et al, 1999). Cytosolic cytochrome c may form the apoptosome with procaspase-9 and apaf-1, and cleave procaspase-9 further causing subsequent caspase-3 activity (Yuan and Horvitz, 2004; Zou et al, 1999). According to the widely accepted mechanism of caspase-dependent apoptosis, cytochrome c release should precede caspase activity. The present data showed peak caspase-3 activity at 12 and 24 h, 7 to 19 h after the first peak of cytochrome c release. The fact that caspase inhibition attenuated the late phase of cytochrome Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

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c release suggests that caspase caused further cytochrome c release after global ischemia. Indeed, prior work has shown that recombinant caspases affect mPT and cause cytochrome c (Marzo et al, 1998; Xia et al, 2002) and AIF release in purified mitochondria (Marzo et al, 1998). Furthermore, caspase inhibition blocks cytochrome c release in purified mitochondria (Xia et al, 2002) or Jurkat cells undergoing apoptosis (Bossy-Wetzel and Green, 1999). During apoptosis caused by gammairradiation and etoposide in human IM-9 multiple myeloma cells, two distinct stages of cytochrome c release were observed (Chen et al, 2000); an early small amount of cytochrome c is released before caspase-9 and -3 activation, which does not cause reduction in ATP and mitochondrial transmembrane potential, and a late phase of cytochrome c release leads to significant loss of ATP and mitochondrial transmembrane potential. Similar to our study, the authors found that caspase inhibition blocks the late stage of cytochrome c release. Taken together, these studies suggest that there is an amplifying feedback effect between caspase and cytochrome c. Blocking this feedback loop may be clinically relevant because it occurs relatively late after transient global ischemia.

Hypothermia and Caspase-Dependent Apoptosis

We previously showed that hypothermia delays cytochrome c release without altering Bcl-2 expression in a focal ischemia model (Yenari et al, 2002), but a fluorometric assay showed no increased caspase activity in that experiment. However, we found that mild hypothermia specifically increased Bcl-2 expression in global ischemia (Zhang et al, 2001). Therefore, we hypothesized that the effect of hypothermia in global and focal ischemia differs, which led to the current study. To our surprise, mild hypothermia completely blocked ischemic cell damage without altering the first phase of cytochrome c release. This result suggests that this early release may not necessarily lead to neuronal death in this model. It is well known that cytochrome c release initiates the subsequent caspase-9 and -3 activity. However, the current study provides evidence that cell death can be prevented even after a small amount of cytochrome c has been released into the cytosol. We also showed that caspase inhibition initiated after the early phase of cytochrome c release blocked the second larger phase of cytochrome c release and attenuated ischemic neuronal loss. Why hypothermia did not block the first phase of cytochrome c release is unclear. It has been found that mild hypothermia merely delays changes in Ca2 þ mobilization (Kristian et al, 1992; Mitani et al, 1991), and the protective effect of mild hypothermia is not proportional to duration of anoxic depolarization (Bart et al, 1998; Sick et al, 1999; Takeda et al, Journal of Cerebral Blood Flow & Metabolism (2005) 25, 1119–1129

2003). In addition, some reports showed that mild hypothermia does not always block extracellular glutamate accumulation (Asai et al, 1998; Simpson et al, 1991), and whether it inhibits glutamate release depends on whether glutamate is measured in the ischemic core or the penumbra (Berger et al, 2002; Huang et al, 1998; Lo et al, 1993). Therefore, it appears in some cases that the acute detrimental biochemical cascades after global ischemia are not appreciably or reliably blocked by hypothermia. These events may cause mitochondrial dysfunction and lead to the early phase of cytochrome c release. A small amount of caspase activity was observed 3 days after hypothermic ischemia, but this low level of caspase activation did not induce ischemic neuronal damage 7 days after ischemia. There may be a threshold for caspases inducing ischemic neuronal damage, or caspases may not cause neuronal death in the absence of other events downstream of caspase activity. In fact, several recent studies of ischemic preconditioning show that caspase activation does not always lead to neuron death (Barone et al, 1998; Garnier et al, 2003; McLaughlin et al, 2003). Actually, a small amount of caspase activation may be essential to protection by preconditioning (Garnier et al, 2003). However, the significance of delayed caspase activity in hypothermic animals not leading to ischemic damage at 1 week after ischemia remains to be determined. In this study, mild hypothermia was induced before ischemia onset. From a therapeutic perspective, it will be important to assess neuroprotection by postischemic hypothermia, which others have shown depends on its time of onset, duration, and depth (Colbourne et al, 1997). We used intraischemic hypothermia in this study because it yields clear neuroprotection (Dietrich et al, 1993), thereby allowing us to explore its protective mechanisms in this model. Whether postischemic hypothermia inhibits the second phase of cytochrome c release and caspase activity is yet to be determined. In addition, the anesthetic we used, isoflurane, has been shown to confer neuroprotection after stroke (Inoue et al, 2004; Kawaguchi et al, 2004). Although isoflurane can reduce ischemic damage, the normothermic and hypothermic groups were exposed to identical periods of isoflurane anesthesia. If isoflurane were protective this would, if anything, lessen the apparent benefit of hypothermia. In conclusion, we show for the first time in an ischemia model that cytochrome c release is biphasic. The second phase of cytochrome c release appears to be caspase-mediated. Hypothermia inhibits the second, but not the first phase of cytochrome c release and attenuates as well as delays caspase activity. These observations suggest potential for antiapoptotic therapies after stroke. It appears that the temporal therapeutic window for targeting cytochrome c release may be relatively long.

Stroke, cytochrome c, and hypothermia H Zhao et al

Acknowledgements The authors thank Beth Hoyte for preparation of the figures, Dr David Schaal for assistance preparing the manuscript, and Dr Paige Bracci (UCSF) for her expert statistical assistance.

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