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Stroke is one of the most debilitating vascular diseases worldwide, which is keeping our healthcare costs as high

as $38.6 billion each year.1 Systemic thrombolysis with intrave-nous tissue-type plasminogen activator and in situ clot retrieval remain the only reperfusion strategies approved by the Food and Drug Administration. However, after nearly 2 decades of tissue-type plasminogen activator use in the United States, most patients with acute ischemic stroke have not benefited from a reperfusion strategy, with <10% of patients actually receiv-ing systemic thrombolysis. This is attributed to tissue-type

plasminogen activator’s contraindications, and most impor-tantly, its narrow therapeutic time window. Furthermore, while a small portion of patients (17%) undergo spontaneous lysis by 6 to 8 hours,2 many patients experience permanent artery occlusion.3 In addition, even if recanalization is successful, outcome is often poor because of reperfusion injury.4

Although energy failure and oxidative stress with reac-tive oxygen species (ROS) generation after ischemia are well-documented pathophysiologies of neural injury, recent research has failed to develop targeted therapies to address

Background and Purpose—Ischemic stroke induces metabolic disarray. A central regulatory site, pyruvate dehydrogeanse complex (PDHC) sits at the cross-roads of 2 fundamental metabolic pathways: aerobic and anaerobic. In this study, we combined ethanol (EtOH) and normobaric oxygen (NBO) to develop a novel treatment to modulate PDHC and its regulatory proteins, namely pyruvate dehydrogenase phosphatase and pyruvate dehydrogenase kinase, leading to improved metabolism and reduced oxidative damage.

Methods—Sprague–Dawley rats were subjected to transient (2, 3, or 4 hours) middle cerebral artery occlusion followed by 3- or 24-hour reperfusion, or permanent (28 hours) middle cerebral artery occlusion without reperfusion. At 2 hours after the onset of ischemia, rats received either an intraperitoneal injection of saline, 1 dose of EtOH (1.5 g/kg) for 2- and 3-hour middle cerebral artery occlusion, 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg in 2 hours) in 4 hours or permanent middle cerebral artery occlusion, and EtOH+95% NBO (at 2 hours after the onset of ischemia for 6 hours) in permanent stroke. Infarct volumes and neurological deficits were examined. Oxidative metabolism and stress were determined by measuring ADP/ATP ratio and reactive oxygen species levels. Protein levels of PDHC, pyruvate dehydrogenase kinase, and pyruvate dehydrogenase phosphatase were assessed.

Results—EtOH induced dose-dependent neuroprotection in transient ischemia. Compared to EtOH or NBO alone, NBO+EtOH produced the best outcomes in permanent ischemia. These therapies improved brain oxidative metabolism by decreasing ADP/ATP ratios and reactive oxygen species levels, in association with significantly raised levels of PDHC and pyruvate dehydrogenase phosphatase, as well as decreased pyruvate dehydrogenase kinase.

Conclusions—Both EtOH and EtOH+NBO treatments conferred neuroprotection in severe stroke by affecting brain metabolism. The treatment may modulate the damaging cascade of metabolic events by bringing the PDHC activity back to normal metabolic levels. (Stroke. 2015;46:492-499. DOI: 10.1161/STROKEAHA.114.006994.)

Key Words: EtOH ◼ ischemia–reperfusion injury ◼ metabolism ◼ pyruvate dehydrogeanse complex ◼ pyruvate dehydrogenase kinase ◼ pyruvate dehydrogenase phosphatase ◼ reactive oxygen species

Ethanol and Normobaric OxygenNovel Approach in Modulating Pyruvate Dehydrogenase Complex After

Severe Transient and Permanent Ischemic Stroke

Xiaokun Geng, MD, PhD*; Omar Elmadhoun, BS*; Changya Peng, MS; Xunming Ji, MD, PhD; Adam Hafeez, BS; Zongjian Liu, PhD; Huishan Du, MD;

Jose A. Rafols, PhD; Yuchuan Ding, MD, PhD

Received August 6, 2014; final revision received October 21, 2014; accepted November 14, 2014.From the China-America Institute of Neuroscience, Luhe Hospital (X.G., X.J., Z.L., H.D., Y.D.) and Department of Neurosurgery, Xuanwu Hospital

(X.J.), Capital Medical University, Beijing, China; Departments of Neurological Surgery (X.G., O.E., C.P., A.H., Y.D.) and Anatomy and Cell Biology (J.A.R.), Wayne State University School of Medicine, Detroit, MI; and Beijing Institute for Brain Disorders, Beijing, China (X.J.).

*Dr Geng and O. Elmadhoun contributed equally.The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.

006994/-/DC1.Correspondence to Yuchuan Ding, MD, PhD, Department of Neurological Surgery, Wayne State University School of Medicine, 550 E Canfield,

Detroit, MI 48201, E-mail yding@med.wayne.edu or Xunming Ji, MD, PhD, Cerebral Vascular Diseases Research Institute (China-America Institute of Neuroscience), Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing 100053, China, E-mail jixm@ccmu.edu.cn

© 2015 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.114.006994

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Geng et al Ethanol and NBO in Severe Stroke 493

these dysfunctions that may confer neuroprotection acutely after stroke. Immediately after brain stroke, a shift from aerobic to anaerobic metabolism occurs in ischemic tissue. Pyruvate dehydrogeanse complex (PDHC), a key element in cellular metabolism, as well as its strategic regulators, is the link between aerobic and anaerobic energy metabolism. It is composed of multiple subunits that altogether convert pyru-vate to acetyl coenzyme A to eventually make ATP needed for different cellular functions.5 PDHC’s vast size and strict regu-lation makes it sensitive to inactivation and downregulation in stroke. Two key regulators of PDHC are pyruvate dehydro-genase kinase (PDK) and pyruvate dehydrogenase phospha-tase (PDP). Notably, PDK actively phosphorylates various sites on the E1α subunit of PDHC resulting in the inhibition of the entire enzyme complex. Conversely, PDP removes the phosphate group off the serine residues and thus activates the enzyme.6 Ultimately, PDHC, PDK, and PDP changes after stroke would provide important indexes to assess oxidative metabolism underlying ischemic injury after stroke.

Previous studies have demonstrated that EtOH decreases brain catabolism,7,8 raising its potential as a clinical neuropro-tectant in ameliorating metabolic dysfunction after stroke. In rats with 2-hour middle cerebral artery occlusion (MCAO), we previously showed that a postischemia moderate but not a lower dose of EtOH reduced brain infarction and improved functional outcome in rats.9 Oxygen administration has been identified as a rational strategy for stroke therapy, and normobaric oxygen (NBO) is readily available, inexpensive and can be quickly applied. However, the time window for NBO is short, and its therapeutic effect is relatively low in both transient and perma-nent focal ischemia.3,10 Considering the brain metabolic depres-sive and protective effects of EtOH, and the beneficial effects of NBO on metabolic stress after stroke, as well as the fact that both agents can be easily delivered through the blood brain bar-rier into the ischemic brain, we aimed at determining the thera-peutic effect of EtOH or EtOH+NBO in stroke by normalizing oxidative metabolism through various regulatory mediators of PDHC. Because an early reperfusion strategy may not be avail-able for most patients, and because the proposed treatments can be effectively administered and delivered to ischemic regions through the collateral circulation that remains patent in patients with stroke,11–15 we used more clinically relevant stroke models with longer ischemia periods (4 versus 2 or 3 hours) or without reperfusion (permanent stroke).

Materials and MethodsSubjectsAll experimental procedures were approved by the Institutional Animal Investigation Committee of Wayne State University in accordance with the National Institutes of Health guidelines for care and use of labora-tory animals. A total of 176 adult, male Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA) were randomly divided into the following groups such as (1) a sham-operated group without MCAO (n=8), (2) 2-hour MCAO (n=8×2), (3) 3-hour MCAO (n=8×2), (4) 4-hour MCAO (n=8×9), and (5) permanent MCAO (28 hours; n=8×8). The 2- and 3-hour MCAO groups were randomly assigned to receive either saline (sham treatment) or an intraperitoneal injection of EtOH (1.5 g/kg) at 2 hours after the onset of ischemia. For the 4-hour MCAO, rats in total 9 groups were randomly assigned to receive 3 different treatments (saline, 1 dose [1.5 g/kg] or 2 doses [1.5 g/kg at 2 hours after

the onset of MCAO followed by 1.0 g/kg in 2 hours] of EtOH), and ani-mals were analyzed at 3 time points (24 hours of reperfusion for infarct volume, 3 and 24 hours of reperfusion for protein, and biochemical measurements). For permanent stroke, rats in 8 groups were randomly assigned to receive 4 treatments (saline, 2 doses of EtOH [1.5 g/kg at 2 hours after the onset of MCAO followed by 1.0 g/kg in 2 hours], 95% NBO [at 2 hours after the onset of ischemia for 6 hours], or EtOH+95% NBO); and were processed for infarct volume and biochemical analy-ses, respectively, at 28 hours after ischemia. The mortality rate was low (<9%) and was about equal in each group. All data were analyzed in a blind manner. Animals were fed the night before.

Middle Cerebral Artery OcclusionRats were subjected to MCAO for either 2, 3, 4, or 28 hours using the intraluminal filament model.16 Reperfusion was achieved by the withdrawal of the filament at each corresponding time point as men-tioned above. Blood PCO

2 and PO

2, and mean arterial pressure were

monitored throughout the procedure. Heating lamps and pads were used to maintain rectal temperature at 36.5 to 37.5°C. See online-only Data Supplement for details.

EtOH TreatmentIn transient ischemic stroke with 2-hour MCAO, the treatment was given immediately before reperfusion both to mimic actual scenarios encountered in clinical settings and to prevent reperfusion injury. In other MCAO groups, all ischemic rats received 1 EtOH dose (1.5 g/kg IP) at 2 hours after ischemia onset (the time point with a confirmed EtOH neuroprotective effect).9 Rats in the 4 hours and permanent MCAO groups received a second EtOH dose (1.0 g/kg) at 2 hours after the first dose.

NBO TreatmentTo receive NBO treatment, rats at 2 hours after the onset of ischemia were placed in a sealed chamber (50×25×25 cm) full of 95% oxygen for 6 hours. To maintain oxygen concentration, an oxygen controller (PRO-OX110; Reming Bioinstruments Co, Redfield, NY) was used and the oxygen flow rate was 2 L/min.17 In addition, carbon dioxide was removed by placing a container of soda lime (Sigma) at the bottom of the chamber.

Combination Treatment With NBO and EtOHAt 2 hours after the onset of ischemia, animals that received IP injections of EtOH as described above were then placed into a sealed chamber full of 95% oxygen to undergo consecutive NBO treatment for 6 hours.

Neurological DeficitThe scoring system proposed by Belayev et al18 was used to exam-ine the severity of deficit in rats before surgery, during MCAO after 24-hour reperfusion or 28-hour MCAO without reperfusion.

Cerebral Infarct VolumeTwenty-four hours after reperfusion or 28 hours after MCAO without reperfusion, brains were obtained from ischemic rats and cut into a 2-mm thick slices and treated with 2, 3, 5-triphenyltetrazolium chlo-ride (Sigma) for staining. An indirect method for calculating infarct volume was used to minimize error caused by edema.9

ADP/ATP RatioBrain metabolism was measured with the BioVision ApoSENSOR Assay Kit (Mountain View, CA), as described by us.8 See online-only Data Supplement.

ROS ProductionROS was detected as described previously by us.17 See online-only Data Supplement.

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494 Stroke February 2015

Protein ExpressionWestern blot was used to detect protein levels of PDHC, PDP, and PDK. Brain tissue samples containing frontoparietal cortex and dor-solateral striatum, the MCA supplied territories, were processed as described previously by us8,19 using primary antibody incubation (PDHC, 1:250; PDP1, 1:1000; PDP2, 1:1000; PDK, 1:1000; Santa Cruz Biotechnology, Dallas, TX) at 4°C. See online-only Data Supplement.

Statistical Analysis (SPSS Software, Version 17, SPSS Inc)All the data were described as mean±SE. Differences among multiple groups were assessed using both the 1- and 2-way ANOVA with a significance level at P<0.05. Post hoc compari-son between groups was detected using the least significant dif-ference method.

ResultsPhysiological ParametersThere were no significant differences in blood pH and PCO

2 in

each group. However, a significantly (P<0.05) increased arte-rial blood PO

2 was found in the NBO-treated groups. Body and

brain temperature remained at ≈37°C. See online-only Data Supplement.

Therapeutic Window of EtOH, NBO, and Combined TreatmentIn ischemic rats with 2- or 3-hour MCAO, a single dose of EtOH (1.5 g/kg) significantly (P<0.01) decreased infarct vol-umes as compared with no-treatment groups (Figure 1A). Only a mild but nonsignificant decrease in infarct volume was obtained using 1 dose of EtOH in the 4 hours and permanent

Figure 1. Infarct volume. A, middle cerebral artery occlusion (MCAO) for 2, 3, and 4 hours, as well as permanent stroke induces substantial infarct vol-ume. EtOH administration significantly (##P<0.01) reduces infarct volume in both the 2- and 3-hour MCAO, but not in the 4 hour and the permanent MCAO groups. B, Whereas in the 4-hour MCAO group, infarct volumes were significantly (##P<0.01) reduced by 2 EtOH doses (1.5 g/kg at 2 hours after the onset of ischemia followed by 1.0 g/kg in 2 hours), treatment with 1 dose of EtOH did not significantly decrease infarct volume. C, In permanent ischemia (28-hour MCAO without reperfusion), 2-dose EtOH (1.5 g/kg at 2 hours after the onset of ischemia followed by 1.0 g/kg in 2 hours), or nor-mobaric oxygen (NBO) administration for 6 hours only slightly decreased (#P<0.05) infarct volume, whereas the combination of EtOH+NBO produced the most sig-nificant (##P<0.01) reduction. Associated with every infarct volume figure is a rep-resentative brain section with and without the treatment. Most of our treatments' effects were observed in the penumbra region, showed by reduced infarction in the peripherally perfused regions.

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Geng et al Ethanol and NBO in Severe Stroke 495

MCAO groups. Furthermore, when 2 doses of EtOH were used, a significant (P<0.01) reduction in infarct volume was observed in the 4-hour MCAO ischemic group (Figure 1B). In the permanent stroke groups, infarct volume (#P<0.05; Figure 1C) was only mildly reduced by either monotherapy with EtOH (although 2 doses) or 95% NBO (Figure 1A and C). The most significant reduction (P<0.01) in brain infarc-tion was obtained when EtOH and 95% NBO were combined (Figure 1C). Examples of 2, 3, 5-triphenyltetrazolium chloride histology show the reduction in penumbral region of ischemic territory supplied by MCA as hypothesized (Figure 1A–C). In addition, compared with control, neurological deficits in both the 2- and 3-hour MCAO groups were decreased significantly (P<0.01) after single EtOH treatment (Figure 2A). However, neither a single EtOH dose in 4-hour MCAO (Figure 2B) nor in tandem EtOH doses in permanent stroke (Figure 2C) were sufficient to reduce neurological deficits (P=0.068). Similarly after permanent MCAO, EtOH+NBO therapy again most effectively (P<0.01) diminished the neurological deficits.

Cerebral Metabolic Disorder and Its AttenuationRats in the 4-hour MCAO group showed a significant (P<0.01) increase in ADP/ATP ratio at 3 and 24 hours of reperfusion (Figure 3A), suggesting a reduced energy production. The 2-dose EtOH therapy was able to further (P<0.01) decrease this ratio. This ratio was also significantly (P<0.01) elevated in permanent stroke (Figure 3B). Conversely, the monotherapy of NBO or EtOH only mildly (P<0.05) reduced this ratio as compared with the no-treatment group. In contrast, EtOH and 95% NBO therapy additively (P<0.01) decreased this ratio.

Oxidative StressAs compared with sham-operated group, ischemia for 4 hours significantly (P<0.01) increased ROS production at 3 and 24 hours of reperfusion (Figure 4A). In tandem EtOH doses significantly (P<0.01) reduced ROS levels. Likewise, a sig-nificant (P<0.01) increase in ROS levels was observed in the permanent stroke (Figure 4B). Neither NBO nor EtOH alone was able to significantly decrease ROS. However, combined treatment induced a large (P<0.01) reduction in ROS levels, suggesting an attenuated oxidative damage.

PDHC and PDK Protein ExpressionCompared to the sham-operated group, 4-hour ischemia fol-lowed by 3-hour reperfusion caused a decrease (P<0.05) in PDHC protein expression (Figure 5A). This decrease was even more significant (P<0.01) at 24-hours reperfusion. EtOH was able to restore PDHC protein expression back to normal levels (P<0.01) at both time points. Permanent stroke significantly (P<0.01) reduced PDHC expression (Figure 5B). Although EtOH or NBO alone were only able to slightly reverse this reduction, EtOH nevertheless raised the level of PDHC expression better than NBO. In contrast, the EtOH+NBO combination substantially (P<0.01) raised PDHC expression. In correlation with decreased PDHC, PDK protein expressions were increased (P<0.01) in ischemic rats with 4-hour MCAO followed by 3- and 24-hour reperfusion, respectively (Figure 5C). In tandem EtOH doses significantly

(P<0.01) decreased PDK expression. Likewise, permanent MCAO significantly (P<0.01) increased PDK (Figure 5D). In addition, the monotherapy with NBO or EtOH only mildly reduced PDK levels. Similarly to its effects on PDHC, EtOH alone induced greater reduction in PDK as compared with NBO alone. As above, the combined treatment was successful in decreasing these levels (P<0.01).

PDP Protein ExpressionCompared to sham-operated control, PDP1 protein expres-sion after 4-hour MCAO was hardly changed at 3 and 24 hours of reperfusion (Figure 6A), whereas EtOH signifi-cantly (P<0.01) increased PDP1 levels. After permanent stroke, PDP1 expression was slightly increased (Figure 6B). Although EtOH but NBO alone slightly altered PDP1 expression, EtOH+NBO combination significantly (P<0.01) increase PDP1. In addition, we assessed another PDP pro-tein level, that of PDP2 (Figure 6C and D). The results fur-ther confirm the beneficial effects of EtOH alone or EtOH in

Figure 2. Neurological deficit. A, Ischemic stroke caused neu-rological deficits. EtOH administration significantly (##P<0.01) reduces these deficits after 2- and 3-hour middle cerebral artery occlusion (MCAO) but not 4 hours and permanent ischemia. B, Afte 4-hour MCAO, neurological deficits were significantly (##P<0.01) reversed by EtOH monotherapy with 2 but not 1 doses. C, Although 2 doses of EtOH and monotherapy of normo-baric oxygen (NBO) did not produce a significant decrease in the deficits, combination therapy with both produced a large reduc-tion (##P<0.01). PMCAO indicates permanent MCAO.

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combination with NBO in both the transient (4-hour MCAO) and permanent stroke.

DiscussionEtOH, NBO, and NeuroprotectionThis study revealed a dose-dependent neuroprotection of EtOH after transient ischemia. In permanent stroke, this neuroprotec-tion was augmented when EtOH was therapeutically combined with NBO. Because reperfusion therapy within a clinically realistic time window is not available to most patients with stroke, a goal of the study was to assess EtOH and NBO neuro-protection under severe ischemic conditions with longer time frames, in contrast to those used in a previous study with mod-erate stroke (2-hour occlusion or less) and shorter therapeu-tic window.9,17 Our analysis after treatment with EtOH alone in transient ischemia or NBO+EtOH with permanent isch-emia improved ATP production and reduced ROS generation. Although the measures here cannot definitely explain the better outcomes, our findings support a role of EtOH in stabilizing dysfunctional metabolic pathways mediated by PDHC, PDK, and PDP in ischemia. In addition, NBO may enhance EtOH effects by ameliorating ischemia-induced anaerobic metabo-lism and attenuating the generation of ROS. In this study, the infarct volume among the brains was variable; the dissected tissues may have included some undamaged tissue, which per-haps would have been rescued by the treatments. In fact, most of our treatments' effects were seen in the penumbra region.

Oxygen has been used in the treatment of stroke as a logical approach to counteract the hypoxic state induced by ischemia.3

Clinically, NBO has been shown to protect the ischemic brain from damage.20–24 However, in both transient and permanent focal ischemia, NBO has a relatively short time window and low thera-peutic effect.3,10 The recent clinical trial for NBO in acute isch-emic stroke was terminated because of unclear efficacy. There was a high mortality rate in the controlled arm of the study that was attributed to external causes (http://clinicaltrials.gov/show/NCT00414726). Thus, it is possible that NBO alone may not fully resolve ischemic injury because of its multifactorial, delete-rious mechanisms. Although EtOH therapeutic potential lies on its ability to ameliorate the metabolic and oxidative distress dur-ing ischemia, especially in the case of short transient ischemia as confirmed by us.8,9 Our present results show that EtOH alone was no longer effective in ischemia without reperfusion. The addition of oxygen as an oxidative compliment is thus deemed essential to confer neuroprotection. Finally, this study also reveals that, although when used alone EtOH effect in permanent stroke was minor, it produced better outcome than NBO on ROS, as well as on PDHC, PDK, and PDP expressions. The mechanism by which EtOH achieves its effect may be because of its ability to induce a hibernation state and minimize oxidative damage by partially inhibiting glycolysis and other metabolic pathways.7,25 These effects in turn may slow down energy depletion and extend the survival time of cells in the penumbra region.26 Thus when meta-bolic dysfunction was inhibited by EtOH, oxygen may be better able to ameliorate the hypoxic state of cells.

PDHC Mechanism in IschemiaPDHC is regarded as a central regulatory site of cellular oxi-dative metabolism in the mitochondrial matrix. PDHC sits

Figure 3. ADP/ATP ratio. A, After 4-hour ischemic stroke with 3- and 24-hour reperfusion, there was a significant (**P<0.01) increase in the ADP/ATP ratio. Two EtOH doses (1.5 g/kg fol-lowed by 1.0 g/kg) were able to significantly (##P<0.01) decrease this ratio. B, After PMCAO, there was a significant (**P<0.01) increase in ADP/ATP ratio. Monotherapy with either EtOH or nor-mobaric oxygen (NBO) alone mildly reduces this ratio (#P<0.05). When used as a combination, EtOH and NBO additively decreased this ratio (##P<0.01). MCAO indicates middle cerebral artery occlusion; and PMCAO, permanent MCAO.

Figure 4. Reactive oxygen species (ROS) production. A, In 4-h ischemic stroke followed by 3- or 24-hour reperfusion, ROS lev-els were significantly (**P<0.01) increased. Two EtOH doses (1.5 g/kg followed by 1.0 g/kg) significantly (##P<0.01) reduced ROS levels. B, In permanent middle cerebral artery occlusion (MCAO), there was a significant (**P<0.01) increase in ROS level. Admin-istered as monotherapy, neither EtOH nor normobaric oxygen (NBO) was able to significantly decrease ROS production. How-ever, combination therapy produced a great reduction (##P<0.01) in ROS levels. PMCAO indicates permanent MCAO.

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Geng et al Ethanol and NBO in Severe Stroke 497

at the cross-roads of the aerobic citric acid cycle, oxidative phosphorylation, anaerobic glycolysis, and gluconeogenesis. PDHC is a multienzyme complex composed of 6 subunits and 2 regulatory kinase molecules, PDK and PDP.5 Any dys-function of PDHC and its regulators would result in meta-bolic crisis, such as a decline in ATP and phosphocreatine, inability to generate nicotinamide adenine dinucleotide and accumulation of lactic acid.5 Specifically, targeted regulation of PDHC occurs primarily via site-specific phosphorylation.6 After ischemic stroke, a severe oxygen deprivation leads to a large impairment of oxidative phosphorylation, thus limiting the production of ATP. This, in turn, raises the production of ROS after reperfusion which exacerbates cell and tissue inju-ries.17,27 Our present results support previous study,5 where large increase in ADP/ATP ratio led to significant rise in ROS production, which in turn exacerbates impairment of PDHC expression. Other studies have shown that PDHC deficiency is related to decreased activity of critical enzymes required for free radical removal, such as MnSOD.28 As such, there may be a spiraling feedback cycle where ROS could inactivate PDHC

which then would further augments ROS production in mito-chondria. Using ATP production as an index here, decreased ROS generation by EtOH in transient or EtOH+NBO in per-manent stroke suggests an overall better cellular metabolic status. We, therefore, would advance that EtOH and NBO may be clinically useful to restabilize cellular metabolism by modulating 2 pathways of ROS damage—the direct pathway for ROS generation and an indirect one for ROS generation via PDHC.

PDHC and Its RegulatorsBecause PDK and PDP are major regulators of PDHC, another major goal of the study was to assess how these regulators might be affected in severe ischemia, and how EtOH and NBO therapies could affect the regulators. Our results on increase in PDK protein expression after ischemia confirm previous stud-ies showing a rise in PDK expression from ischemia as a signal of low energy state pushing the cell to use fatty acids or ketone bodies as a major energy source.29 Our therapy decreased the levels of PDK protein, which led to disinhibition of PDHC

Figure 5. Pyruvate dehydrogeanse complex (PDHC) and pyruvate dehydrogenase kinase (PDK) protein expressions. A, PDHC expres-sion was decreased (*P<0.05) in 4-hour ischemia followed by 3- and 24-hour reperfusion as compared with the sham-operated group referenced as 1. Treatment with 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg) significantly (##P<0.01) increased PDHC protein expres-sion at both time points. B, Permanent MCAO caused a significant (**P<0.01) reduction in PDHC protein expression. A slight (#P<0.05) increase in expression of PDHC was observed in the EtOH-only and normobaric oxygen (NBO)-only treated groups. However, combi-nation of EtOH+NBO largely increased (##P<0.01) PDHC expression. C, There was a significant (**P<0.01) increase in PDK expression from rats with 4-hour ischemia followed by 3- and 24-hour reperfusion. Using 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg) led to a significant (##P<0.01) reduction at both time points. D, After permanent middle cerebral artery occlusion (MCAO), there was a significant (**P<0.01) increase in PDK protein levels. EtOH but not NBO alone slightly (#P<0.05) reduced this expression. The combined treatment significantly (##P<0.01) reversed the reduction in PDK expression. Representative immunoblots are illustrated. PMCAO indicates perma-nent MCAO.

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by increasing its expression, thus helping to stabilize the cell after ischemic insult. In addition, we observed a small increase in the expression of PDP1 and PDP2 after ischemia, even before treatment. These findings stand in contrast to the PDP isoenzyme declines previously observed after traumatic brain injury.30 Such increases could be a mild compensatory mechanism whereby the cell attempts to self-modulate and stabilize the concentration of PDHC, although this compensa-tory mechanism seems to be insufficient. Our therapy (EtOH or EtOH+NBO), however, was able to further increase PDP levels beyond its compensatory physiological levels, leading to neuroprotection. Taken together, these results strongly sup-port the concept that anaerobic metabolism after stroke may have been stabilized through the effects of PDHC expres-sion by EtOH or EtOH+NBO which led to decreased PDK, whereas simultaneously increasing PDP levels.

Clinical Potential of EtOH and NBO TherapyCollateral perfusion, widely recognized to remain functional after stroke, may exert a dramatic effect on the time course

of ischemic injury, stroke severity, imaging findings, as well as therapeutic opportunities and subsequent neurological out-comes after stroke.11–15 Because EtOH and NBO are widely available, readily cross the blood brain barrier and easily dif-fuse through the collateral circulation into ischemic regions, even before reperfusion is established, their clinical potential as therapeutics is apparent. Future implementation of EtOH and NBO in a clinical setting may move us closer toward the development of an efficacious neuroprotective therapy.

Although the primary focus here is centered on treatment effects on ischemic brain tissues, additional mechanistic studies are needed to assess the correlation between functional outcomes and the regulatory roles of PDP and PDK. At a more fundamen-tal level, future studies will also aim to determine the cause/effects relations between EtOH/NBO outcomes and PDHC and its regulators in ischemic as well as nonischemic brain tissues.

Sources of FundingThis work was partially supported by Wayne State University Neurosurgery Fund, American Heart Association Grant-in-Aid

Figure 6. Pyruvate dehydrogenase phosphatase (PDP) protein expression. A, Compared to the sham-operated group referenced as 1, there was no change in PDP1 level in rats with 4-hour ischemia followed by 3- or 24-hour reperfusion. EtOH significantly (##P<0.01) increased the expression of PDP1 to levels more than its normal physiological ones, at both reperfusion time points. B, After permanent stroke, there was a small increase in the expression of PDP1 in the stroke group; EtOH but not normobaric oxygen (NBO) slightly further increased (#P<0.05) these levels. Combined (EtOH+NBO) treatment largely (##P<0.01) increased the protein expression. C, There was a small (#P<0.05) increase in PDP2 levels in 4-hour ischemia followed by 3- or 24-hour reperfusion. PDP2 levels were further increased (##P<0.01) by ETOH. D, Again, a slight (#P<0.05) increase was observed in permanent stroke after monotherapy of EtOH and NBO. This increase was significantly (##P<0.01) enhanced by the NBO+EtOH combination. Representative immunoblots are illustrated. MCAO indi-cates middle cerebral artery occlusion; and PMCAO, permanent MCAO.

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(14GRNT20460246), National Basic Research Program of China (973 Program, No. 2011CB707804), and National Outstanding Youth Science Fund of China (No. 81325007).

DisclosuresNone.

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by guest on June 27, 2016http://stroke.ahajournals.org/Downloaded from

Huishan Du, Jose A. Rafols and Yuchuan DingXiaokun Geng, Omar Elmadhoun, Changya Peng, Xunming Ji, Adam Hafeez, Zongjian Liu,

Dehydrogenase Complex After Severe Transient and Permanent Ischemic StrokeEthanol and Normobaric Oxygen: Novel Approach in Modulating Pyruvate

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1

SUPPLEMENTAL MATERIAL

Materials & Methods

MCA Occlusion (with additional information). Rats were subjected to MCAO for either

2, 3, 4, or 28 h using the intraluminal filament mode1. Briefly a 4.0 nylon suture with a blunted

tip coated with poly-L-lysine was inserted into the right external carotid artery and lodged in the

narrow proximal anterior cerebral artery to block the MCA at its origin. Reperfusion was

achieved by the withdrawal of the filament at each corresponding time point as mentioned above.

Blood pCO2 and pO2, mean arterial pressure (MAP), as well as rectal and brain temperature were

monitored throughout the procedure. In addition, heating lamps and pads were utilized to

maintain rectal temperature at 36.5-37.5 °C.

ADP/ATP Ratio (with additional information). Brain metabolism was measured with

the BioVision ApoSENSOR Assay Kit (CA, USA)2. Briefly right cerebral hemispheres of the

rats were extracted and homogenized in cold PBS buffer. Next, the sample (10 µL) was

transferred into a luminometer plate and 100 µL of the Nucleotide Releasing Buffer was added.

The mixture was then incubated for 10 min at room temperature with gentle shaking and brain

ATP levels in the brain were measured by adding 1 µL of the ATP Monitoring Enzyme into the

brain cell lysate. A luminometer (DTX 880 Multimode Detector, Beckman Coulter) was used to

read the samples after 1 min (Data A). After 10 mins, ADP levels were measured again (Data

B). One µL of ADP Converting Enzyme was then added and the samples were read once more

after 1 min (Data C). ADP/ATP ratio was calculated as: (Data C – Data B)/Data A.

ROS Production (with additional information). The method for detection of ROS was

described previously by us3. This method tests H2O2 levels with hydrogen peroxidase linked to a

fluorescent compound. Brain samples taken from the animals were diluted to 10 mg/ml based on

protein concentration (BCA method) and incubated for 30 min after 100 µg/ml of digitonin

addition. H2O2 levels in brain homogenates were determined using 50 µM Amplex red, 0.1 U/ml

horse radish peroxidase (HRP), and respiratory substrates (4 mM pyruvate, 2 mM malate, 2 mM

glutamate, and 0.8 µM complex V inhibitor oligomycin) at 37ºC on a DTX-880 multimode

detector.

Protein Expression (with additional information). Western Blot analysis was used to

detect protein levels of PDHC, PDP, and PDK. Briefly, extracted protein from the brain issue of

fronto-parietal cortex and striatum were loaded onto a single 10% sodium dodecyl sulfate-

polyacrylamide gel for electrophoresis. Forty µg of protein was loaded per well. Samples were

transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). Primary

antibody incubation (PDHC, 1:250; PDP1, 1:1000; PDP2, 1:1000; PDK, 1:1000, Santa Cruz

Biotechnology, Dallas, Texas) was then carried out overnight at 4°C. Secondary antibody

incubation (anti-goat, anti-rabbit, 1:1250; 1:5000; 1:5000; 1:5000, Santa Cruz Biotechnology,

Dallas, Texas) was done for one hour at room temperature. An ECL system was used to detect

immunoreactive bands by luminescence and to quantify protein expression profiles, with the

relative density of Western blot images being obtained by using an image analysis program

(ImageJ 1.48, National Institutes of Health, USA). As a reference, the mean amount of protein

expression from the sham-operation group was assigned a value of 1. The expressions of target

proteins were represented as fold-differences compared to the control.

2

Results

Physiological parameters (Tables Ⅰ and Ⅱ). There were no significant difference in blood pH

and pCO2 in each group. However, a significantly (P<0.05) increased arterial blood pO2 was

found in the NBO-treated groups. Body and brain temperature remained at about 37 °C.

Table Ⅰ. Physiologic Parameters during Transient MCA Occlusion.

Stroke

(2h)

Stroke (2h)

and EtOH

(1.5g/kg)

Stroke(3h)

Stroke (3h)

and EtOH

(1.5g/kg)

Stroke

(4h)

Stroke (4h)

and EtOH

(1.5g/kg)

Stroke (4h)

and EtOH

(1.5+1.0g/kg)

MAP

Pre MCAO 88.9±2.9 84.6±3.7 87.4±2.8 83.3±4.2 88.8±3.7 86.1±3.2 81.7±3.9

Pre reperfusion 88.1±2.3 85.1±2.4 86.6±2.3 87.0±2.6 83.5±3.8 89.3±3.1 90.0±4.5

2h after reperfusion 80.2±3.1 84.5±2.6 86.8±2.7 83.8±3.5 82.9±4.4 88.7±3.6 85.1±2.5

pH

Pre MCAO 7.40±0.02 7.38±0.02 7.39±0.02 7.38±0.01 7.38±0.03 7.36±0.02 7.40±0.02

Pre reperfusion 7.41±0.03 7.36±0.02 7.40±0.02 7.39±0.02 7.38±0.03 7.35±0.03 7.39±0.02

2h after reperfusion 7.39±0.02 7.39±0.02 7.38±0.03 7.37±0.02 7.35±0.03 7.40±0.02 7.41±0.03

PO2

Pre MCAO 134.6±5.5 130.9±6.4 137.0±4.2 140.9±7.1 132.2±4.9 133.6±6.4 135.9±5.2

Pre reperfusion 131.1±6.6 136.9±3.7 133.9±4.8 131.6±6.1 134.6±5.8 135.9±4.0 139.1±6.3

2h after reperfusion 129.2±9.9 137.1±8.2 141.1±6.9 135.2±4.8 134.7±4.7 130.1±3.5 137.0±5.5

PCO2

Pre MCAO 44.5±1.2 45.5±2.1 47.0±2.1 43.9±2.2 48.0±4.1 45.5±2.9 48.0±2.0

Pre reperfusion 43.3±3.7 42.8±1.5 44.8±2.2 46.7±1.9 42.4±2.6 44.0±1.7 43.0±3.7

2h after reperfusion 41.1±5.7 49.3±2.3 43.0±1.0 46.6±4.5 45.2±2.3 42.1±3.5 47.8±4.3

3

Table Ⅱ. Physiologic Parameters During Permanent MCA Occlusion. Stroke

Stroke and

EtOH (1.5g/kg)

Stroke and EtOH

(1.5+1.0g/kg)

Stroke and

NBO

Stroke and EtOH

(1.5+1.0g/kg)/NBO

MAP

Pre MCAO 84.2±2.1 87.6±3.1 87.4±2.2 90.1±2.6 84.8±2.3

2h after MCAO 89.1±3.3 82.6±2.7 85.5±2.7 83.3±4.2 87.0±2.6

8h after MCAO 86.2±2.9 83.6±2.2 89.1±3.7 84.3±2.8 82.2±2.9

pH

Pre MCAO 7.39±0.02 7.35±0.05 7.36±0.02 7.40±0.03 7.38±0.02

2h after MCAO 7.41±0.02 7.36±0.02 7.37±0.01 7.37±0.03 7.39±0.02

8h after MCAO 7.35±0.02 7.37±0.01 7.40±0.03 7.35±0.02 7.40±0.01

PO2

Pre MCAO 134.9±5.7 138.9±6.1 133.9±4.3 132.9±7.5 132.5±5.2

2h after MCAO 131.5±6.7 134.9±4.4 138.3±5.1 134.9±6.1 141.6±7.4

8h after MCAO 139.1±7.7 135.8±6.2 129.1±7.8 419.6±19.1* 434.1±19.9*

PCO2

Pre MCAO 45.5±1.6 47.1±1.9 45.3±2.1 48.0±2.0 44.4±1.8

2h after MCAO 43.9±2.7 47.8±1.5 44.8±2.2 43.3±2.5 46.0±2.2

8h after MCAO 48.1±4.3 43.9±2.2 47.4±1.9 43.1±2.3 46.4±1.9

Physiologic parameters of Mean Arterial Pressure (MAP), blood PCO2, PO2, and pH were

recorded before, during, and after permanent MCA occlusion. * indicates P<0.01 as compared to

other groups.

Reference

1. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion

without craniectomy in rats. Stroke. 1989;20:84-91

2. Kochanski R, Peng C, Higashida T, Geng X, Huttemann M, Guthikonda M, et al.

Neuroprotection conferred by post-ischemia ethanol therapy in experimental stroke: An inhibitory

effect on hyperglycolysis and nadph oxidase activation. J Neurochem. 2013;126:113-121

3. Geng X, Fu P, Ji X, Peng C, Fredrickson V, Sy C, et al. Synergetic neuroprotection of normobaric

oxygenation and ethanol in ischemic stroke through improved oxidative mechanism. Stroke.

2013;44:1418-1425