Address correspondence and reprints requests to Yuchuan Ding, MD, PhD, Department of Neurological Surgery Wayne State University School of Medicine 550 E Canfield, Detroit, MI 48201, USA. Email: firstname.lastname@example.org
Ethanol provides neuroprotection following ischemia/reperfusion. This study assessed ethanol's effect on hyperglycolysis and NADPH oxidase (NOX) activation. Adult, male Sprague–Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Three sets of experiments were conducted to determine ethanol's effect on (i) conferring neuroprotection by measuring infarct volume and neurological deficits 24 h post reperfusion; (ii) cerebral glucose metabolism and lactic acidosis by measuring brain and blood glucose concentrations and protein expression of glucose transporter 1 and 3 (GLUT1, GLUT3), phosphofructokinase (PFK), as well as lactic acidosis by measuring lactate dehydrogenase (LDH), and lactate; and (iii) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation by detecting enzymatic activity and subunit expression at 3 h after reperfusion. When administered upon reperfusion, ethanol (1.5 g/kg) reduced infarct volume by 40% (p < 0.01) and neurological deficits by 48% at 24 h post reperfusion while reducing (p < 0.01) elevations in glycolytic protein expression and lactate levels during early reperfusion (3 h). Ethanol increased the reductions in cerebral glucose concentration at 3 h post reperfusion by 64% (p < 0.01) while enhancing (p < 0.01) post stroke blood glucose concentration, suggesting a reduced cellular glucose uptake and utilization. Ethanol decreased (p < 0.01) stroke-induced NOX activation by reducing enzymatic activity and gp91phox expression by 45% and 38%, respectively. Post-ischemia ethanol treatment exerts neuroprotection through attenuation of hyperglycolysis and associated NOX activation. Because of the lack of associated hypoglycemia and selectivity toward decreasing cerebral metabolism, further investigation of ethanol's use as a post-stroke therapy, especially in the context of hyperglycemia, seems warranted.
Thrombolytics are currently the only FDA approved treatment for stroke. However, because of their limited therapeutic time window, prompt administration benefits only apply to a small percentage of patients, demonstrating the need for additional therapeutic options (Hacke et al. 2004). Moderate alcohol consumption has been shown to exhibit neuroprotection by preconditioning the brain against possible future ischemic insults (Wang et al. 2007), but few studies have addressed the neuroprotective potential of ethanol when administered after the onset of ischemia. We recently demonstrated that administration of a moderately high dose of ethanol (1.5 g/kg) at up to 4 h after ischemia significantly decreases brain infarct volume and improves functional outcome in a rat ischemia/reperfusion model (Wang et al. 2012). However, mechanisms underlying ethanol-induced neuroprotection remain to be elucidated.
An increase in glucose uptake and metabolism relative to the rate of oxygen utilization, also known as hyperglycolysis, has been demonstrated within the penumbra of the ischemic brain (Sako et al. 1985, Yao et al. 1995, Tohyama et al. 1998). Hyperglycolysis is believed to be detrimental to the survival of the penumbra as it leads to lactic acidosis and overproduction of reactive oxygen species (ROS) (Parsons et al. 2002, Kruyt et al. 2010). During ischemia/ reperfusion, excess utilization of glucose, especially in the context of hyperglycemia, is associated with an increase in lactic acidosis ultimately leading to reduced penumbral salvage (Parsons et al. 2002, Vannucci et al. 2005). Furthermore, activation of NADPH oxidase (NOX), a superoxide-producing enzyme found within leukocytes and brain cells has also been shown to exacerbate ischemia/reperfusion injury through the overproduction of deleterious ROS (Tang et al. 2012). NOX is dependent on glucose metabolism, and increased availability and catabolism of glucose has been shown to further increase ROS production by NOX (Suh et al. 2008). Taken together, these studies suggest that rapid restoration and subsequent uptake and utilization of glucose by previously ischemic tissue can be harmful. To elucidate mechanisms underlying ethanol-induced neuroprotection, we sought to determine the beneficial effect of ethanol in decreasing lactic acidosis and NOX activity through suppression of hyperglycolysis following ischemia/reperfusion injury. Therefore, in this study, we measured both brain and blood glucose concentrations and expression of several key glycolytic proteins including glucose transporter 1 and 3 (GLUT1, GLUT3) and phosphofructokinase (PFK). The degree of lactic acidosis was assessed by measuring cerebral lactate concentration and lactate dehydrogenase (LDH) expression. Enzymatic activity of NOX and expression of proteins that comprise its catalytic site were also measured. ADP/ATP ratios were measured to assess the overall metabolic viability of cerebral tissue.
Materials and methods
All experiments were approved by the Institutional Animal Investigation Committee of our institution and were in accordance with National Institutes of Health guidelines for care and use of laboratory animals. A total of 48 adult (280–300 g) male Sprague–Dawley rats (Charles River, Wilmington, MA, USA) were randomly divided into a sham-operated group and two stroke groups (n = 16 per group), including a saline-treated group and an ethanol-treated group receiving 1.5 g/kg of ethanol [the dose found to be most efficacious in our previous study (Wang et al. 2012)] after 2 h of MCAO followed by 3 h (24 rats for biochemical assays, n = 8 per group) or 24 h (24 rats for infarct/behavioral analysis, n = 8 per group) of reperfusion. The data were analyzed in a researcher-blinded manner.
Focal cerebral ischemia
Rats were subject to a 2 h MCAO using the intraluminal filament model (Longa et al. 1989). Briefly, a 4-0 nylon suture with blunted tip coated with poly-l-lysine was inserted into the right external carotid artery and lodged in the narrow proximal anterior cerebral artery resulting in the blockage of the MCA at its origin. Two hours after the MCA occlusion, reperfusion was initiated by withdrawing the filament. No MCA occlusion was performed in the sham-operation group. Physiologic parameters including blood pCO2, pO2, mean arterial pressure as well as rectal and brain temperature were monitored during the procedure. Heating lamps and pads were used to maintain a consistent rectal temperature between 36.5°C and 37.5°C.
Rats in the experimental stroke groups were administered either ethanol (1.5 g/kg) diluted to 3.0 mL in normal saline or normal saline (3.0 mL) by intraperitoneal injection immediately after ischemia. The pharmacokinetics of an intraperitoneal injection of 1.5 g/kg ethanol has been previously studied in rats, and this dose has been shown to establish a blood concentration of approximately 150 mg/dL at 30 min, which then declines to approximately 90 mg/dL at 3 h post injection (da-Silva et al. 1996; Walker & Ehlers, 2009). Animals were killed at 3 h post reperfusion for molecular analyses or at 24 h post reperfusion for infarct volume analysis and neurobehavioral testing.
Measurement of infarct volume
Twenty-four hours after reperfusion, both untreated and ethanol-treated stroke group rats (n = 8 per group) were injected with 120 mg of pentobarbital and the forebrain was quickly removed and sliced into 2-mm-thick coronal sections. Six coronal brain sections were cut from freshly obtained cleaned tissue and stained with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St Louis, MO, USA). All slices were incubated for 30 min in a 2% solution of TTC at 37°C and fixed in 10% paraformaldehyde solution. To minimize error introduced by edema, an indirect method for calculating infarct volume was used (Wang et al. 2012). Infarct volume was expressed as a percentage in comparison with the non-infarcted contralateral cerebral hemisphere.
Rats in both the untreated and ethanol-treated stroke groups (n = 8 per group) were examined based on the scoring system proposed by Belayev et al. (1996), which is composed of the postural reflex test described by Bederson et al. (1986). The upper body posture was assessed while rat was suspended by its tail with scoring as follows: 0, no observable deficit; 1, limb flexion during hang test; 2, deficit on lateral push and the forelimb placing test by De Ryck et al. (1989). Assessed forelimb placing responses to visual, tactile, and proprioceptive stimuli with scoring as follows: 0, complete immediate placing; 1, incomplete and/or delayed placing (< 2 s); 2, absence of placing. All neurological evaluations were done by a researcher blinded to the animal groupings. Scoring was performed using a scale from 0 (normal) to 12 (maximal deficits).
Cerebral and blood glucose measurements
The concentrations of glucose were detected using Glucose Assay Kits (BioVision Research Products, Mountain View, CA, USA) according to manufacturer's protocols. Cerebral glucose measurements were recorded at 3 h following reperfusion. Right cerebral hemispheres, including frontoparietal cortex and striatum supplied by MCA in the rats, were extracted and homogenized in cold phosphate-buffered saline (PBS) buffer. Two microliters of homogenized brain was transferred into a luminometer plate and 46 μL of Glucose Assay Buffer and 2 μL of Glucose Enzyme Mix were also added. The mixture was incubated at 37°C for 30 min. Measurements were performed using a DTX-880 multimode detector (Beckman Coulter, Brea, CA, USA) at absorbance of 570 nm. Blood glucose measurements were recorded at three time points: (i) prior to ischemic onset; (ii) at the end of the 2 h ischemic period, immediately prior to occlusive filament removal; and (iii) at 3 h following reperfusion.
Brain metabolism was measured using the BioVision ApoSENSOR Assay Kit. Right cerebral hemispheres of the rats were extracted and homogenized in cold PBS buffer. Ten microliters of the homogenized brain was transferred into a luminometer plate and 100 μL of the Nucleotide Releasing Buffer was added to the sample which was incubated for 10 min at 20°C with gentle shaking. ATP levels in the brain were measured by adding 1 μL of the ATP Monitoring Enzyme into the brain cell lysate. The samples were read in a luminometer (DTX 880 Multimode Detector, Beckman Coulter) after 1 min (Data A). ADP levels were measured by reading the samples again after 10 min (Data B). One microliter of ADP Converting Enzyme was then added and the samples were read again after 1 min (Data C). ADP/ATP ratio was calculated as: (Data C−Data B)/Data A.
Brain lactate levels were detected at 3 h following reperfusion using the Lactate Assay Kit (BioVision Research Products) according to manufacturer's protocol. Right cerebral hemispheres were extracted and homogenized in Lactate Assay Buffer and 46 μL was added to each luminometer plate along with 2 μL of Lactate Substrate Mix and 2 μL of Lactate Enzyme Mix. Samples were incubated at 20°C for 30 min. Measurements were performed using a DTX-880 multimode detector at O.D. 450 nm.
NADPH oxidase activity
Right cerebral hemispheres were processed as previously described (Liu et al. 2008). Briefly, samples were homogenized in PBS at pH 7.4 (120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 2.2 mM CaCl2, 0.15 mM Na2HPO4, 0.4 mM KH2PO4, 20 mM HEPEs, 5 mM NaHCO3, and 5.5 mM glucose) with phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Twenty microliters of homogenate was added to a 96-well luminescence plate then, 80 μL PBS with 6.25 μM lucigenin was added to each well. The reaction was initiated by the addition of NADPH (100 μM). Luminescence was recorded every 30 s for 5 min using the DTX-880 multimode detector.
Western analysis was used to assess protein expression of GLUT1, GLUT3, PFK, LDH, gp91phox and p22phox. Cerebral hemisphere (right) samples consisting of frontoparietal cortex and striatum were processed using the desired antibodies, which included primary polyclonal rabbit anti-PFK (1 : 2000; Santa Cruz Biotechnology, Inc.), primary polyclonal rabbit anti-GLUT1 (1 : 2000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), primary monoclonal mouse anti-GLUT3 (1 : 1000; Santa Cruz Biotechnology, Inc.), primary polyclonal rabbit anti-LDH (1 : 200; Santa Cruz Biotechnology, Inc.), primary polyclonal goat anti-gp91phox (Santa Cruz Biotechnology, Inc.), primary goat anti-p22phox (1 : 2000; Santa Cruz Biotechnology, Inc.). Equal protein loading was confirmed and adjusted by intracellular protein β-actin (goat polyclonal anti-β-actin antibody, 1 : 1000, Santa Cruz Biotechnology, Inc.). Targeted antigens were visualized using standard chemical luminescence methods (Amersham ECL, GE Healthcare BioSciences, Piscataway, NJ, USA). Quantification of relative target protein expression was performed using the program ImageJ 1.42 (NIH, Bethesda, MD, USA).
All values are expressed as means ± SE. Statistical significance was determined by analysis of variance supplemented by post hoc test (Duncan multiple range, SPSS software, Version 17, IBM, Armonk, NY, USA) for comparison of multiple groups. Infarct volume between two groups was analyzed by Student's t-test. A non-parametric Krukal–Wallis test was used for behavioral studies. A value of p < 0.05 was considered statistically significant.
No significant discrepancies in pO2, pCO2, or blood pH were noted in ischemic rats either receiving or not receiving ethanol treatment. Body and brain temperature remained at approximately 37°C throughout the course of the experiments. No hypothermic action was observed.
Infarct volume and neurobehavioral outcome
Infarct volume percentage was significantly (p < 0.01) reduced by ethanol treatment (1.5 g/kg) at 24 h post reperfusion. The ethanol-treated group (n = 8, 27.6 ± 2.0%) showed ~40% reduction in infarct in comparison with the untreated stroke group (n = 8, 45.2 ± 6.7%) (Fig. 1a). Neurobehavioral testing performed at 24 h post reperfusion showed significant (p < 0.01) improvement by 48% in neurological score in the ethanol-treated group compared with the untreated stroke group (4.4 ± 0.3 vs. 8.4 ± 0.7; Fig. 1b, n = 8).
Effect of ethanol treatment on hyperglycolysis
Cerebral and blood glucose concentration
A significant (n = 8, F[2,15] = 8.7, p < 0.01) reduction in cerebral glucose levels was seen in the untreated stroke group at 3 h after reperfusion compared with control. This decrease was partially but significantly (p < 0.01) ameliorated by post-stroke ethanol treatment (Fig. 2a). Blood glucose levels were significantly (n = 8, F[5,36] = 4.4, p < 0.05) increased during ischemia (Fig. 2b) as well as 3 h after ischemia (n = 8, F[2,15] = 11.9, p < 0.05) compared with the control levels, suggesting a hyperglycemic condition after stroke. In the ethanol-treated group, blood glucose levels were significantly (p < 0.01) higher compared with the untreated stroke group at 3 h following reperfusion, suggesting a reduced cellular glucose uptake and utilization.
GLUT1 and GLUT3
Protein expressions of the two major glucose transporters found in cerebral tissue were measured using western blot analysis. A significant (n = 8, F[2,16] = 44.8, p < 0.01) increase in protein expression of GLUT1 (Fig. 2) and GLUT3 (n = 8, F[2,15] = 42.2, p < 0.01; Fig. 3) was seen at 3 h after reperfusion compared with the control group. Ethanol treatment significantly (n = 8 per group, p < 0.01) attenuated the increases in protein expression of both GLUT1 and GLUT3 compared with the untreated stroke group.
Protein expression of PFK, the major rate-limiting enzyme of glycolysis, was measured using western blot analysis. A significant (n = 8, F[2,20] = 13.6, p < 0.01) elevation in PFK expression, measured at 3 h after reperfusion, was observed in the untreated stroke group (Fig. 3). Ethanol treatment significantly (p < 0.01) attenuated this elevation in PFK expression.
The overall metabolic viability of cerebral tissue was assessed by measuring the ADP/ATP ratio. The ratio was significantly (n = 8, F[2,23] = 17.8, p < 0.01) elevated in the untreated stroke group (Fig. 4) at 3 h after reperfusion compared with control, indicating a decrease in production and/or an increase in ATP utilization. Ethanol treatment significantly (p < 0.01) decreased the ratio (Fig. 4). This is indicative of preservation of ATP levels by ethanol following ischemia/reperfusion injury.
Effect of ethanol treatment on lactic acidosis
The end product of anaerobic metabolism was measured to assess levels of lactic acidosis following ischemia/reperfusion. Lactate concentration measured at 3 h following reperfusion was significantly (n = 8, F[2,20] = 11.8, p < 0.01) elevated compared with the control group (Fig. 5a).This increase in lactate concentration was partially but significantly (p < 0.01) ameliorated by ethanol treatment.
LDH protein expression
Western blot analysis was used to measure the protein expression of LDH, the important enzyme involved in anaerobic glycolysis. A significantly (n = 8, F[2,20] = 41.2, p < 0.01) increased level of LDH expression (Fig. 5b) was seen in the untreated stroke group compared with control. This increase was significantly (p < 0.01) suppressed by ethanol treatment.
Effect of ethanol treatment on NADPH oxidase
Enzymatic activity of NOX was significantly (n = 8, F[2,15] = 6.5, p < 0.01) increased compared with control at 3 h after reperfusion in ischemic rats (Fig. 6a). This increase in NOX activity was partially, but significantly (p < 0.01) attenuated in the ethanol-treated group compared with the untreated stroke group.
NOX protein expression
Expression of gp91phox and p22phox, two proteins that comprise NOX, were measured using western blot. Protein levels of gp91phox in the untreated stroke group were significantly (n = 8, F[2,16] = 12.3, p < 0.01) elevated compared with the control group at 3 h after ischemia (Fig. 6b). Ethanol treatment significantly (p < 0.01) suppressed this increase in the gp91phox expression compared with the untreated stroke group at 3 h after ischemia. No significant differences were seen in p22phox expression in the untreated stroke group compared with control (n = 8, F[2,11] = 1.2, p = 0.18) (Fig. 6b) and ethanol treatment did not have a significant effect on p22phox expression compared with the untreated stroke group (p = 0.78).
In this study, ethanol provided significant neuroprotection as shown by reductions in both infarct volume and neurological deficits while also decreasing key metabolic proteins involved in glucose uptake and utilization following ischemia/reperfusion injury. This was associated with an increase in both cerebral and blood glucose concentrations in the ethanol-treated group compared with the untreated stroke group. Furthermore, a decrease in lactic acidosis and NOX activation with subsequent preservation of energy production as shown by the ADP/ATP ratio was observed.
Numerous models of ischemia/reperfusion injury have demonstrated elevations in glucose utilization at up to 6 h after the onset of reperfusion (Sako et al. 1985, Yao et al. 1995, Nedergaard et al. 1986, Shiraishi et al. 1989). Hyperglycolysis is defined as an increase in glucose uptake and metabolism relative to the rate of oxygen utilization and is believed to be detrimental because it leads to uncoupling of glycolysis and oxidative phosphorylation and a resultant increase in lactic acidosis (Sako et al. 1985, Parsons et al. 2002, Kruyt et al. 2010, Kelly et al. 2000). Acute ethanol intoxication has a well-documented depressive effect on cerebral metabolism and has specifically been shown to reduce both glucose utilization and ATP consumption even at low doses in healthy subjects (Volkow et al. 1990, Volkow et al. 2006). Moderately high doses have also been shown to attenuate hyperglycolysis and confer neuroprotection in traumatic brain injury (Kelly et al. 2000). This study suggests that ethanol (1.5 g/kg) ameliorates hyperglycolysis following ischemia/reperfusion injury as demonstrated by elevated cerebral glucose concentrations with corresponding decreases in GLUT1, GLUT3, PFK, lactate and LDH in animals receiving ethanol on reperfusion. We chose to measure the concentrations of these proteins at 3 h following onset of reperfusion, because it was well within the time interval of hyperglycolysis, which as stated above, has been shown to be present up to 6 h following reperfusion in animal models. Our previous study demonstrated ethanol-induced neuroprotection when ethanol was administered up to 4 h following ischemic onset. It is likely that we would also see significant changes in protein concentrations if measurements were performed following ethanol therapy at any time point within the previously established hyperglycolytic time window. GLUT1 is predominantly found in capillary endothelial cells and the astrocyte end feet that form the blood–brain barrier (BBB) and serves as the predominant control over basal glucose transport (Simpson et al. 2007), while GLUT3 is the major glucose transporter expressed in neurons (Maurer et al. 2006). The levels of expression of both transporters are regulated according to regional rates of cerebral glucose utilization, and elevations of GLUT1 and 3 have been shown at the gene and protein levels after hypoxia/ischemia (Vannucci et al. 1998). Because transport of glucose across the BBB into neurons and glia has been shown to be a significant rate-limiting step in glycolysis (Vannucci et al. 2005), decreased expression of GLUT1 and 3 supports the notion that glucose uptake is depressed by ethanol. PFK is the major rate-limiting enzyme in glycolysis and is up-regulated during times of metabolic demand (Lowry et al. 1964, Minchenko et al. 2003). The decreased expression of PFK in ethanol-treated animals suggests a reduction of hyperglycolysis. In addition, ATP breakdown products such as ADP, AMP, and Pi stimulate PFK, and under conditions of ischemia/reperfusion, PFK is maximally stimulated because of falling ATP levels and increased accumulation of ADP (Vannucci et al. 2005, Bachelard 1970). The increased cerebral glucose concentration combined with the decreased expression of PFK in ethanol-treated animals suggests improvement of ATP levels following ischemia, which ultimately leads to a decrease in demand for glucose metabolism. The low ADP/ATP ratio seen in rats treated with ethanol is consistent with metabolic viability and is therefore suggestive of a balance between glycolysis and oxidative phosphorylation, whereas high ratios are seen in cells that are forced to undergo apoptosis or necrosis because of energy failure (Bradbury et al. 2000). Furthermore, lactate concentration and LDH expression were both reduced by ethanol. LDH converts pyruvate to lactate during conditions of hypoxia, and accumulation of lactate is believed to exacerbate acidosis and cell death (Rossignol et al. 2003, Siesjo 1988). Taken together, these results indicate that ethanol attenuates the increase in glucose uptake and utilization following stroke resulting in decreased lactic acidosis and preservation of energy production. Because we used cerebral hemisphere homogenates, we were unable to distinguish hyperglycolytic activity among varying cell types within various brain regions. It is likely that hyperglycolysis is occurring predominantly in astrocytes because of their greater propensity to elevate their glycolytic rate at elevated glucose concentrations (Hertz 2008). Future studies are certainly indicated to determine precisely how ethanol impacts specific neuronal cell types.
In our study, we observed transient hyperglycemia both during and after ischemia in animals that did not have elevated blood glucose levels prior to the initial ischemic onset. The cause of the increase in blood glucose is thought to be because of an increase in catecholamine and corticosteroid release brought on by activation of the hypothalamic–pituitary–adrenal (HPA) axis during ischemic stress (Kruyt et al. 2010, Vanhorebeek et al. 2006). Corticosterone-mediated inhibition of glucose uptake in neurons has been shown to correlate with adverse outcomes following stroke by increasing ischemia-induced neuronal death (Sapolsky et al. 1996, Sugo et al. 2002). Ethanol's neuroprotective effect in this study appears to be multifactorial, consisting of not only decreased glucose uptake into neurons but also decreased glycolytic catabolism and attenuation of NOX during reperfusion. Thus, it is unlikely that ethanol's protective effect is through an increase in the stress hormone response following stroke.
Hyperglycemia has a well-documented adverse effect on ischemia/reperfusion injury, and increased blood glucose levels can perpetuate hyperglycolysis (Kruyt et al. 2010, Kagansky et al. 2001, Li et al. 2001). It has been suggested that anaerobic glycolysis under hyperglycemic conditions leads to the accumulation of lactic acid and dysfunctional pH regulation, both of which are believed to contribute to increased brain injury (Kruyt et al. 2010, Katsura et al. 1992, Siesjo 1988). Hyperglycemia also positively correlates with elevations in cerebral lactate concentration and reduced salvage of the penumbra after ischemia (Parsons et al. 2002). Transient hyperglycemia following stroke has been documented in not only diabetics but also those without any pre-existing history of hyperglycemia, thus making it a challenging obstacle for clinicians to treat (Capes et al. 2001). However, tight glycemic control with insulin is a controversial treatment strategy because of the associated risk of hypoglycemia (Kruyt et al. 2010). This suggests that the treatment strategy directed at slowing cerebral glucose uptake and metabolism, rather than controlling blood glucose concentration, may be a viable approach for neuroprotection in stroke-induced hyperglycemia. Interestingly, acute ethanol administration has been shown to worsen hyperglycemia in healthy subjects (Ting & Lautt 2006), and increases cerebral blood flow to both the pre-frontal and temporal cortices (Volkow et al. 1988), which would suggest a greater delivery of glucose during reperfusion. This previous study was consistent with this study showing a greater increase in blood glucose concentration in animals that were treated with ethanol after stroke compared with those that were not. The fact that this level of hyperglycemia observed in this study actually decreased brain injury and lactic acidosis caused by ischemia/reperfusion lends further credence to ethanol's mechanism of attenuating the uptake and utilization of glucose by cerebral tissues rather than tightly regulating blood glucose levels. Our results suggest that slowing cerebral glucose uptake and metabolism could be an alternative treatment strategy for hyperglycemia-enhanced brain injury in stroke. Because of the lack of hypoglycemia associated with ethanol and its selectivity toward decreasing brain glucose uptake and catabolism, further investigation of its use as a therapeutic modality during and after ischemia/reperfusion injury is warranted.
Lactic acidosis is not the only proposed mechanism by which hyperglycemia worsens ischemia/reperfusion injury. Several studies have shown that hyperglycemia still exacerbates injury in experimental circumstances when lactic acid is pH buffered or does not accumulate (Suh et al. 2008, Venables et al. 1985, Rytter et al. 2003). NOX, a superoxide-producing enzyme found within leukocytes and brain cells, has been shown to also exacerbate ischemia/reperfusion injury (Tang et al. 2012). NOX is dependent on glucose metabolism, specifically the hexose monophosphate shunt, which supplies the NADPH necessary for enzymatic activity (Suh et al. 2008). Reperfusion in the presence of glucose has been shown to increase neuronal NOX activity (Tang et al. 2012, Suh et al. 2008), and our results are consistent with this observation in rats undergoing stroke, but not receiving ethanol. When glucose is restored to cerebral tissue during reperfusion, it supplies NOX by serving as requisite electron donor for reperfusion-induced neuronal superoxide production through its metabolism via the hexose monophosphate shunt to generate NADPH (Suh et al. 2008). Inhibition of NOX has been shown to decrease brain injury following ischemia/reperfusion (Walder et al. 1997, Chen et al. 2009). In our study, ethanol treatment significantly decreased NOX activity during reperfusion as well as suppressed expression of the key protein that comprises its catalytic site (gp91phox) (Ago et al. 2011). While it has been demonstrated that NOX can be inhibited in knock-out mice deficient in gp91phox (Chen et al. 2009), our results suggest that ethanol may provide a direct inhibitory effect on NOX activation by blocking gp91phox expression. As glucose is necessary for NOX activation, it is plausible that ethanol may also indirectly inhibit NOX by attenuating the uptake and metabolism of glucose. NADPH is a necessary cofactor for NOX activation and a decrease in NADPH implies a decrease in production through the hexose monophosphate shunt. The hexose monophosphate shunt, like glycolysis, is a metabolic pathway dependent on both glucose uptake and metabolism. By attenuating hyperglycolysis, ethanol may decrease the production of NADPH through the hexose monophosphate shunt. NADPH is a necessary cofactor for NOX as it transfers its electron to O2 to create O2- (Sumimoto, 2008). Activity of the catalytic subunit, gp91phox, is therefore dependent on the presence of NADPH and it is surmisable that decreased production of NADPH would result in decreased enzymatic activity of NOX. This would also explain why no significant decrease in p22phox, a non-catalytic subunit that is ubiquitously expressed in non-phagocytic cells (Rytter et al. 2003), was observed in the ethanol-treated group. In this study, we did not distinguish the cell types or locations within the ischemic brain where NOX activity was greatest; however, NOX has been identified within microglia, cerebral vasculature, astrocytes, and neurons, and it is likely that increased NOX activity in all of these cell types in addition to infiltrating leukocytes contributes to reperfusion-mediated injury following ischemia (Tang et al. 2012). Further characterization of NOX-containing cells within the brain in future studies is warranted as selective targeting of cells with increased NOX activity following stroke would be of therapeutic interest.
Despite the well-studied pharmacological parameters of ethanol intoxication on brain metabolism, only recently have studies assessed its therapeutic potential in treating ischemia/reperfusion injury. Ethanol's ability to attenuate hyperglycolysis and NOX activation without the associated risk of hypoglycemia could make it an appealing treatment modality for the first several hours after ischemia/reperfusion injury especially in the context of hyperglycemia. Further studies are required to assess the precise mechanism by which ethanol preserves mitochondrial ATP production in the presence of decreased glucose catabolism.
This work was supported by the American Heart Association and Wayne State University Neurosurgery Fund to YD.
Disclosure/conflict of interest
The authors of this article have no conflicts of interest regarding the information published above.