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Published in final edited form as: Brain Res. 2007 Dec 7;1194:73–80. doi: 10.1016/j.brainres.2007.11.061

Effect of Chronic Alcohol Consumption on Brain Damage Following Transient Focal Ischemia

Hong Sun 1, Honggang Zhao 1, Glenda M Sharpe 1, Denise M Arrick 1, William G Mayhan 1
PMCID: PMC2275899  NIHMSID: NIHMS41757  PMID: 18191819

Abstract

Chronic alcohol consumption impairs cerebral vasoreactivity, and thus may result in an increase in ischemic brain damage. The goal of this study is to examine the influence of chronic alcohol consumption on transient focal ischemia-induced brain damage. Sprague-Dawley rats were divided into two groups, a control group and an alcohol group. Eight weeks after being fed a liquid diet with or without alcohol, responses of parietal pial arterioles to systemic hypoxia and hypercapnia were measured using a cranial window technique. In separate experiments, rats were subjected to right middle cerebral artery occlusion (MCAO) for 2 hours under ketamine/xylazine or isoflurane anesthesia. Regional cerebral blood flow (rCBF) was monitored through a Laser-Doppler flow probe attached to the lateral aspect of the skull. Neurological evaluation and ischemic lesion were assessed 24-hour after reperfusion. Dilation of pial arterioles in response to hypoxia and hypercapnia was significantly reduced in alcohol-fed rats. Alcohol-fed rats had significantly larger infarct volumes and worse neurological outcomes than nonalcoholfed rats under ketamine/xylazine or isoflurane anesthesia. In addition, rCBF measurement indicated that alcohol-fed rats had less regulatory rebound increase in rCBF after the initial drop in rCBF at the onset of MCAO. Our findings suggest that chronic alcohol consumption exacerbates transient focal ischemia-induced brain damage. Increased ischemic brain damage during alcohol consumption may be related to an impaired cerebral vasoreactivity.

INTRODUCTION

Stroke is a leading cause of mortality and a major cause of long-term disability. Alcohol is one of the most abused drugs, and chronic consumption of alcohol and binge drinking are major contributing factors to hemorrhagic and ischemic stroke [5,15-17,19,20,23,27]. In addition, previous studies [31,33,52,53,56] have shown that chronic alcohol consumption induces oxidative stress, and impairs cerebral vasodilation, and thus may potentiate ischemic stroke-induce brain damage.

Few studies have investigated the effects of alcohol on outcome of cerebral ischemic stroke. Clinical trials found a strong positive relationship between alcohol consumption and risk of mortality from stroke [19], cerebral infarction was more common at younger ages in heavy drinkers compared with social drinkers [57], and ex-drinkers (women) had an elevated risk of dying from ischemic stroke[17]. In experimental studies, acute alcohol has been reported to worsen brain edema following focal ischemia [60], increase focal ischemia-induced infarct [50], have no effect on transient global ischemia-induced loss of CA1 pyramidal neurons [8], and reduce the extent of transient global ischemia-induced CA1 pyramidal neuron loss [45]. In addition, Mandybur et al found that chronic alcohol consumption increased infarct volume and mortality in gerbils subjected to permanent ligation and sectioning of the right common carotid artery [30], and Favalli et al found an exacerbated permanent focal ischemia-induced cerebral damage in 24 h alcohol withdrawn rats [13]. Recently, recanalization with drugs and devices during acute ischemic stroke has been clinically shown to be effective for treating ischemic stroke [49]. Thus, transient focal ischemia may be used to model the clinical situation in which there is a prolonged ischemia with arterial recanalization. However, no studies that we are aware of have investigated the influence of alcohol consumption on brain damage in transient focal ischemic model.

Our first goal was to determine whether in vivo reactivity of cerebral arterioles in response to systemic hypoxia and hypercapnia is altered during chronic alcohol consumption. Our second goal was to examine whether chronic alcohol consumption exacerbates transient focal ischemia-induced brain damage. A 2-hour MCAO/24-hour reperfusion model was used to compare ischemic brain damage between nonalcohol-fed and alcohol-fed rats. Outcome of ischemic stroke is associated with the regulatory capacity of cerebral blood flow (CBF) during ischemia [40,42,48]. Thus, rCBF of middle cerebral artery (MCA) supply border was compared between nonalcohol-fed and alcohol-fed rats during ischemia.

RESULTS

Response of pial arterioles to systemic hypoxia and hypercapnia

A 5-min period of breathing 7.5% O2/92.5% N2 gas mixture induced systemic hypoxia (blood PO2: 56.6 ± 1.9 mmHg in nonalcohol-fed (n=7) and 52.6 ± 3.3 mmHg (n=7) in alcohol-fed rats (P > 0.05)). A 5-min period of breathing 10% CO2 induced systemic hypercapnia (blood PCO2: 52.0 ± 1.7 mmHg in nonalcohol-fed and 52.3 ± 1.4 mmHg in alcohol-fed rats (P > 0.05)). Before hypoxia and hypercapnia, baseline diameter of pial arterioles was 43 ± 1 μm in nonalcohol-fed and 44 ± 3 μm in alcohol-fed rats (P > 0.05). Hypoxia and hypercapnia dilated pial arterioles by 29 ± 3% and 22 ± 2%, respectively, in nonalcohol-fed, but by only 12 ± 2% and 5 ± 2%, respectively, in alcohol-fed rats (P < 0.05) (Fig. 1).

Figure 1.

Figure 1

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Effect of chronic alcohol consumption on response of parietal pial arterioles to systemic hypoxia and hypercapnia. Values are means ± SE. *P< 0.05 vs. nonalcohol-fed rats.

MCAO under ketamine/xylazine anesthesia

Body weight was similar in nonalcohol-fed and alcohol-fed rats (nonalcohol-fed (n=10): 413 ± 9 g vs. alcohol-fed (n=10): 410 ± 4 g). There were significant increases in total lesion, cortical infarct, and subcortical infarct in the alcohol-fed group compared with nonalcohol-fed group (Fig. 2). Representative samples of TTC-stained brain sections are shown in Fig. 3. Consistently, the neurological deficit was significantly greater in alcohol-fed rats (Fig. 4). Occlusion of right middle cerebral artery produced an equivalent decrease in rCBF at the point of MCAO onset in both nonalcohol-fed and alcohol-fed rats. However, alcohol-fed rats had less rCBF regulatory rebound increase than nonalcohol-fed rats during 30-min observation period (Fig. 5).

Figure 2.

Figure 2

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Effect of chronic alcohol consumption on brain damage at 24 hours after a 2-hour right MCAO in rats anethetized with ketamine/xylazine or isoflurane. Total lesion is the sum of intermediate staining and infarct lesion. Values are means ± SE. *P < 0.05 vs. nonalcohol-fed rats. #P < 0.05 vs. rats anethetized with ketamine/xylazine.

Figure 3.

Figure 3

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Representative 2 mm-thick, TTC-stained coronal sections of the brain from nonalcohol-fed and alcohol-fed rats subjected to a 2-hour MCAO/24-hour reperfusion under ketamine/xylazine or isoflurane anesthesia.

Figure 4.

Figure 4

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Effect of chronic alcohol consumption on neurological deficit scores at 24-hour after a 2-hour right MCAO in rats anesthetized with ketamine/xylazine or isoflurane. Values are means ± SE. □*P < 0.05 vs. nonalcohol-fed rats. #P < 0.05 vs. alcohol-fed rats anesthetized with ketamine/xylazine.

Figure 5.

Figure 5

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Effect of chronic alcohol consumption on ipsilateral parietal cerebral blood flow during first 30 minutes of a 2-hour right MCAO in rats anesthetized with ketamine/xylazine. Values are means ± SE. *P < 0.05 vs. nonalcohol-fed rats.

MCAO under isoflurane anesthesia

Body weight was similar in nonalcohol-fed and alcohol-fed rats (nonalcohol-fed (n=7): 408 ± 7 g vs. alcohol-fed (n=7): 405 ± 8 g). There were significant increases in total lesion and cortical infarct in the alcohol-fed group. The subcortical infarct in the alcohol-fed rats was similar to that of nonalcohol-fed rats (Fig. 2). Representative samples of TTC-stained brain sections are shown in Fig. 3. Similarly, the neurological dysfunction was significantly increased in alcohol-fed rats compared with nonalcohol-fed rats (Fig. 4). Isoflurane did not alter total lesion, but reduced cortical infarct in both nonalcohol-fed and alcohol-fed rats and subcortical infarct in alcohol-fed rats as compared with the ketamine/xylazine-anesthetized groups (Fig. 2). In addition, isoflurane did not alter neurological score in nonalcohol-fed rats, but significantly improved neurological deficit in alcohol-fed rats. Occlusion of right middle cerebral artery produced a similar decrease in rCBF at MCAO onset in both nonalcohol-fed and alcohol-fed rats. However, a decrease in rCBF regulatory rebound increase was found in alcohol-fed rats during 30-min observation period (Fig. 6).

Figure 6.

Figure 6

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Effect of chronic alcohol consumption on ipsilateral parietal cerebral blood flow during first 30 minutes of a 2-hour right MCAO in rats anesthetized with isoflurane. Values are means ± SE. *P < 0.05 vs. nonalcohol-fed rats.

DISCUSSION

This is the first study to determine the influence of chronic alcohol consumption on transient focal ischemia-induced brain damage, and examine potential mechanisms that account exacerbated ischemic brain damage during chronic alcohol consumption. There are three new findings from this study. First, the response of cerebral arterioles to systemic hypoxia and hypercapnia is impaired during alcohol consumption. Second, chronic alcohol consumption exacerbates transient focal ischemia-induced brain damage. Third, chronic alcohol consumption reduces the regulatory rebound increase of rCBF during ischemia.

An inverse correlation has been demonstrated between infarct volume and absolute rCBF [11,12]. Nagai et al reported that alcohol ingestion had a poor influence on the microcirculation in patients with ischemic cerebrovascular diseases [41]. In the present study, we found that regulation of rCBF was significantly reduced during ischemia in alcohol-fed rats. In addition, chronic alcohol consumption significantly reduced the response of pial arterioles to systemic hypoxia and hypercapnia. Pial arterioles account for nearly 50% of vascular resistance in the cerebral circulation [34,35]. There is general agreement between studies that have examined responses of pial arterioles to those that examined parenchymal arterioles [9,25]. Since blood flow appears to vary with the cube of the diameter of a vessel [36], modest changes in diameter of pial arterioles can greatly alter cerebral blood flow. Based upon this evidence, we suggest that impaired cerebral vasoreactivity during chronic alcohol consumption results in a reduced regulatory capacity of rCBF, and thus contributes to exacerbated ischemic brain damage. Although rebound increase in rCBF is considered the result of dilation of ACA-MCA network, other possibilities cannot be excluded, such as that dilation of MCA itself around the suture to provide an increased in flow.

Two additional mechanisms may be involved in exacerbated cerebral ischemic damage during alcohol consumption. First, alcohol consumption induces oxidative stress. Previous studies, including ours [52,53,56], suggest that alcohol consumption induces oxidative stress [1,39,43,51,59] by augmenting production of reactive oxygen species (ROS) and diminishing antioxidant capacity. Oxidative stress plays an important role during reperfusion when oxygen delivery to previously ischemic tissue resumes. Due to the high reactivity, ROS provoke damage to lipids, DNA, and proteins, leading to neuronal death. ROS also contribute to the breakdown of the blood-brain barrier and brain edema [14,26]. Second, alcohol consumption induces glutamate excitotoxicity. Glutamate and GABA are the major excitatory and inhibitory neurotransmitters, respectively, and neurons using these neurotransmitters constitute over 80% in the brain. Glutamate is known to play a predominant role in the pathogenesis of ischemic brain damage. This excitatory amino acid is released at high concentrations in hypoperfused brain tissue. In this area, glutamate overactivates its receptors leading to a massive influx of Ca2+ that activates a variety of catabolic processes that subsequently produce cell death. In addition, the augmented intracellular Ca2+ further promotes an increase in extracellular glutamate, thus propagating the excitotoxicity. The overactivation of these receptors, mainly NMDA subtype, further leads to Na+, Ca2+, Cl−, and H2O accumulation, cellular swelling, and cytotoxic edema. Chronic alcohol consumption leads a homeostatic up-regulation of glutamate receptors and down-regulation of GABA receptors [6,21,22,47,55]. Previous studies have shown that ischemia-induced release of glutamate was attenuated during chronic alcohol [13,37], although chronic alcohol consumption enhanced NMDA-induced excitotoxicity in neuronal culture [8]. However, Favalli et al found an exacerbated cerebral damage coupled with increased glutamate and aspartate release following focal ischemia in 24 h alcohol withdrawn rats [13]. Thus, this mechanism may account for enhanced ischemic neuronal damage during alcohol withdrawal. In the present study, to avoid acute effect of alcohol and anesthetic accidents during the surgical procedures we removed diet including alcohol from the animals for 8 hours before the experiments. Thus, effect of alcohol withdrawal might be involved in mechanisms of increased ischemic brain damage. In the present study, two anesthetic methods were used to evaluate transient focal ischemia-induced brain damage during chronic alcohol consumption. Isoflurane has been demonstrated to have neuroprotective effect against ischemic damage by inhibiting glutamate-mediated excitotoxicity and ischemic neuronal depolarization [10,18,24,44,46,58], which are responsible for rapid neuronal death during the ischemic and early reperfusion periods. In the present study, infarct size was significantly reduced in isoflurane-anesthetized rats compared with ketamine/xylazine-anesthetized rats. However, total ischemic lesion volume in both nonalcohol-fed and alcohol-fed rats was not affected by isoflurane. In addition, cortical infarct volume was larger in alcohol-fed rats compared with nonalcohol-fed rats under isoflurane anesthesia. Thus, it appears that increased total ischemic lesion during chronic alcohol consumption may be not related to increased glutamate-mediated excitotoxicity.

It is not clear whether chronic alcohol consumption alters postischemic cerebral vasoreactivity. Cerebral ischemia produces early vascular abnormalities, hyperemia and delayed hypoperfusion, during reperfusion. Previous studies have shown that ischemia produces a decrease in dilation of cerebral arterioles to endothelial nitric oxide synthase (eNOS)-dependent agonist [32], neuronal nitric oxide synthase (nNOS)-dependent agonist [4] and CGRP [29]. In addition, function of ATP-sensitive potassium channel and inward rectifier potassium channel in cerebral vascular smooth muscle cells is impaired after cerebral ischemia/reperfusion [2,3]. A recent study found a reduced dilation of cerebral arterioles to adenosine and ATP in a 2-hour MCAO/24-hour reperfusion model. Impaired cerebral vasodilation may contribute to reduced reperfusion of the cerebral circulation during reperfusion. Previously we have shown that chronic alcohol consumption impairs several important vasodilator pathways including eNOS-dependent, nNOS-dependent, and potassium channel activation-mediated in cerebral arterioles under normal condition via an increased release of oxygen radicals [52-54,56]. Although cerebral vasoreactivity during reperfusion was not measured in the present study, we believe that a worse postischemic cerebral vasoreactivity might exist in alcohol-fed rats.

In summary, the present study defined the influence of chronic alcohol consumption on transient focal ischemia-induced brain damage. Although the precise cellular mechanisms underlying exacerbated cerebral ischemic damage during alcohol consumption remain to be further determined, the present findings suggest that impaired vasoreactivity may be involved in alcohol consumption-induced increase in ischemic cerebral damage.

EXPERIMENTAL PROCEDURE

Experimental diets

All procedures were in accordance with the “Principle of Laboratory Animal Care” (NIH publication No. 86−23, revised 1985) and were approved by the Institutional Animal Care and Use Committee. We used male Sprague-Dawley rats. At 2 months of age (body weight 200 to 220 g), the rats were singly housed and divided into two groups, a nonalcohol-fed group (n=24) and an alcohol-fed group (n=24). We fed rats liquid diets (Dyets, Bethlehem, PA) for 8−12 weeks. These diets have been used extensively to study the chronic effects of alcohol in rats [28,31,38]. The nonalcohol rats were given a liquid diet that contained 1.0 kcal/ml, of which 35% were derived from fat, 47% were derived from carbohydrates, and 18% were derived from protein. Rats in the alcohol fed groups were given a liquid diet that contained 1.0 kcal/ml, of which 35% were derived from fat, 11% were derived from carbohydrates, 18% were derived from protein, and 36% were derived from ethanol. We gradually introduced ethanol into the diet over a 5-day period. The total daily volume of diet fed to the nonalcohol-fed rats was based on the consumption of diet by the alcohol-fed rats, and thus the daily consumption of diet was similar in the nonalcohol-fed and alcohol-fed rats. To avoid acute effect of alcohol and anesthetic accidents during the surgical procedures, rats were fasted for 8 hours before the experiments.

Cerebral Vascular Reactivity

On the day of the experiment, rats (nonalcohol-fed: n=7; alcohol-fed: n=7) were anesthetized, and a tracheotomy was performed. The rats were ventilated mechanically with room air and supplemental oxygen. A catheter was placed into a femoral vein for injection of supplemental anesthesia, and a femoral artery was cannulated for measurement of arterial blood pressure and to obtain a blood sample for the measurement of arterial blood gas.

To visualize the microcirculation of the cerebrum, a craniectomy was prepared over the left parietal cortex. The cranial window was suffused with artificial cerebrospinal fluid (2 ml/min) that was bubbled with 95% nitrogen and 5% carbon dioxide. Temperature of the suffusate was maintained at 37±1°C. The cranial window was connected via a three-way valve to a pump, which was allowed for infusion of agonists and antagonists into the suffusate. This method maintained a constant temperature, pH, PCO2 and PO2 of the suffusate during infusion of drugs. Diameter of pial arteriolar was measured using a video image-shearing device.

Cerebral vessels were superfused with artificial cerebral spinal fluid for one hour before testing responses of arterioles. To examine the effect of chronic alcohol consumption on dilation of pial arterioles in response to hypoxia and hypercapnia, we measured responses of pial arterioles at 5-min after started inhaling a gas mixture of 7.5% O2/92.5% N2 (hypoxia), or a gas mixture of 10% CO2 (hypercapnia). In each rat only one pial arteriole was studied. At same time, arterial blood gas was measured.

Transient Focal ischemic Brain Damage

In separate experiments, rats were anesthetized with ketamine/xylazine (100/15 mg/kg i.p.) or isoflurane (induced by 5% and maintained by 2% isoflurane in 30% oxygen-balance nitrogen). Body temperature was maintained at 37°C throughout the procedures using a rectal temperature regulated heating pad. To measure rCBF of MCA supply boarder, a laser Doppler flow probe (PeriFlux System 5000, Perimed) was attached to the right side of the dorsal surface of the skull 1−2 mm caudal and 5−6 mm lateral to bregma. The blood flow at this area is directly provided by MCA, and potentially supplied by anterior cerebral artery (ACA) through ACA-MCA anastomoses [7]. MCAO onset will induce a rapid drop of rCBF. However, dilation of those arterial anastomoses in response to hypoxia will produce a rebound increase in rCBF. We believe that the magnitude of rebound increase in rCBF represents cerebral vasoreactivity of this area during MCAO.

A catheter was placed into a femoral artery for measurement of arterial blood pressure and to obtain a blood sample for the measurement of arterial blood gas.

Occlusion of the middle cerebral artery was induced using the intraluminal suture occlusion technique. The right common and external carotid arteries were exposed and ligated. A 3−0 monofilament nylon suture was prepared by rounging its tip by heating and coating with poly-L-lysine. The MCA was occluded by inserting filament from the basal part of the external carotid artery and advanced cranially into the internal carotid artery to the point where the middle cerebral artery branched off from the internal artery. The MCAO was determined by a rapid drop in regional cerebral blood flow (rCBF) to the cerebral hemisphere. Regional CBF was assessed for 30 min following placement of the suture. After occluding the right MCA for 2 hours, reperfusion was initiated by removing the suture from the internal carotid artery. Rats were allowed to recover for 24 hours. At the end of recovery time, rats were assessed for neurological deficits on a 24-point scale (Table 1). Rats were then anesthetized with thiobutabarbital sodium (Inactin) (150 mg/kg body weight, i.p.) and exsanguinated. The brain was quickly removed and placed in ice-cold sterile saline for 5 min, and cut into six 2-mm coronal sections. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma). Slice images were digitalized, the ischemic lesion was evaluated using Kodak Molecular Imaging Software. Dark stain indicates viable tissue, and complete lack of stain is defined as infarct lesion. Total lesion is specified as the sum of intermediate staining and infarct lesion. Total and Infarct lesions corrected for cerebral edema were expressed as percentage of the contralateral hemisphere.

Table 1.

Neurological evaluation system

0 1 2 3
Spontaneous activity (5 min) No movement Slight movement Touches 1 or 2 sides of cage Touches 3 or 4 sides of cage
Symmetry of movement Left side: no movement Left side: slight movement Left side: moves slowly Both side: move symmetrically
Response to vibrissae touch No response on left side Weak response on left side Symmetrical response on left side
Floor walking No walking Walks in circles only Curvilinear path Straight path
Beam walking Falls off of beam Hugs beam Stands on beam Walks on beam
Symmetry of forelimbs (outstretching while held by tail) Left side: no movement, no outreaching Left side: slight movement to outreach Left side: moves and outreaches less than right side Symmetrical outreach
Climbing wall of wire cage Fails to climb Left side is weak Normal climbing
Reaction to touch on either side of trunk No response on left side Weak response on left side Symmetrical response
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Statistical analysis

For comparison of the various treatments, results were compared using a two-way repeated measure ANOVA with Tukey's post hoc test. Values are means ± SEM. A p value of 0.05 or less was considered to be significant.

ACKNOWLEDGMENTS

This study was supported by National Institutes of Health Grants DA 14258, HL79587, AA 11288, a Scientist Development Grant from the American Heart Association (0635052N), and funds from the University of Nebraska Medical Center.

Footnotes

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