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Does alcohol-induced blood cell agglutination cause brain damage?
Journal of the Neurological Sciences, 1986, 72:43-48 Elsevier
43
JNS 2600
Does Alcohol-Induced Blood Cell Agglutination Cause Brain Damage? Stephen C. Phillips Department of Anatomy, Monash University, Clayton, Victoria 3168 (Australia) (Received, 14 May, 1985) (Revised, received 9 August, 1985) (Accepted 14 August, 1985)
SUMMARY
After 3 weeks of alcohol intoxication, the brains of rats were searched with lightand electron microscopy for degenerating nervous tissue and agglutination of erythrocytes in the blood vessels. There was no sign of degeneration of nerve cells or synapses in the cerebral cortex, hippocampus, cerebellum, midbrain or hindbrain. No histological sections showed blood vessels with erythrocytes inside them. It is concluded that the agglutination of red blood cells seen in the conjunctivae of intoxicated human alcoholics is not necessarily an indication that vascular congestion is also occurring in the brain of such patients, nor that this is the primary mechanism of alcohol-related brain damage.
Key words:
Alcohol
- Blood
cells - Brain - Degeneration
INTRODUCTION
A biophysical mechanism by which human abuse of alcohol might cause brain damage has come to light in the finding that patients, after consuming alcohol, showed disturbed blood flow in the conjunctiva of the eye (Moskow et al. 1968). At the highest concentration of blood alcohol (49-71 mmol/l) there was a complete cessation of flow in an unspecified proportion of blood vessels, due to agglutination of red blood cells,
This work was supported financially by the Australian Associated Brewers. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
44 and rupture of some of the smaller vessels. Moreover, these authors claimed that within this concentration range, the number of blood vessels in statis with no flow through them (due to "sludging") correlated well with the blood alcohol level. No statis was observed at a blood alcohol level below 46 mmol/1. With the aid of a mathematical model, Pennington and Knisely (1973) suggest that such circulatory effects of ethanol can lead to localised areas of anoxia in the brain resulting in mental deterioration and morphological damage of the neural tissue. Moskow et al. (1968) have quoted Courville's (1966) autopsy finding that brains from alcoholics "contain many congested small vessels"; however, this is an inconclusive statement because the blood vessels would have been filled with cells due to the cessation of systemic blood flow at the time of death. In a series of survival-time experiments which are less easy to interpret, Pennington and Knisely (1973) injected rabbits with lethal doses of alcohol (9 g/kg) and found that those rabbits which had been pre-medicated with heparin (1000 U/kg) survived a significantly longer period after alcohol administration than did nonheparinized rabbits. The authors attribute this time difference to the delayed cell agglutination and delayed reduction of blood flow in the heparinized rabbits, as seen in the conjunctiva of these animals. It remains, however, that no direct evidence has been presented to show that "sludging" after alcohol administration in humans or animals occurs in the brain or damages brain tissue. Experimental work on laboratory animals permits control of blood alcohol levels and makes possible histological examination of brain tissue at optimal times for the detection of ultrastructural changes with the electron microscope and axon and axon terminal degeneration with the light microscope using a specific silver impregnation stain (Nauta technique) modified by Fink and Heimer (1967). We have employed such histological techniques to search the brains of rats for blood cell agglutination and brain pathology after exposure to ethanol for long time periods. MATERIALSAND METHODS Six male Sprague-Dawley rats (300-400 g) received ethanol by being placed inside an airtight chamber, the air supply to which contained a concentration of cane spirit (Commonwealth Sugar Refineries) which could be regulated. A description of the experimental apparatus has been published previously (Phillips and Cragg 1982). All animals received a constant supply of ethanol vapour 24 h/day for 3 weeks, During the last 5 consecutive days, for 9 h/day, the air concentration of alcohol was increased such that the animals were unable to stand on their feet or even to fight themselves if pushed over onto their side. Blood samples were taken from the tip of the tail during both the light ethanol treatment period and severe treatment periods. From each blood sample, 0.1 ml of whole blood was added to 0.9ml of cold 2~o TCA, centrifuged and the supernatant assayed for its alcohol content using a routine enzymatic (Sigma) method. At the end of the 3 week treatment period, all 6 alcohol-treated animals and 6 age-matched control rats were killed by chloroform overdose and the whole body intracardially perfused with 100 ml of 4% paraformaldehyde in 0.1 M sodium phos-
45 phate buffer (pH 7.4), at a pressure not exceeding the normal systolic blood pressure (120 mmHg) of the rat. The brains and livers were immediately removed and stored in the fixative for 1-2 weeks. Parasagittal sections of brain tissue (30 #m thick) within 1 mm of the midline were cut on a freezing microtome and stained either with 0.002~o toluidine blue in 0.1 M sodium phosphate buffer overnight, or with a modified Fink and Heimer silver stain (Anker and Cragg 1974). Small blocks of brain were secondarily fixed in osmium tetroxide, embedded in Araldite, and silver-grey sections stained with lead citrate and examined in a JEOL 100S electron microscope. For liver histology, 30-#m frozen sections of liver were stained with either Dunn's stain to reveal red blood cells, or with Wright's stain. RESULTS Blood alcohol levels of 20, 28 and 38 mmol/l were recorded from 3 separate animals during the light ethanol treatment, and 96 mmol/1 and 108 mmol/1 recorded during the severe treatment period. There was no change in body weight of the animals over the 3-week ethanol exposure period although age-matched untreated male rats of the same strain continued to increase their body weight by 25 g. Despite the high blood alcohol levels, the tongue and footpads remained pink in colour, suggesting that cyanosis had not occurred. Pieces of liver were removed from 3 of the 6 rats immediately after perfusion, and histological sections (each with a surface area of 1 cm 2) showed no sign of any blood cells in 2 of the animals, while the third did contain a few red blood cells in 3 of the 6 histological sections stained. The cerebellar Purkinje cell count for ethanol-exposed animals was 1805 + 30 (mean + SE) per histological section, and 1799 + 35 per histological section for controis. Histological sections stained with the Fink-Heimer technique were clear of degeneration in those areas examined (all layers of the cerebral cortex, hippocampus, cerebellum, midbraln and hindbrain) in all 6 alcohol-treated animals. Concurrently, stained sections from a positive control animal (subjected to a heat lesion of the cerebral cortex) were intensely impregnated with silver granules. To confh'rn the Fink-Heimer result, pieces of the cerebral cortex and cerebeUar cortex were examined under the electron microscope. There was no sign of degeneration in the ultrastructure and no accumulation of macrophages. Synaptic contacts (Fig. 1) were indistinguishable from those present in control material. Endothelial cells of blood vessels were normal in nuclear and cytoplasmic appearance with no sign of loss of continuity of the cell membrane. Five sections (each at least 100 #m apart) were examined from each animal and no blood vessels were observed to contain blood cells. Perfusion of tissues with fLxativeis essential for good quality electron microscopy histology, however since the pressure of the perfusate never exceeded systolic blood pressure (see Methods section), the fixative would not have flushed out those blood vessels blocked by blood cell agglutination. Thus the appearance in histological sections of empty blood vessels is representative of uninterrupted free-flowing blood in those vessels in the living animal.
46
Fig. 1. Electron micrographs of rat cerebral cortex after 3 weeks of ethanol intoxication. A: Endothelial cell in molecular layer ( × 10000). B: Edge of a nerve cell in close proximity to a blood vessel separated by neuropil containing abundant synapses with normal appearance ( × 25 000).
47 DISCUSSION The concentrations of alcohol that we have produced in our rats are similar to or greater than those observed in the human subjects used by Moskow et al. (1968) at the time sludging was seen in the conjunctiva. No damage was seen in the form of loss of cells or degenerating axons or axon terminals in light microscopy sections taken from our animals so treated, and electron microscopy showed no evidence of abnormal structure of synapses or associated neuropil. The maximum dose of alcohol which our animals received (104 mmol/l) repeatedly on 5 consecutive days is rarely expected to be exceeded by a human alcoholic. It therefore seems unlikely that cerebral microvascular sludging after alcohol consumption would produce a loss of neurons unless human nerve cells are more easily damaged than those of rats. Pennington and Knisely (1973) suggest that anoxic death oflocalised brain tissue can result around blood vessels where sludging has occurred. However, such sludging is presumed to persist only for as long as the blood alcohol level is high (above 46 mmol/1), yet repeated bouts of asphyxiation leading to blood oxygen desaturation to 44 mm Hg for a total of 101 min over a 3-h period in rats with a blood alcohol level of 110 mmol/1, does not lead to permanent structural damage in the brain (Cragg and Phillips 1984). It is known that a proportion of human alcoholics with demonstrable brain disturbances possess liver disease (Fox et al. 1976; Acker et al. 1982) and the resulting metabolic disturbances may represent a biochemical threat to the brain in the long term. Reduced cerebral blood flow is a possible acute biophysical effect of ethanol which can be tested with radiological techniques (Caille et al. 1978; Drayer et al. 1980). Experimental studies with rats indicate that both cerebral blood flow and oxygen consumption are reduced during an acute intoxicating period, but these effects are less marked after repeated intoxications over 3 or 4 days (Hemmingsen and Barry 1979). At clinically observed levels, alcohol has been found to induce spasms of the cerebral vasculature in rats (Altura et al. 1983), but it has not been demonstrated that this leads to nerve tissue damage. Even a brain tissue concentration of 3.8~o (3080rag/g) ethanol maintained for 1 h does not produce cerebral degeneration in the adult rat detectable with the electron microscope (Phillips et al. 1981). It is known that the blood vessels of the brain are morphologically and functionally distinguishable from blood vessels elsewhere in the body (Rapoport, 1976). Thus, Moskow et al. (1973) may be in error to have assumed that their observations of leaking of the endothelium and subsequent haemorrhages in the conjunctiva are a reflection of events concurrently occurring in the brain. Indeed, both acute (1 h) and chronic (6 months) exposure of alcohol to rats does not significantly alter the permeability of the blood-brain barrier to sucrose (Phillips and Cragg 1982). Moreover, in a study of the histological changes produced by dextran-induced experimental red cell agglutination in rabbits, Stalker (1967) found sites of hypoxic damage in the liver and heart muscle to be "present in every 2 or 3 low-power fields", yet in the same animals, changes in the brain "were so slight that they were not useful indicators of damage". It remains that although the human alcoholic does display brain disturbances in radiological scans (Fox et al. 1976) and postmortem histology (Harper 1982), there is no direct evidence that blood cell agglutination is the principle mediator of these changes.
4~ ACKNOWLEDGEMENT
The author expresses his thanks towards Dr. Brian Cragg for his contribution to this work. REFERENCES Acker, W., E. J. Aps, S. K. Majumbar, G. K. Shaw and A. D. Thomson (1982) The relationship between brain and liver damage in chronic alcoholic patients, J. Neurol. Neurosurg. Psychol., 45: 984-987. Altura, B.M., B.T. Altura and A. Gebrewold (1983) Alcohol-induced spasms of cerebral blood vessels - Relation to cerebrovascular accidents and sudden death, Science, 220:331-333. Anker, R.L. and B.G. Cragg (1974) Development of the extrinsic connections of the visual cortex in the cat, J. Comp. Neurol., 154: 29-42. Caille, J., P. Constant, J. Billerey and A. Renou (1978) Variations in the measurement of regional blood volume by CAT, Computerized Tomography, 2: 63-68. Courville, C.B. (1966) Effects of Alcohol on the Nervous System of Man, San Lucus, Los Angeles, CA. Cragg, B.G. and S.C. Phillips (1984) A search for brain damage in a rat model of alcoholic sleep apnoea, Exp. Neurol., 80: 218-226. Drayer, B.P., D. Gur, S.K. Wolfson and E.E. Cook (1980) Abnormality of the xenon blood:brain co-efficient and blood flow in cerebral infarction - - An in vivo assessment using transmission computerized tomography, Radiology, 135: 349-354. Fink, R. P. and L. Heimer (1967)Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Res., 4: 369-374. Fox, J.H., R.G. Ramsey, M.S. Huckman and A.E, Proske (1976) Cerebral ventricular enlargement - Chronic alcoholics examined by computerized tomography, J. Amer. Med. Assoc., 236: 365-368. Harper, C. (1982) Neuropathology of brain damage caused by alcohol, Med. J. Aust., 2: 277-282. Hemmingsen, R. and D.I. Barry (1979) Adaptive changes in cerebral blood flow and oxygen consumption during ethanol intoxication in the rat, Acta Physiol. Scand., 106: 249-255. Moskow, H.A., R.C. Pennington and M.H. Knisely (1968) Alcohol, sludge, and hypoxic areas of nervous system, liver and heart, Microvasc. Res., 1: 174-185. Pennington, R.C. and M.H. Knisely (1973) Experiments aimed at separating the mechanical circulatory effects of ethanol from specific chemical effects, Ann. N.Y. Acad. Sci., 215: 356-365. Phillips, S.C. (1981) Does ethanol damage the blood-brain barrier?, J. Neurol. Sci., 50: 81-87. Phillips, S.C. and B.G. Cragg (1982) Weakening of the blood-brain barrier by alcohol-related stresses in the rat, J. Neurol. Sci., 54: 271-278. Phillips, S. C., B. G. Cragg and S.C. Singh (1981) The short-term toxicity of ethanol to neurons in rat cerebral cortex tested by topical application in vivo, and a note on a problem in estimating ethanol concentrations in tissue, J. Neurol. Sci., 49: 353-361. Rapoport, S.I. (1976) The Blood-Brain Barrier in Physiology and Medicine, Raven Press, New York, NY. Stalker, A. L. (1967) Histological changes produced by experimental erythrocytes aggregation, J. Path. Bact.. 93: 203-212.
43
JNS 2600
Does Alcohol-Induced Blood Cell Agglutination Cause Brain Damage? Stephen C. Phillips Department of Anatomy, Monash University, Clayton, Victoria 3168 (Australia) (Received, 14 May, 1985) (Revised, received 9 August, 1985) (Accepted 14 August, 1985)
SUMMARY
After 3 weeks of alcohol intoxication, the brains of rats were searched with lightand electron microscopy for degenerating nervous tissue and agglutination of erythrocytes in the blood vessels. There was no sign of degeneration of nerve cells or synapses in the cerebral cortex, hippocampus, cerebellum, midbrain or hindbrain. No histological sections showed blood vessels with erythrocytes inside them. It is concluded that the agglutination of red blood cells seen in the conjunctivae of intoxicated human alcoholics is not necessarily an indication that vascular congestion is also occurring in the brain of such patients, nor that this is the primary mechanism of alcohol-related brain damage.
Key words:
Alcohol
- Blood
cells - Brain - Degeneration
INTRODUCTION
A biophysical mechanism by which human abuse of alcohol might cause brain damage has come to light in the finding that patients, after consuming alcohol, showed disturbed blood flow in the conjunctiva of the eye (Moskow et al. 1968). At the highest concentration of blood alcohol (49-71 mmol/l) there was a complete cessation of flow in an unspecified proportion of blood vessels, due to agglutination of red blood cells,
This work was supported financially by the Australian Associated Brewers. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
44 and rupture of some of the smaller vessels. Moreover, these authors claimed that within this concentration range, the number of blood vessels in statis with no flow through them (due to "sludging") correlated well with the blood alcohol level. No statis was observed at a blood alcohol level below 46 mmol/1. With the aid of a mathematical model, Pennington and Knisely (1973) suggest that such circulatory effects of ethanol can lead to localised areas of anoxia in the brain resulting in mental deterioration and morphological damage of the neural tissue. Moskow et al. (1968) have quoted Courville's (1966) autopsy finding that brains from alcoholics "contain many congested small vessels"; however, this is an inconclusive statement because the blood vessels would have been filled with cells due to the cessation of systemic blood flow at the time of death. In a series of survival-time experiments which are less easy to interpret, Pennington and Knisely (1973) injected rabbits with lethal doses of alcohol (9 g/kg) and found that those rabbits which had been pre-medicated with heparin (1000 U/kg) survived a significantly longer period after alcohol administration than did nonheparinized rabbits. The authors attribute this time difference to the delayed cell agglutination and delayed reduction of blood flow in the heparinized rabbits, as seen in the conjunctiva of these animals. It remains, however, that no direct evidence has been presented to show that "sludging" after alcohol administration in humans or animals occurs in the brain or damages brain tissue. Experimental work on laboratory animals permits control of blood alcohol levels and makes possible histological examination of brain tissue at optimal times for the detection of ultrastructural changes with the electron microscope and axon and axon terminal degeneration with the light microscope using a specific silver impregnation stain (Nauta technique) modified by Fink and Heimer (1967). We have employed such histological techniques to search the brains of rats for blood cell agglutination and brain pathology after exposure to ethanol for long time periods. MATERIALSAND METHODS Six male Sprague-Dawley rats (300-400 g) received ethanol by being placed inside an airtight chamber, the air supply to which contained a concentration of cane spirit (Commonwealth Sugar Refineries) which could be regulated. A description of the experimental apparatus has been published previously (Phillips and Cragg 1982). All animals received a constant supply of ethanol vapour 24 h/day for 3 weeks, During the last 5 consecutive days, for 9 h/day, the air concentration of alcohol was increased such that the animals were unable to stand on their feet or even to fight themselves if pushed over onto their side. Blood samples were taken from the tip of the tail during both the light ethanol treatment period and severe treatment periods. From each blood sample, 0.1 ml of whole blood was added to 0.9ml of cold 2~o TCA, centrifuged and the supernatant assayed for its alcohol content using a routine enzymatic (Sigma) method. At the end of the 3 week treatment period, all 6 alcohol-treated animals and 6 age-matched control rats were killed by chloroform overdose and the whole body intracardially perfused with 100 ml of 4% paraformaldehyde in 0.1 M sodium phos-
45 phate buffer (pH 7.4), at a pressure not exceeding the normal systolic blood pressure (120 mmHg) of the rat. The brains and livers were immediately removed and stored in the fixative for 1-2 weeks. Parasagittal sections of brain tissue (30 #m thick) within 1 mm of the midline were cut on a freezing microtome and stained either with 0.002~o toluidine blue in 0.1 M sodium phosphate buffer overnight, or with a modified Fink and Heimer silver stain (Anker and Cragg 1974). Small blocks of brain were secondarily fixed in osmium tetroxide, embedded in Araldite, and silver-grey sections stained with lead citrate and examined in a JEOL 100S electron microscope. For liver histology, 30-#m frozen sections of liver were stained with either Dunn's stain to reveal red blood cells, or with Wright's stain. RESULTS Blood alcohol levels of 20, 28 and 38 mmol/l were recorded from 3 separate animals during the light ethanol treatment, and 96 mmol/1 and 108 mmol/1 recorded during the severe treatment period. There was no change in body weight of the animals over the 3-week ethanol exposure period although age-matched untreated male rats of the same strain continued to increase their body weight by 25 g. Despite the high blood alcohol levels, the tongue and footpads remained pink in colour, suggesting that cyanosis had not occurred. Pieces of liver were removed from 3 of the 6 rats immediately after perfusion, and histological sections (each with a surface area of 1 cm 2) showed no sign of any blood cells in 2 of the animals, while the third did contain a few red blood cells in 3 of the 6 histological sections stained. The cerebellar Purkinje cell count for ethanol-exposed animals was 1805 + 30 (mean + SE) per histological section, and 1799 + 35 per histological section for controis. Histological sections stained with the Fink-Heimer technique were clear of degeneration in those areas examined (all layers of the cerebral cortex, hippocampus, cerebellum, midbraln and hindbrain) in all 6 alcohol-treated animals. Concurrently, stained sections from a positive control animal (subjected to a heat lesion of the cerebral cortex) were intensely impregnated with silver granules. To confh'rn the Fink-Heimer result, pieces of the cerebral cortex and cerebeUar cortex were examined under the electron microscope. There was no sign of degeneration in the ultrastructure and no accumulation of macrophages. Synaptic contacts (Fig. 1) were indistinguishable from those present in control material. Endothelial cells of blood vessels were normal in nuclear and cytoplasmic appearance with no sign of loss of continuity of the cell membrane. Five sections (each at least 100 #m apart) were examined from each animal and no blood vessels were observed to contain blood cells. Perfusion of tissues with fLxativeis essential for good quality electron microscopy histology, however since the pressure of the perfusate never exceeded systolic blood pressure (see Methods section), the fixative would not have flushed out those blood vessels blocked by blood cell agglutination. Thus the appearance in histological sections of empty blood vessels is representative of uninterrupted free-flowing blood in those vessels in the living animal.
46
Fig. 1. Electron micrographs of rat cerebral cortex after 3 weeks of ethanol intoxication. A: Endothelial cell in molecular layer ( × 10000). B: Edge of a nerve cell in close proximity to a blood vessel separated by neuropil containing abundant synapses with normal appearance ( × 25 000).
47 DISCUSSION The concentrations of alcohol that we have produced in our rats are similar to or greater than those observed in the human subjects used by Moskow et al. (1968) at the time sludging was seen in the conjunctiva. No damage was seen in the form of loss of cells or degenerating axons or axon terminals in light microscopy sections taken from our animals so treated, and electron microscopy showed no evidence of abnormal structure of synapses or associated neuropil. The maximum dose of alcohol which our animals received (104 mmol/l) repeatedly on 5 consecutive days is rarely expected to be exceeded by a human alcoholic. It therefore seems unlikely that cerebral microvascular sludging after alcohol consumption would produce a loss of neurons unless human nerve cells are more easily damaged than those of rats. Pennington and Knisely (1973) suggest that anoxic death oflocalised brain tissue can result around blood vessels where sludging has occurred. However, such sludging is presumed to persist only for as long as the blood alcohol level is high (above 46 mmol/1), yet repeated bouts of asphyxiation leading to blood oxygen desaturation to 44 mm Hg for a total of 101 min over a 3-h period in rats with a blood alcohol level of 110 mmol/1, does not lead to permanent structural damage in the brain (Cragg and Phillips 1984). It is known that a proportion of human alcoholics with demonstrable brain disturbances possess liver disease (Fox et al. 1976; Acker et al. 1982) and the resulting metabolic disturbances may represent a biochemical threat to the brain in the long term. Reduced cerebral blood flow is a possible acute biophysical effect of ethanol which can be tested with radiological techniques (Caille et al. 1978; Drayer et al. 1980). Experimental studies with rats indicate that both cerebral blood flow and oxygen consumption are reduced during an acute intoxicating period, but these effects are less marked after repeated intoxications over 3 or 4 days (Hemmingsen and Barry 1979). At clinically observed levels, alcohol has been found to induce spasms of the cerebral vasculature in rats (Altura et al. 1983), but it has not been demonstrated that this leads to nerve tissue damage. Even a brain tissue concentration of 3.8~o (3080rag/g) ethanol maintained for 1 h does not produce cerebral degeneration in the adult rat detectable with the electron microscope (Phillips et al. 1981). It is known that the blood vessels of the brain are morphologically and functionally distinguishable from blood vessels elsewhere in the body (Rapoport, 1976). Thus, Moskow et al. (1973) may be in error to have assumed that their observations of leaking of the endothelium and subsequent haemorrhages in the conjunctiva are a reflection of events concurrently occurring in the brain. Indeed, both acute (1 h) and chronic (6 months) exposure of alcohol to rats does not significantly alter the permeability of the blood-brain barrier to sucrose (Phillips and Cragg 1982). Moreover, in a study of the histological changes produced by dextran-induced experimental red cell agglutination in rabbits, Stalker (1967) found sites of hypoxic damage in the liver and heart muscle to be "present in every 2 or 3 low-power fields", yet in the same animals, changes in the brain "were so slight that they were not useful indicators of damage". It remains that although the human alcoholic does display brain disturbances in radiological scans (Fox et al. 1976) and postmortem histology (Harper 1982), there is no direct evidence that blood cell agglutination is the principle mediator of these changes.
4~ ACKNOWLEDGEMENT
The author expresses his thanks towards Dr. Brian Cragg for his contribution to this work. REFERENCES Acker, W., E. J. Aps, S. K. Majumbar, G. K. Shaw and A. D. Thomson (1982) The relationship between brain and liver damage in chronic alcoholic patients, J. Neurol. Neurosurg. Psychol., 45: 984-987. Altura, B.M., B.T. Altura and A. Gebrewold (1983) Alcohol-induced spasms of cerebral blood vessels - Relation to cerebrovascular accidents and sudden death, Science, 220:331-333. Anker, R.L. and B.G. Cragg (1974) Development of the extrinsic connections of the visual cortex in the cat, J. Comp. Neurol., 154: 29-42. Caille, J., P. Constant, J. Billerey and A. Renou (1978) Variations in the measurement of regional blood volume by CAT, Computerized Tomography, 2: 63-68. Courville, C.B. (1966) Effects of Alcohol on the Nervous System of Man, San Lucus, Los Angeles, CA. Cragg, B.G. and S.C. Phillips (1984) A search for brain damage in a rat model of alcoholic sleep apnoea, Exp. Neurol., 80: 218-226. Drayer, B.P., D. Gur, S.K. Wolfson and E.E. Cook (1980) Abnormality of the xenon blood:brain co-efficient and blood flow in cerebral infarction - - An in vivo assessment using transmission computerized tomography, Radiology, 135: 349-354. Fink, R. P. and L. Heimer (1967)Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Res., 4: 369-374. Fox, J.H., R.G. Ramsey, M.S. Huckman and A.E, Proske (1976) Cerebral ventricular enlargement - Chronic alcoholics examined by computerized tomography, J. Amer. Med. Assoc., 236: 365-368. Harper, C. (1982) Neuropathology of brain damage caused by alcohol, Med. J. Aust., 2: 277-282. Hemmingsen, R. and D.I. Barry (1979) Adaptive changes in cerebral blood flow and oxygen consumption during ethanol intoxication in the rat, Acta Physiol. Scand., 106: 249-255. Moskow, H.A., R.C. Pennington and M.H. Knisely (1968) Alcohol, sludge, and hypoxic areas of nervous system, liver and heart, Microvasc. Res., 1: 174-185. Pennington, R.C. and M.H. Knisely (1973) Experiments aimed at separating the mechanical circulatory effects of ethanol from specific chemical effects, Ann. N.Y. Acad. Sci., 215: 356-365. Phillips, S.C. (1981) Does ethanol damage the blood-brain barrier?, J. Neurol. Sci., 50: 81-87. Phillips, S.C. and B.G. Cragg (1982) Weakening of the blood-brain barrier by alcohol-related stresses in the rat, J. Neurol. Sci., 54: 271-278. Phillips, S. C., B. G. Cragg and S.C. Singh (1981) The short-term toxicity of ethanol to neurons in rat cerebral cortex tested by topical application in vivo, and a note on a problem in estimating ethanol concentrations in tissue, J. Neurol. Sci., 49: 353-361. Rapoport, S.I. (1976) The Blood-Brain Barrier in Physiology and Medicine, Raven Press, New York, NY. Stalker, A. L. (1967) Histological changes produced by experimental erythrocytes aggregation, J. Path. Bact.. 93: 203-212.