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Ethanol and pentobarbital in combination increase blood-brain barrier permeability to horseradish peroxidase
Brain Research. 443 (1988) 12-20 Elsevier
12 BRE 13331
Ethanol and pentobarbital in combination increase blood-brain barrier permeability to horseradish peroxidase P.A. Stewart 1, E.M. Hayakawa 1 and P.L. Carlen 2 IDepartraent of Anatomy, Univers;.tyof Toronto, Toronto, Ont. (Canada) and 2Addiction Research Foundation, Clinical Institute, Department of Physiology and Medicine (Neurology), University of Toronto and Playfair Neuroscience lnstit,~te, Toronto, Ont. (Canada) (Accepted 18 August 1987)
Key words: Blood-brain barrier; Ethanol; Pentobarbital; Vascular permeability; Capillary ultrastructure
The structure and function of the blood-brain barrier (BBB) is determined mainly by the characteristics of brain capillary endothelial membranes. Lipophilic drugs that modify the cell membrane might be anticipated to alter the BBB. We investigated the effect of acute ethanol in combination with either a barbiturate or a non-barbiturate anesthetic on the ability of the rat BBB to exclude circulating horseradish peroxidase (HRP). Rats were injected into the peritoneal cavity with ethanol plus either a barbiturate (pentobarbital) or a non-barbiturate (ketamine hydrochioride) anesthetic. HRP was subsequently injected transcardially 30 s prior to decapitation. In the ethanol plus barbiturate-treated rats focal leakage of HRP caused peroxidase levels in the cerebral cortex to be about 8-fold higher than in ethanol plus ketamine hydrochloride-treated rats. UltrastructuraUy endothelial cells in leaking vascular segments were infiltrated with HRP and, in some eases, they were lysed so that the structural integrity of the blood-brain interface was Ios~:. Lysed segments were accompanied by staining of the adjacent basal lamina with HRP, and edematous astrocytic endfeet. These results show that ethanol plus a barbiturate anesthetic causes breakdown in the BBB by structurally damaging brain capillary endothelial cells. Whether the daniage is caused by the expansion and lysing of the cell membrane by these two lipophilic drugs, or by increased intracellular calcium to toxic levels is not yet known.
INTRODUCTION The term 'blood-brain harrier' (BBB) includes both functional and structural characteristics of the cerebral capillary endothelium that collectively allow the blood-brain interface to act as a selectively permeable membrane (for review, see Betz and Goldsteinl). Most hydrophilic molecules are not able to move passively across the barrier in significant amounts. However, certain nutritionally important molecules, that would be expected to be excluded on the basis of size and charge (e.g. hexoses, amino acids, nucleic acid precursors), cross the BBB selectively via membrane-bound transport molecules (reviewed by Pardridge24). The restrictive property of the BBB is the result of the continuous epithelial-like arrangement of the capillary endothelial cells. They are joined by contin-
uous tight junctions that prevent blood-borne molecules from passing between them 2'9'34. Endothelial vesicles, that are thought to be involved in non-specific vascular permeability in other tissues 6'7Aa'4°, are rare in brain capillary endothelium unde'r normal conditions. Fenestrae, that form pores in capillary walls in highly permeable vessels are absent in brain capillaries except in areas where the BBB is deftcient 6'8"1°, In all 3 of these presumptive permeability routes the cell memb~aiLe is the major structural component. The integrity of the BBB is dependent on the structural characteristics of brain capillary endothelial cells and their membranes. Drugs that alter membrane structural characteristics could hypothetically alter non-specific BBB permeability. Previous studies on the effect of ethanol on the BBB are seemingly inconsistent. Acute ethanol has been reported to
Correspondence.. P.A. Stewart, Department of Anatomy, University of Toronto, Toronto, Ont., M5S IA8 Canada. 0006-8993/,°,8/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
3 compromise the BBB under some circumstances ~s" 20.28.29but not others 3A7"27. The circumstances under which ethanol damages the barrier include additional stress such as starvat',on-s. specifically thiamine deficiency29, although neither of these alone affects the BBB. In two stud~.es showing a deleterious effect of ethanol on the Br3B 2°'28 a barbiturate anesthetic was also used. In contrast, in studies showing no effect of ethanol on the BBB ~7"27"35.a variety of non-barbiturate anesthetics were used. Barbiturates have a well recognized, but poorly understood, synergistic effect when used with alcohol. The objectives of the present study were to determine whether alcohol in combination with a barbiturate anesthetic causes any change in the integrity of the BBB and, if ~-o, what the ultrastrurtu,":! basis of this change is. The integrity of the B~B wa.,, assessed by its ability to exclude a circulating vascular tracer, horseradish peroxidase (HRP). HRP "loes not cross the intact BBB significantly and has the added advantages that its concentration can be measured spectrophotometricaily32 and it can be visualized in the light and electron microscopes. MATERIALS AND METHODS
Procedure Adult Wistar rats 200-300 g were obtained from Charles River, Canada. Rats were injected with varying doses of ethanol from 0 to 4 g/kg in a 25% solution (100% ethanol in Ringer's) into the peritoneal cavity 30 min prior to sacrifice. Ten min prior to sacrifice the animals were anesthetized with 65 mg/kg sodium pentobarbital (Somnotol ®, M.T.C. Pharmaceuticals, Mississauga, Ont.) into the peritoneal cavb ty. Alcohol alone did not anesthetize the animals to the point at which they could undergo opening of the chest cavity for withdrawal of blood and injection of HRP. A second, non-barbiturate anesthetic therefore was used in these animals. A combination of 100 mg/kg ketamine hydrochloride (Rogarsetic ®, Rogar/STB, London, Ont.) was injected intramuscularly in one thigh, plus 5 mg/kg accr~romazine (Atravet ®, Ayerst Labs., Montreal, Que.) intramuscularly in the other thigh. At the time of sacrifice the chest cavity was opened and approximately 1 ml of blood withdrawn from the right ventricle for serum alcohol determination. 5600
units of either type VI (Sigma, St. Louis, MO) or type II (Boehringer Mannheim) HRP dissolved in 0.2-0.3 ml 0.9% NaC! were injected into the left ventricle and the rats were decapitated 30 s later. Care was taken during the injection of HRP to insure that the injection was done slowly to avoid a sudden increase in intravascular pressure in the brain that might rupture blood vessels and lead to BBB disruption through trauma. Care was also taken to avoid mechanical damage to the brains when they were removed from the cranial cavities. One cerebral hemisphere was cut coronaily into two equally sized pieces and fixed by immersion in 2.5% glutaraldehyde, 0.6% glucose in 0.1 M phosphate buffer at pH 7.2. The parietal cortex plus a small amount of underlying white matter was oat from the other hemisphere and was homogenized in a 9x volume of 0.9% NaCI plus 5x volume of 1% Triton X - I ~ solution for HRP extraction (see below for assay).
Assessment of BBB permeability qhe permeability of the BBB in parietal cortex under experimental conditions was assessed by measuring the tissue peroxidase activity after HRP injection and comparing this to the activity in control animals. HRP activity in the cortical extracts was measured using a modification of the method of Raymond et al. 3:. Briefly, after homogenization the samples were kept at 4 °C for 30 min and then centrifuged 5 min at 12,500 g. Fifty/~l of supernatant were added to 3 ml of a 0.05% solution of diamino benzidine (DAB) plus 1% H20 2 in a cuvette, and the rate of change in absorption compared to that in a blank (0.9% saline + DAB + H20,) was measured using a Beckman Du-8 spectrophotometer for 130 s. Peroxidase a¢',ivity was expressed as the change in absorption/g wet wt./min. To determine the sites of BBB breakdown, peroxidase activity in sections of brain was visualized. Coronal slices 100/~m thick were cut from the parietal region of the fixed hemispheres using a vibratome. Peroxidase activity was demonstrated using she cob~lb glucose oxidase method described by Itoh et al. ~s
Electron microscopy Perfusion fixation is the method of choice for obtaining good tissue preservation for examination in the electron microscope. In our experience however, consistently good perfusion can only be obtained if
14 the vasculature is first cleared of blood with saline before infusion of fixative. Such treatment washes out much of the HRP that had extravasated during the 30-s circulation time and we were unable to locate the leaking vascular segments using this technique. To overcome this problem we used a rapi~ immersion fixation technique as follows: the basin of a vibratome was filled with fixative (2.5% glutaraldehyde, 0.6% glucose in 0.1 M phosphate buffer at pH 7.2). Brains from experimental and control rats were rapidly removed from the crania, glued to a vibratome chuck and immediately immersed in the fixative in the vibratome. Five to ten lf~0-gm coronal sections were cut within a few minutes of sacrifice of the animal. These sections were placed in fresh fixative for 2-3 h and then in two changes of 0.1 M phosphate buffer at pH 7.2. The sections were reacted for peroxidase activity as outlined above. Tissue fragments containing leaking vascular segments were identified using a dissecting microscope and cut out of the section, postfixed with osmium tetroxide and stained en bloc in 2% uranyl ace'~ate. The tissues were dehydrated in a graded series of ethanols, embedded in epon and sectioned on aa ultramicrotome. Half-micron sections were stained in To|uidine blue and examined in the light microscope. Thin sections of selected blocks were cut and mounted on slot grids coated with formvar, left unstained or stained lightly with uranyl acetate and examined using a Hitachi 7000 electron microscope.
Circulation time was, therefore, doubled to adjust for this and it was assumed that the HRP would perfuse the entire vascular tree in the brain and then be replaced by blood. We expected, therefore, to find HRP to be absent from the vascular lumena and present only in areas whe,e it had leaked through a compromised barrier into the brain parenchyma. What we found was that a significant amount of HRP remained in the iumena in both experimental and control rats (Fig. 1). In brain from rats that had not been injected with HRP, peroxidase activity can be found on!y in red blood cells (Fig. 1A). In the HRP-injected rats peroxidase activity was seen both in the red blood cells and also along the edges of the vessels (Fig. 1B). Presumably HRP binds to the surface layers of the endothelial cells. This 'retained' HRP contributed to the high peroxidase levels in HRP-injected brain compared with uninjected control brain (Table I). Effect o f ethanol and pentobarbital on B B B permeability (Table I) Endogenous levels of brain peroxidase were not affected by either pentobarbitai or alcohol. Peroxidase activity in cerebral cortex after 30 s of HRP circulation was the same in pentobarbital alone-treated rats as in ethanol plus ketamine hydrochloridetreated rats. Ethanol plus pentobarbital together
TABLE I Determination o f blood alcohol levels Serum alcohol was measured by gas chromatography using the method of Jain 19. A serum sample of one hundred ~1 was diluted with 100/~1 water containing isopropyl alcohol as an internal standard. This was injected directly into a ~;as chromatographic column (Halcomid M-18, 6' × 1/4") at an oven temperature of 108 °C.
Effect of pentobarbital, pentobarbital plus ethanol, and ketamine plus ethanol on BBB permeability to horseradish peroxidase in rat parietal cortex
Values expressed as means + S.E.M. Treatment
Pentobarbital
Dosage
65 mg/kg
no HRP
Peroxidase activity in cortex (AA/g wet wt. ~rain)
0.72 _+0.18 n = 5
RESULTS
Pentobarbital + Ethanol no HRP
65 mg/kg 4 g/kg
0.84 _+0.36 n=4
Distribution o f H R P After injection the HRP was allowed to ciculate for 30 s. The transit time for a bolus of solution injected into the carotid artery in rats is approximately 15 s 1-'. however cerebral blood flow is slowed by barbiturates 36.
Pentobarbital
65 mg/kg
10.85 + 1.51 n
=5
Ketamine Atravet/Ethanol
100 mg/kg 4 g/kg
11.75 + 1.21 n=5
Pentobarbital Ethanol
65 mg/kg 4 g/kg
82.73 + 30 n = 10
15
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Fig. 1. Peroxidase localization in rat brain. A: endogenous peroxidase activity primarily in red blood cells. B: after 30 s of H R P circulation peroxidase activity coats the walls of the vessels, x 100.
caused an 8-fold increase in the peroxidase levels of the cortex in HRP-injected rats.
tion was rejected on two g.",3unds; the rats whose heart rates durin,, HRP injection were observed to be slow or erratic did not correlate with high cortical
Effect of varying doses of ethanol in the tgresence of pentobarbital (Fig. 2) Ethanol in doses ranging from 0.2 to 4 g/kg was injected i.p. and the rats were subsequently anesthetized with 65 mg/kg pentobarbital as outlined above. Serum alcohol levels were measured and correlated with changes in cortical peroxidase. From Fig. 2 it can be seen that cortical peroxidase levels increased with increasing levels of serum ethanol. At high serum alcohol level,; brain peroxidase activity became highly variabie, in rats treated with ethanol and ketarnine/atravet the variation was considerably smaller. Cerebral blood flow is known to be depressed by both ethanol" and barbiturates 3<'. We questioned, therefore, whether low cerebral blood flow would result in slow clearance of HRP from the vasculature and, consequent high peroxidase activity. This explana-
250-
200-
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Fig. 2. Pero×idase activity in cerebral cortex after 30 s of HP.P circulation in rats treated with var~ing doses of ethanol plus either pentobarbital (filled triangles) or ketamine h vdrochloride (open triangles). The slope of the regression line for ethanol plus pentobarbilal is significantly greater than zero (P < (I.01). The slope of the • ,gression line for ethanol plus ket-
amine hydrochloride is zero.
16 peroxidase activity, and high peroxidase activity did correlate with the density of leaking segments (data not shown).
Ultrastructure of leaking vascular segments Leakage was not uniform but occurred at discrete loci (Fig. 3). At the light microscope level leakage seemed to be confined to penetrating arterioles, however using the electron microscope we also found abnormalities at the capillary level (see below). Occasional HRP-infiltrated endothelial cells were seen in rats treated with pentobarbital alone and with alcohol and a ketamine-atravet combination, however, these cells were always structurally intact. In contrast, in the alcohol plus pentobarbitai-treated rats vessel walls in the areas of leakage could be seen to have undergone a dramatic change. Many of the endothelial cells were grossly infiltrated and deeply stained by HRP (F'_.g.4). Furthermore, many of these
Fig. 3. Focal leakage of HRP in brain microvessels of a rat treated with ethanol (4 g/kg) and pentobarbital (65 mg/kg). xl19.
were lysed (Fig. 5) with consequent loss of continuity of the blood-brain interface: Staining of the basal lamina (Fig. 5) and swelling of the perivascular astrocytes (Fig. 4, asterisks) always accompanied such segments. Clearly, this loss of integrity of the endothelial layer is responsible for the leakage described in light micrographs. Endothelial cells in nonleaking segments in the same rat appeared normal (Fig. 6) although the membranes were not well demonstrated. Membranes expanded by anesthetics tend to twist and are difficult to visualize in the electron microscope (P. Seeman, personal communication), DISCUSSION
There are several known mechanisms by which the BBB breaks down, including osmotic stress, hypertension, trauma, inflammation and anoxia. None of these mechanisms is a likely explanation for the breakdown that we have observed in the presence of both ethanol and pentobarbital. Alcohol increases serum osmolarity by about 100150 mOsm 33. We calculated the expected rise in osmolarity due to the highest circulating levels of alcohol that we u~ed (approximately 100 mM/i) using the method in Redetzki et al. 33 and found that an additional 108 mOsm would be added to the normal value of approximately 290, for a total osmolarity of about 400 mOsm. Since osmotically induced barrier breakdown requires approximately 16013 mOsm at the blood-endothelial cell interface 31, and since alcohol rapidly partitions into the tissues so that its serum concentration drops, we eliminated this as a possible explanation for our results. Hypertension is similarly unlikely. Bath pentobarbitai 36 and ethanol 16 lower cerebral blood flow and blood pressure. Furthermore, 1 ml of blood was withdrawn prior to HRP injection and this would contribute further to hypotension. The slowing in cerebral blood flow may well have increased the retained intravascular HRP and thereby contribute to the slightly higher peroxidase activity in the pentobarbitaltreated rats at zero ethanol (Fig. 2). Decreased cerebral blood flow, however, is unlikely to explain the 8fold increase in peroxidase activity, and would not give rise to leaking vascular segments (Fig. 3) that we found in the ethanol plus pentobarbital-treated rats. Anoxia damages endothelial cells and leads to
17
Fig. 4. Brain capillary profile from an ethanol plus pentobarbital-treated rat. The endothelial cell (En) is infiltrated and deeply stained with HRP. The cell is structurally intact and HRP staining does not extend into the basal lamina (BLL Edematous astrocytic endfeet (asterisks) can be seen in association with the vessel wall. x 18,900.
BBB breakdown 26, however, at least 40 min of complete ischemia are required for this effect. In our rats, brain oxygenation may have been decreased due to the slowing of cerebral blood flow (see above) but they continued to have good color and robust respiration throughout the experimental period, and so clearly were not suffering from the profound anoxia that is required for barrier breakdown. Ultrastructural examination of the vessels in the leaking segments showed that the tracer (HRP) does not cross the vessel wall via one or more of the accepted permeability routes, but grossly infiltrates the endothelial cell. These cells appear similar to those damaged by stab wounds 25. In several cases the endothelial cell was clearly damaged (Fig. 5), resulting in a loss of continuity of the endothelial layer and consequent loss of integrity of the BBB.
We questioned whether The cell lysis occurred in the living animal, or whether it occurred during fixation as a result of prior weakening of the cells by ethanol plus pentobarbital. Although we cannot answer this directly, recent data show that, in ethanol plus pentobarbital-treated rats, the size of the extravasation spots and the total peroxidase activity of the brain increase with increasing HRP circulation time (Farrell and Stewart, unpublished results). This observation argues that _I3BB dantage occurs in the living animal and is not secondary to aldehyde treatment. The cellular mechanism that causes such damage to the endothelial cell is not clear. Both ethanol and barbiturates insert into the cell membrane and expand it and, if present in high enough concentrations, such drugs can also lyse the membrane 3'~. The obser-
18
Fig. 5. A iysed endothelial cell from a leaking segment in an ethanol plus pentobarbitai-treated rat brain. The luminal membrane seems to have disappeared (expected position indicated by arrowheads) and the cytoplasm is fragmented and heavily infiltrated with HRP. The basal lamina (BL) is also stained. Membrane profiles (asterisks) may represent the fragments of the lysed endothelial membrane (Er, erythrocyte), x43,224.
vation that H R P is able to infiltrate some endothelial cells in rats treated with pentobarbi~al alone or with alcohol and ketamine/atravet, suggests that both
Fig. 6. Portion of the wall of a capillary from a non-leaking segment in the brain of a rat treated with ethanol plus pentobarbital. HRP can be seen in the lumen but does not infiltrate the endothelial cell (En). x31,781.
pentobarbital and ethanol individually disrupt the endothelial membrane sufficiently to allow at least transient pores to develop that are large enough to admit HRP. In rats treated with ethanol and pentobarbital in combination, the disruption of the membrane may be sufficient to lyse it and cause destruction of the endothelial cell. If this is the case, the results reported here would predict that alcohol in combination with other highly lipid-soluble anesthetics might have a similar effect. Alternatively, endothelial cell lysis may be due to raised intracellular calcium. High intracelluar calcium levels are toxic to some cells 5"37. Ethanol (100 mM) is known to kill rat hepatocytes by increasing intracellular calcium 3~. Ethanol and barbiturates both tend to block inward calcium currents 4 presumably due to the inactivating action of raised intraceilular
19
c a l c i u m II . E t h a n o l also inhibits c a l c i u m influx in syna p t o s o m e s 21 as d o b a r b i t u r a t e s 22. E t h a n o l is also
cellular c a l c i u m to toxic levels.
k n o w n to disturb i n t r a c e l l u l a r c a l c i u m ion h o m e o s t a -
ACKNOWLEDGEMENTS
sis 14'-'3 w h i c h s h o u l d result in r a i s e d i n t r a c e l l u l a r ion c o n c e n t r a t i o n as has b e e n m e a s u r e d by P o z o s a n d
This w o r k w a s s u p p o r t e d by the M e d i c a l R e s e a r c h
O a k e s 3°. B a s e d o n t h e a b o v e we h y p o t h e s i z e t h a t an
C o u n c i l of C a n a d a . T h e a u t h o r s are g r a t e f u l to Mrs.
a l t e r n a t i v e m e c h a n i s m for t h e d e s t r u c t i v e a c t i o n o f
K. H a y a k a w a for excellent t e c h n i c a l assistance, to
e t h a n o l a n d p e n t o b a r b i t a l o n the b l o o d - b r a i n bar-
Dr. B. K a p u r for m e a s u r i n g s e r u m e t h a n o l levels and
rier m a y be due to t h e i r synergistically raising intra-
to Dr. P. S e e m a n for his helpful discussions.
REFERENCES
14 Garrett, K.M. and Ross, D.H., Effects of in vivo ethanol administration on Ca ~+/Mg + ÷ ATPase and ATP-dependent Ca ++ uptake activity in synaptosomal membranes, Neurochem. Res., 8 (1983) 1013-1028. 15 Gulati, A., Nath, C., Shanker, K. and Srimal, R.C., Effect of alcohols on the permeability of the blood-brain barrier, Phannacol. Res. Commun., 17 (1985)85-93. 16 Hemmingsen, R. and Barry, D.I., Adaptive changes in cerebral blood flow and oxygen consumption during ethanol intoxication in the rat, Acta Physiol. Scand.. 106 (1979) 249-255. 17 Hemmingsen, R., Hertz, M.M. and Bolwig, T.G., Integrity of the blood-brain barrier during ethanol intoxication and withdrawal in the rat: normal glucose transfer and permeability to Na + and Cl-, Stroke, 11 (1980) 141-144. 18 Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, A. and Sugimoto, T., Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method, Brain Research. 175 (1979) 341-346. 19 Jain, N.C., Direct blood injection method for gas chromatographic determination of alcohols and other volatile compounds, Clin. Chem., 17 (1971) 82-85. 20 Lee, J.C., Effect of alcohol injections on the blood-brain barrier, Q. J. Stud. Alcohol.. 23 (1962)4-15. 21 Leslie, S.W., Barr, E. and Chandler, L.J., Comparison of voltage-dependent 45 Ca ++ uptake rates by synaptosomes isolated from rat brain regions, J. Neurochem., 41 (1983) 1601-1605. 22 Leslie, S.W., Friedman, M.B., Wilcox, R.E. and EIrond, S.V., Acute and chronic effects of barbiturates on depolarization-induced calcium influx into rat synaptosomes, Brain Research, 185 (1980) 409-417. 23 Michaelis, M.L., Kitos, T.E, and Tehan, T., Differential effects of ethanol on two synaptic membrane Ca ÷÷ transport systems, Alcohol, 2 (1985) 129-132. 24 Pardridge, W.M., Transport of nutrients and hormones through the blood-brain barrier, Fed. Proc. Fed. Am. Soc. Exp. Biol.. 43 (1984) 201-204. 25 Persson, L. and Hansson, H.-A., Reversible blood-brain barrier dysfunction to peroxidasc after a small stab wound in the rat cerebral cortex, Acta Neuropathol.. 35 (1976) 333-342. 26 Petito, C.K., Pulsinelli, W.A., Jacobson, G. and Plum, F., Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction, J. Neuropathol. Exp. Neurol., 41 (1982)423-436. 27 Phillips, S.C., Does ethanol damage the blood-brain bar-
1 Betz, A.L. and Goldstein, G.W., Brain capillaries. Structure and function. In A. Lajtha (Ed.), Handbook of Neuroci~emistrv, 2nd edn., Plenum, London, 1984, pp. 465-484. 2 Brightman, M.W. and Reese, T.S., Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol., 40 (1969) 648-677. 3 Burov. Y.V., Borisenko, S.A., Kiia:~maa, K., Lekhtosalo, J. and Mannisto, P., Effect of a single injection of ethanol on permeability of the blood-brain barrier for ~4C-tyrosine, 14C-DOPA and horseradish peroxidase, Bull. Exp. Biol. Med., 101 (1986) 341-343. 4 Carlen, P.L., Gurevich, N. and O'Beirne, M., Electrophysiological evidence for increased calcium mediated potassium conductance by low-dose sedative-hypnotic drugs. In R.P. Rubin, G.B. Weiss and J.W. Putney (Eds.), Calcium in Biological Systems. Plenum, New York, 1985, pp. 193-200. 5 Chizzonite, R.A. and Zak, R., Calcium induced cell death: susceptibility of cardiac myocytes is age dependent, Science, 213 (1981) 508-511. 6 Coomber. B.L. and Stewart, P.A., Morphometric analysis of CNS microvascular endothelium, Microvasc. Res., 30 (1985) 99-115. 7 Coomber, B.I.. and Stewart, P.A., Three dimensional reconstruction of vesicles in endothelium of blood-brain barrier versus highly permeable microvesseis, Anat. Rec., 215 (1986) 256-261. 8 Dempsey, E.W., Neural and vascular ultrastructure of the area postrema in the rat, J. Comp. Neurol., 150 (1973) 1 7 7 - 2(10. 9 Dermietzel, R., Junctions in the central nervous system of the cat. IV. !nterendothe!ia! junctions of cerebral blood vessels from selected areas of brain, Cell Tissue Res., 164 (1975) 45-62. 10 Dermietzel, R. and Leibstein, A.G., The microvascular pattern and perivascular linings of the area postrema, Cell Tissue Res., 186 (1978) 97-110. 11 Eckert, R. and Chad, J.E., Inactivation of calcium channels, Prog. Biophys. Mol. Biol., 44 (1984)215-267. 12 Fenstermacher, J.D., Current models of blood-brain transfer, Trends Neurosci., 8 (1985) 449-453. 13 Frokjaer-Jensen, J., The plasmalemmal vesicular system in capillary endothelium. In K. Mebmer and F. Hammersen (Eds.), Structure and Function of Endothelial (?ells, Progress in Applied Microcirculation. Vol. !, Karger, Basel, 1983, pp. 17-34.
20 rier?, J. Neurol. Sci., 50 (1981)81-87. 28 Phillips, S.C. and Cragg, B.G,, Weakening of the bloodbrain barrier by alcohol related stresses in the rat, J. Neurol. Sci., 54 (1982) 271-278. 29 Phillips, S.C. and Cragg, B.G., Blood-brain barrier dysfunction in thiamine-deficient, alcohol treated rats, Acta Neuropathol., 62 (1984) 235-241. 30 Pozos, R.S. and Oakes, S.G., The effects of ethanol on the electrophysiology of calcium channels. In M. Galanter (Ed.), Developments in Alcohoi~rn, Vol. 5, Plenum, New York, 1987, pp. 327-345. 31 Rapoport, S.I., Hod, M. and Klatzo, I., Testing of a hypothesis for osmotic opening of the blood-brain barrier, Am. J. Physiol., 223 (1972) 323-331. 32 Raymond, J.J., Robertson, D.M., Dinsdale, H.B. and Nag, S., Pharmacological modification of blood-brain barrier permeability following a cold lesion, Can. J. Neurol. Sci., 11 (1984) 447-451. 33 Redetzki, H.M., Koerner, T.A., Hughes, J.R. and Smith, A.G., Osmometry in the evaluation of alcohol intoxication, Clin. Toxicol., 5 (1972) 343-363.
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12 BRE 13331
Ethanol and pentobarbital in combination increase blood-brain barrier permeability to horseradish peroxidase P.A. Stewart 1, E.M. Hayakawa 1 and P.L. Carlen 2 IDepartraent of Anatomy, Univers;.tyof Toronto, Toronto, Ont. (Canada) and 2Addiction Research Foundation, Clinical Institute, Department of Physiology and Medicine (Neurology), University of Toronto and Playfair Neuroscience lnstit,~te, Toronto, Ont. (Canada) (Accepted 18 August 1987)
Key words: Blood-brain barrier; Ethanol; Pentobarbital; Vascular permeability; Capillary ultrastructure
The structure and function of the blood-brain barrier (BBB) is determined mainly by the characteristics of brain capillary endothelial membranes. Lipophilic drugs that modify the cell membrane might be anticipated to alter the BBB. We investigated the effect of acute ethanol in combination with either a barbiturate or a non-barbiturate anesthetic on the ability of the rat BBB to exclude circulating horseradish peroxidase (HRP). Rats were injected into the peritoneal cavity with ethanol plus either a barbiturate (pentobarbital) or a non-barbiturate (ketamine hydrochioride) anesthetic. HRP was subsequently injected transcardially 30 s prior to decapitation. In the ethanol plus barbiturate-treated rats focal leakage of HRP caused peroxidase levels in the cerebral cortex to be about 8-fold higher than in ethanol plus ketamine hydrochloride-treated rats. UltrastructuraUy endothelial cells in leaking vascular segments were infiltrated with HRP and, in some eases, they were lysed so that the structural integrity of the blood-brain interface was Ios~:. Lysed segments were accompanied by staining of the adjacent basal lamina with HRP, and edematous astrocytic endfeet. These results show that ethanol plus a barbiturate anesthetic causes breakdown in the BBB by structurally damaging brain capillary endothelial cells. Whether the daniage is caused by the expansion and lysing of the cell membrane by these two lipophilic drugs, or by increased intracellular calcium to toxic levels is not yet known.
INTRODUCTION The term 'blood-brain harrier' (BBB) includes both functional and structural characteristics of the cerebral capillary endothelium that collectively allow the blood-brain interface to act as a selectively permeable membrane (for review, see Betz and Goldsteinl). Most hydrophilic molecules are not able to move passively across the barrier in significant amounts. However, certain nutritionally important molecules, that would be expected to be excluded on the basis of size and charge (e.g. hexoses, amino acids, nucleic acid precursors), cross the BBB selectively via membrane-bound transport molecules (reviewed by Pardridge24). The restrictive property of the BBB is the result of the continuous epithelial-like arrangement of the capillary endothelial cells. They are joined by contin-
uous tight junctions that prevent blood-borne molecules from passing between them 2'9'34. Endothelial vesicles, that are thought to be involved in non-specific vascular permeability in other tissues 6'7Aa'4°, are rare in brain capillary endothelium unde'r normal conditions. Fenestrae, that form pores in capillary walls in highly permeable vessels are absent in brain capillaries except in areas where the BBB is deftcient 6'8"1°, In all 3 of these presumptive permeability routes the cell memb~aiLe is the major structural component. The integrity of the BBB is dependent on the structural characteristics of brain capillary endothelial cells and their membranes. Drugs that alter membrane structural characteristics could hypothetically alter non-specific BBB permeability. Previous studies on the effect of ethanol on the BBB are seemingly inconsistent. Acute ethanol has been reported to
Correspondence.. P.A. Stewart, Department of Anatomy, University of Toronto, Toronto, Ont., M5S IA8 Canada. 0006-8993/,°,8/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
3 compromise the BBB under some circumstances ~s" 20.28.29but not others 3A7"27. The circumstances under which ethanol damages the barrier include additional stress such as starvat',on-s. specifically thiamine deficiency29, although neither of these alone affects the BBB. In two stud~.es showing a deleterious effect of ethanol on the Br3B 2°'28 a barbiturate anesthetic was also used. In contrast, in studies showing no effect of ethanol on the BBB ~7"27"35.a variety of non-barbiturate anesthetics were used. Barbiturates have a well recognized, but poorly understood, synergistic effect when used with alcohol. The objectives of the present study were to determine whether alcohol in combination with a barbiturate anesthetic causes any change in the integrity of the BBB and, if ~-o, what the ultrastrurtu,":! basis of this change is. The integrity of the B~B wa.,, assessed by its ability to exclude a circulating vascular tracer, horseradish peroxidase (HRP). HRP "loes not cross the intact BBB significantly and has the added advantages that its concentration can be measured spectrophotometricaily32 and it can be visualized in the light and electron microscopes. MATERIALS AND METHODS
Procedure Adult Wistar rats 200-300 g were obtained from Charles River, Canada. Rats were injected with varying doses of ethanol from 0 to 4 g/kg in a 25% solution (100% ethanol in Ringer's) into the peritoneal cavity 30 min prior to sacrifice. Ten min prior to sacrifice the animals were anesthetized with 65 mg/kg sodium pentobarbital (Somnotol ®, M.T.C. Pharmaceuticals, Mississauga, Ont.) into the peritoneal cavb ty. Alcohol alone did not anesthetize the animals to the point at which they could undergo opening of the chest cavity for withdrawal of blood and injection of HRP. A second, non-barbiturate anesthetic therefore was used in these animals. A combination of 100 mg/kg ketamine hydrochloride (Rogarsetic ®, Rogar/STB, London, Ont.) was injected intramuscularly in one thigh, plus 5 mg/kg accr~romazine (Atravet ®, Ayerst Labs., Montreal, Que.) intramuscularly in the other thigh. At the time of sacrifice the chest cavity was opened and approximately 1 ml of blood withdrawn from the right ventricle for serum alcohol determination. 5600
units of either type VI (Sigma, St. Louis, MO) or type II (Boehringer Mannheim) HRP dissolved in 0.2-0.3 ml 0.9% NaC! were injected into the left ventricle and the rats were decapitated 30 s later. Care was taken during the injection of HRP to insure that the injection was done slowly to avoid a sudden increase in intravascular pressure in the brain that might rupture blood vessels and lead to BBB disruption through trauma. Care was also taken to avoid mechanical damage to the brains when they were removed from the cranial cavities. One cerebral hemisphere was cut coronaily into two equally sized pieces and fixed by immersion in 2.5% glutaraldehyde, 0.6% glucose in 0.1 M phosphate buffer at pH 7.2. The parietal cortex plus a small amount of underlying white matter was oat from the other hemisphere and was homogenized in a 9x volume of 0.9% NaCI plus 5x volume of 1% Triton X - I ~ solution for HRP extraction (see below for assay).
Assessment of BBB permeability qhe permeability of the BBB in parietal cortex under experimental conditions was assessed by measuring the tissue peroxidase activity after HRP injection and comparing this to the activity in control animals. HRP activity in the cortical extracts was measured using a modification of the method of Raymond et al. 3:. Briefly, after homogenization the samples were kept at 4 °C for 30 min and then centrifuged 5 min at 12,500 g. Fifty/~l of supernatant were added to 3 ml of a 0.05% solution of diamino benzidine (DAB) plus 1% H20 2 in a cuvette, and the rate of change in absorption compared to that in a blank (0.9% saline + DAB + H20,) was measured using a Beckman Du-8 spectrophotometer for 130 s. Peroxidase a¢',ivity was expressed as the change in absorption/g wet wt./min. To determine the sites of BBB breakdown, peroxidase activity in sections of brain was visualized. Coronal slices 100/~m thick were cut from the parietal region of the fixed hemispheres using a vibratome. Peroxidase activity was demonstrated using she cob~lb glucose oxidase method described by Itoh et al. ~s
Electron microscopy Perfusion fixation is the method of choice for obtaining good tissue preservation for examination in the electron microscope. In our experience however, consistently good perfusion can only be obtained if
14 the vasculature is first cleared of blood with saline before infusion of fixative. Such treatment washes out much of the HRP that had extravasated during the 30-s circulation time and we were unable to locate the leaking vascular segments using this technique. To overcome this problem we used a rapi~ immersion fixation technique as follows: the basin of a vibratome was filled with fixative (2.5% glutaraldehyde, 0.6% glucose in 0.1 M phosphate buffer at pH 7.2). Brains from experimental and control rats were rapidly removed from the crania, glued to a vibratome chuck and immediately immersed in the fixative in the vibratome. Five to ten lf~0-gm coronal sections were cut within a few minutes of sacrifice of the animal. These sections were placed in fresh fixative for 2-3 h and then in two changes of 0.1 M phosphate buffer at pH 7.2. The sections were reacted for peroxidase activity as outlined above. Tissue fragments containing leaking vascular segments were identified using a dissecting microscope and cut out of the section, postfixed with osmium tetroxide and stained en bloc in 2% uranyl ace'~ate. The tissues were dehydrated in a graded series of ethanols, embedded in epon and sectioned on aa ultramicrotome. Half-micron sections were stained in To|uidine blue and examined in the light microscope. Thin sections of selected blocks were cut and mounted on slot grids coated with formvar, left unstained or stained lightly with uranyl acetate and examined using a Hitachi 7000 electron microscope.
Circulation time was, therefore, doubled to adjust for this and it was assumed that the HRP would perfuse the entire vascular tree in the brain and then be replaced by blood. We expected, therefore, to find HRP to be absent from the vascular lumena and present only in areas whe,e it had leaked through a compromised barrier into the brain parenchyma. What we found was that a significant amount of HRP remained in the iumena in both experimental and control rats (Fig. 1). In brain from rats that had not been injected with HRP, peroxidase activity can be found on!y in red blood cells (Fig. 1A). In the HRP-injected rats peroxidase activity was seen both in the red blood cells and also along the edges of the vessels (Fig. 1B). Presumably HRP binds to the surface layers of the endothelial cells. This 'retained' HRP contributed to the high peroxidase levels in HRP-injected brain compared with uninjected control brain (Table I). Effect o f ethanol and pentobarbital on B B B permeability (Table I) Endogenous levels of brain peroxidase were not affected by either pentobarbitai or alcohol. Peroxidase activity in cerebral cortex after 30 s of HRP circulation was the same in pentobarbital alone-treated rats as in ethanol plus ketamine hydrochloridetreated rats. Ethanol plus pentobarbital together
TABLE I Determination o f blood alcohol levels Serum alcohol was measured by gas chromatography using the method of Jain 19. A serum sample of one hundred ~1 was diluted with 100/~1 water containing isopropyl alcohol as an internal standard. This was injected directly into a ~;as chromatographic column (Halcomid M-18, 6' × 1/4") at an oven temperature of 108 °C.
Effect of pentobarbital, pentobarbital plus ethanol, and ketamine plus ethanol on BBB permeability to horseradish peroxidase in rat parietal cortex
Values expressed as means + S.E.M. Treatment
Pentobarbital
Dosage
65 mg/kg
no HRP
Peroxidase activity in cortex (AA/g wet wt. ~rain)
0.72 _+0.18 n = 5
RESULTS
Pentobarbital + Ethanol no HRP
65 mg/kg 4 g/kg
0.84 _+0.36 n=4
Distribution o f H R P After injection the HRP was allowed to ciculate for 30 s. The transit time for a bolus of solution injected into the carotid artery in rats is approximately 15 s 1-'. however cerebral blood flow is slowed by barbiturates 36.
Pentobarbital
65 mg/kg
10.85 + 1.51 n
=5
Ketamine Atravet/Ethanol
100 mg/kg 4 g/kg
11.75 + 1.21 n=5
Pentobarbital Ethanol
65 mg/kg 4 g/kg
82.73 + 30 n = 10
15
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Fig. 1. Peroxidase localization in rat brain. A: endogenous peroxidase activity primarily in red blood cells. B: after 30 s of H R P circulation peroxidase activity coats the walls of the vessels, x 100.
caused an 8-fold increase in the peroxidase levels of the cortex in HRP-injected rats.
tion was rejected on two g.",3unds; the rats whose heart rates durin,, HRP injection were observed to be slow or erratic did not correlate with high cortical
Effect of varying doses of ethanol in the tgresence of pentobarbital (Fig. 2) Ethanol in doses ranging from 0.2 to 4 g/kg was injected i.p. and the rats were subsequently anesthetized with 65 mg/kg pentobarbital as outlined above. Serum alcohol levels were measured and correlated with changes in cortical peroxidase. From Fig. 2 it can be seen that cortical peroxidase levels increased with increasing levels of serum ethanol. At high serum alcohol level,; brain peroxidase activity became highly variabie, in rats treated with ethanol and ketarnine/atravet the variation was considerably smaller. Cerebral blood flow is known to be depressed by both ethanol" and barbiturates 3<'. We questioned, therefore, whether low cerebral blood flow would result in slow clearance of HRP from the vasculature and, consequent high peroxidase activity. This explana-
250-
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Fig. 2. Pero×idase activity in cerebral cortex after 30 s of HP.P circulation in rats treated with var~ing doses of ethanol plus either pentobarbital (filled triangles) or ketamine h vdrochloride (open triangles). The slope of the regression line for ethanol plus pentobarbilal is significantly greater than zero (P < (I.01). The slope of the • ,gression line for ethanol plus ket-
amine hydrochloride is zero.
16 peroxidase activity, and high peroxidase activity did correlate with the density of leaking segments (data not shown).
Ultrastructure of leaking vascular segments Leakage was not uniform but occurred at discrete loci (Fig. 3). At the light microscope level leakage seemed to be confined to penetrating arterioles, however using the electron microscope we also found abnormalities at the capillary level (see below). Occasional HRP-infiltrated endothelial cells were seen in rats treated with pentobarbital alone and with alcohol and a ketamine-atravet combination, however, these cells were always structurally intact. In contrast, in the alcohol plus pentobarbitai-treated rats vessel walls in the areas of leakage could be seen to have undergone a dramatic change. Many of the endothelial cells were grossly infiltrated and deeply stained by HRP (F'_.g.4). Furthermore, many of these
Fig. 3. Focal leakage of HRP in brain microvessels of a rat treated with ethanol (4 g/kg) and pentobarbital (65 mg/kg). xl19.
were lysed (Fig. 5) with consequent loss of continuity of the blood-brain interface: Staining of the basal lamina (Fig. 5) and swelling of the perivascular astrocytes (Fig. 4, asterisks) always accompanied such segments. Clearly, this loss of integrity of the endothelial layer is responsible for the leakage described in light micrographs. Endothelial cells in nonleaking segments in the same rat appeared normal (Fig. 6) although the membranes were not well demonstrated. Membranes expanded by anesthetics tend to twist and are difficult to visualize in the electron microscope (P. Seeman, personal communication), DISCUSSION
There are several known mechanisms by which the BBB breaks down, including osmotic stress, hypertension, trauma, inflammation and anoxia. None of these mechanisms is a likely explanation for the breakdown that we have observed in the presence of both ethanol and pentobarbital. Alcohol increases serum osmolarity by about 100150 mOsm 33. We calculated the expected rise in osmolarity due to the highest circulating levels of alcohol that we u~ed (approximately 100 mM/i) using the method in Redetzki et al. 33 and found that an additional 108 mOsm would be added to the normal value of approximately 290, for a total osmolarity of about 400 mOsm. Since osmotically induced barrier breakdown requires approximately 16013 mOsm at the blood-endothelial cell interface 31, and since alcohol rapidly partitions into the tissues so that its serum concentration drops, we eliminated this as a possible explanation for our results. Hypertension is similarly unlikely. Bath pentobarbitai 36 and ethanol 16 lower cerebral blood flow and blood pressure. Furthermore, 1 ml of blood was withdrawn prior to HRP injection and this would contribute further to hypotension. The slowing in cerebral blood flow may well have increased the retained intravascular HRP and thereby contribute to the slightly higher peroxidase activity in the pentobarbitaltreated rats at zero ethanol (Fig. 2). Decreased cerebral blood flow, however, is unlikely to explain the 8fold increase in peroxidase activity, and would not give rise to leaking vascular segments (Fig. 3) that we found in the ethanol plus pentobarbital-treated rats. Anoxia damages endothelial cells and leads to
17
Fig. 4. Brain capillary profile from an ethanol plus pentobarbital-treated rat. The endothelial cell (En) is infiltrated and deeply stained with HRP. The cell is structurally intact and HRP staining does not extend into the basal lamina (BLL Edematous astrocytic endfeet (asterisks) can be seen in association with the vessel wall. x 18,900.
BBB breakdown 26, however, at least 40 min of complete ischemia are required for this effect. In our rats, brain oxygenation may have been decreased due to the slowing of cerebral blood flow (see above) but they continued to have good color and robust respiration throughout the experimental period, and so clearly were not suffering from the profound anoxia that is required for barrier breakdown. Ultrastructural examination of the vessels in the leaking segments showed that the tracer (HRP) does not cross the vessel wall via one or more of the accepted permeability routes, but grossly infiltrates the endothelial cell. These cells appear similar to those damaged by stab wounds 25. In several cases the endothelial cell was clearly damaged (Fig. 5), resulting in a loss of continuity of the endothelial layer and consequent loss of integrity of the BBB.
We questioned whether The cell lysis occurred in the living animal, or whether it occurred during fixation as a result of prior weakening of the cells by ethanol plus pentobarbital. Although we cannot answer this directly, recent data show that, in ethanol plus pentobarbital-treated rats, the size of the extravasation spots and the total peroxidase activity of the brain increase with increasing HRP circulation time (Farrell and Stewart, unpublished results). This observation argues that _I3BB dantage occurs in the living animal and is not secondary to aldehyde treatment. The cellular mechanism that causes such damage to the endothelial cell is not clear. Both ethanol and barbiturates insert into the cell membrane and expand it and, if present in high enough concentrations, such drugs can also lyse the membrane 3'~. The obser-
18
Fig. 5. A iysed endothelial cell from a leaking segment in an ethanol plus pentobarbitai-treated rat brain. The luminal membrane seems to have disappeared (expected position indicated by arrowheads) and the cytoplasm is fragmented and heavily infiltrated with HRP. The basal lamina (BL) is also stained. Membrane profiles (asterisks) may represent the fragments of the lysed endothelial membrane (Er, erythrocyte), x43,224.
vation that H R P is able to infiltrate some endothelial cells in rats treated with pentobarbi~al alone or with alcohol and ketamine/atravet, suggests that both
Fig. 6. Portion of the wall of a capillary from a non-leaking segment in the brain of a rat treated with ethanol plus pentobarbital. HRP can be seen in the lumen but does not infiltrate the endothelial cell (En). x31,781.
pentobarbital and ethanol individually disrupt the endothelial membrane sufficiently to allow at least transient pores to develop that are large enough to admit HRP. In rats treated with ethanol and pentobarbital in combination, the disruption of the membrane may be sufficient to lyse it and cause destruction of the endothelial cell. If this is the case, the results reported here would predict that alcohol in combination with other highly lipid-soluble anesthetics might have a similar effect. Alternatively, endothelial cell lysis may be due to raised intracellular calcium. High intracelluar calcium levels are toxic to some cells 5"37. Ethanol (100 mM) is known to kill rat hepatocytes by increasing intracellular calcium 3~. Ethanol and barbiturates both tend to block inward calcium currents 4 presumably due to the inactivating action of raised intraceilular
19
c a l c i u m II . E t h a n o l also inhibits c a l c i u m influx in syna p t o s o m e s 21 as d o b a r b i t u r a t e s 22. E t h a n o l is also
cellular c a l c i u m to toxic levels.
k n o w n to disturb i n t r a c e l l u l a r c a l c i u m ion h o m e o s t a -
ACKNOWLEDGEMENTS
sis 14'-'3 w h i c h s h o u l d result in r a i s e d i n t r a c e l l u l a r ion c o n c e n t r a t i o n as has b e e n m e a s u r e d by P o z o s a n d
This w o r k w a s s u p p o r t e d by the M e d i c a l R e s e a r c h
O a k e s 3°. B a s e d o n t h e a b o v e we h y p o t h e s i z e t h a t an
C o u n c i l of C a n a d a . T h e a u t h o r s are g r a t e f u l to Mrs.
a l t e r n a t i v e m e c h a n i s m for t h e d e s t r u c t i v e a c t i o n o f
K. H a y a k a w a for excellent t e c h n i c a l assistance, to
e t h a n o l a n d p e n t o b a r b i t a l o n the b l o o d - b r a i n bar-
Dr. B. K a p u r for m e a s u r i n g s e r u m e t h a n o l levels and
rier m a y be due to t h e i r synergistically raising intra-
to Dr. P. S e e m a n for his helpful discussions.
REFERENCES
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