- Email: [email protected]
Protective effects of etomidate in hypoxic-ischemic brain damage in the rat. A morphologic assessment
76, 181-195
EXPERIMENTALNEUROLOGY
(1982)
Protective Effects of Etomidate in Hypoxic-lschemic Brain Damage in the Rat. A Morphologic Assessment Jos VAN REEMPTS,
Laboratories
MARCEL BORGERS, JAN VAN CARLO HERMANS’
of Cell Biology
Received
August
and Applied Pharmacology, B-2340 Beerse. Belgium 14, 1981;
revision
received
EYNDHOVEN,
Janssen
November
AND
Pharmaceutics,
10, 1981
Cerebral damage was induced in rats by intermittent exposure to pure nitrogen subsequent to right carotid artery ligation (“Levine preparation”). After a survival period of 24 h all animals developed typical ischemic lesions in the parietal cortex and to a lesser extent in the hippocampus of the hemisphere ipsilateral to the interrupted blood flow. The contralateral hemispheres were almost devoid of damage. The morphologic appearance of the brain tissue of animals pretreated with etomidate at a dose sufficient to induce hypnosis during the nitrogen exposures, resembled that of normoxic animals, suggesting that this drug preserved the structural integrity of the brain tissue. A hypnotic dose of thiopental also reduced neuronal damage to some extent. When exposure to nitrogen started after the rat’s complete recovery from hypnosis, etomidate still significantly suppressed the formation of lesions. These results indicate that etomidate, in addition to its hypnotic and anticonvulsant activities, is very effective in preventing cerebral damage induced by a severe hypoxic-ischemic insult and that the hypnotic effect does not seem to be essential in assuring this protection. There are several possible mechanisms by which this drug exerts its protective effects.
INTRODUCTION Little or no histologic data are available on pharmacological protection against brain cell damage. One major reason for this is the paucity of suitable models in which reproducible brain damage can be induced in the ’ Correspondence should be Janssen Pharmaceutics, B-2340 of Dr. A. Wauquier, Professor Mr. L. Wouters for statistical their technical assistance, and
addressed to Mr. Van Beerse, Belgium. The R. Reneman, and Mr. evaluation, to Mr. L. to Mrs. D. Verkuringen
Reempts, Laboratory of Cell Biology, authors appreciate the valuable advice T. Jageneau. They are also grateful to Leijssen and Mrs. M. Van de Ven for for typing the manuscript.
181 0014-4886/82/040181-15$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
182
VAN
REEMPTS
ET AL.
conscious rat. The hypoxic-ischemic rat model, previously described by Levine (14) was shown to be a reliable model for this purpose. In this “Levine preparation” one hemisphere of the forebrain is made more vulnerable to hypoxia by the ligation of a common carotid artery. Intermittent exposure of such animals to pure nitrogen induces progressive damage to the hemisphere whose blood supply has been altered. A comprehensive histologic description of the nature and distribution of early and late stages of nerve cell damage in this model was given by Brown and Brierley (5), making the Levine preparation especially suitable for the present morphologic study. Etomidate, R-( +) ethyl- 1-( 1-phenylethyl)- 1H-imidazole-5carboxylate, is a potent, short-acting, and safe nonbarbiturate hypnotic ( 11, 12) with prominent anticonvulsant and brain protective activities (26, 27, 29). The aim of this study was to add histologic evidence to these previously reported effects. MATERIALS
AND
METHODS
A total of 52 nonfasted male Wistar rats of approximately 250 g was used in this study. Carotid Artery Ligation. The right common carotid artery was dissected out under ether anesthesia. Blood flow in this artery was interrupted by cutting the vessel between two ligatures. Nitrogen Exposure. After a recuperation period of 3 to 4 h, each animal was exposed to pure nitrogen in a tightly closed Perspex chamber continuously flushed with nitrogen at a flow of 9 liters/min. Each exposure lasted 1 min and was followed by artificial ventilation using a rubber bulb, standardized to three periods of 10 inflations. The exposures were repeated nine times within a standard period of 40 min. The onset of convulsions and apnea was recorded. After the last nitrogen exposure all rats were housed in groups for 24 h. During that period water and food pellets were freely available. Treatment. The “Levine” rats were divided into six groups as shown in Table 1. Apart from these groups, four rats with only a ligated artery served as control. They were not exposed to nitrogen and remained untreated. Fixation. At the end of the 24-h survival period the animals were anesthetized with ether. After injection of heparin (0.15 ml; 5000 IU/ml) into the tail vein, the chest was opened, and the descending aorta clamped. Intracardiac perfusion was carried out through the left ventricle during 5 min with Karnovsky’s fixative (2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer 0.1 M, pH 7.4) at room temperature. After decapitation, the brain was left in the skull for at least 1 h, while immersed in
PROTECTION
AGAINST
HYPOXIC TABLE
183
BRAIN DAMAGE
1
Treatment Schedule of “Levine” Rats Exposed Intermittently to Pure Nitrogen after Ligation of the Right Common Carotid Artery Time of injection
Group”
Treatment
Dose S.C. (w/kg)
Before carotid ligation
I II III IV V VI
None Etomidate* Etomidate Etomidate Etomidate Thiopental’
10 10 2.5 2.5 20
-10 min -10 min -10 min
Before nitrogen exposure -10 -1 -10 -1 -10
min h min h min
a N = 8 for each group. * Etomidate; R 16 659, Janssen Pharmaceutics. ’ Thiopental; Abbott.
the same fixative. Using a stereotaxic apparatus, a 2-mm transverse slice was cut corresponding to the plane A2.2-A4.2 of the atlas of De Groot (8). After an overnight stay in the same fixative at room temperature, 200pm sections were cut with an Oxford Vibratome. Sections were postfixed in 1% 0~0~ buffered with Verona1 acetate, 0.05 M, pH 7.4 and impregnated in 0.5% uranium acetate. The parietal cortex and the hippocampus were excised, dehydrated in a graded series of ethanol, and routinely embedded in LX 112 epoxy resin (Ladd Ind.). Histologic Evaluation. Toluidine blue-stained sections (2-pm thickness) from selected regions of the right and left cortex and hippocampus were completely screened at the highest light microscope magnification. The occurrence of the four types of ischemic cell change as described by Brown and Brierley (5) was evaluated. Type I changes (microvacuolation), type II changes (ischemic cell change with or without incrustations), type III changes (severe cell change), and type IV changes (cell loss) were listed according to a three-point scale whereby 1 = occasionally cells involved, 2 = several, and 3 = many cells involved. The scores were given separately for lamina III, laminae IV-V, and lamina VI of the cortex and for the CAl, CA3, and CA4 layers of the hippocampus. Dark neurons or hydropic astrocytes, mainly due to incomplete fixation, were not taken into account. To obtain a global picture of cortical damage, the scores of the different layers were added for each particular type of change, a score of 9 thus indicating the maximum degree of damage (Fig. 10).
184
VAN REEMPTS
ET AL.
FIGS. l-3. Ipsilateral cortex of control rats with interrupted carotid flow and not exposed to nitrogen.
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
185
RESULTS All rats survived the hypoxic-ischemic insult. Only in a few rats was the perfusion fixation incomplete in some foci of the brain. These rats however, were not eliminated because the fixation artefacts, which were limited to the formation of dark cells, hydropic cells, and cells with a slightly different nuclear chromatin pattern, could easily be distinguished from the changes induced by hypoxic ischemia (Fig. 3). Ligated Control Rats. Four rats in which the common carotid artery was ligated, but which were not exposed to nitrogen were completely devoid of lesions 24 h later (Figs. l-3). The histologic appearance of both hemispheres was similar and comparable to that of normal rats, indicating an adequate fixation even in the hemisphere to which carotid flow was interrupted. Hypoxic-Ischemic Rats, Untreated. After 35 to 45 s of nitrogen exposure, all hypoxic-ischemic animals developed convulsions followed by apnea. Gasping started within 1 min. Reanimation became more difficult with increasing number of nitrogen exposures, so that additional periods of artificial ventilation were required. Twenty-four hours after exposure to the nitrogen all animals showed lesions varying in severity and extent. The lesions were most prominent in the ipsilateral neocortex (laminae III and VI). They were found only occasionally in the ipsilateral hippocampus. Except for two rats, mild lesions also were found in the contralateral neocortex. Four types of cell changes were encountered (Figs. 4-6). In type I changes (microvacuolation), the cells exhibited a dark cytoplasm filled with small vacuoles. The nucleus was densely stained with an almost normal chromatin pattern. These cells were occasionally found. Type II changes (ischemic cell change mostly with incrustations) were seen in all untreated rats. These cells were considerably shrunken, their nuclei were pyknotic and the cytoplasm and cell processes were very dense and clumped. Type III changes (severe cell change) were characterized by swelling of the nuclei, loss of the cytoplasmic density and occurrence of large membranous vacuolation in and around the cell body. Type IV changes (cell loss) were FIG. 1. Survey of the different cortical layers (I-VI) to show the absence of damage in the neuronal as well as the vascular compartments (X90). FIG. 2. Detail of lamina III. Neuronal cells (arrow) and astrocytes (arrow-head) appeared completely normal. Capillaries (c) were widely distended and perivascular spaces could not be recognized (X460). FIG. 3. Cortical area showing incomplete fixation. Changes consisted mainly of dark neurons (D) and hydropic astrocytes (H) which could easily be distinguished from hypoxic-ischemic changes (X570).
186
VAN
FIGS. 4-6.
Ipsilateral
REEMPTS
cortex
ET AL.
of hypoxic-ischemic
untreated
rats.
PROTECTION
AGAINST
HYPOXIC
TABLE
187
BRAIN DAMAGE
2
Number of Animals Showing the Various Types of Ischemic Brain Damage in the Ipsilateral Cortex Treatment” Etomidatc 2x 10 Wk& 8x., -10 min
Etomidate 1 x 10 w/W SC. -1 h
Etomidatc 2 x 2.5 me/b? s.c., -10 min
Etomidate I x 2.5 w/4’ s.c., -I h
Thiopental 2 x 20 w/k& s.c., -10 min
2 “.S.
2 n.s.
scare
NOM
21 Pd
"
n.s.
5~ 11 (ischemic cell change)
21
8
1
2
P
-
0.001
0.007
"3.
0.007
n.s.
Type III (severe cell change)
21 P
6 -
0 0.007
0 0.007
1 0.041
I 0.041
3 “3
Type IV (all loss)
bl P
4
0
0
0
I
2 "S.
Type of cell change
Type1 (microvacuolation)
0
I
tl.8.
I “A
n.s. 6
n.s.
2
"3.
Ncxr. Smre L I, ‘N = 8 for each group. b Bolus injections given 10 min before carotid artery ligation and again IO min &fore ’ Bolus injection given once at 1 h before nitrogen exposure. ‘P = Fisher two-tailed exact probability test; KS. = not significant.
Its.
nitrogen
6
exposure.
seen in a large part of the brains of rats that also showed type III damage. The latter two types of cell change involved both neurons and astrocytes and were responsible for the typical spongioid state of large regions of the brain. Perivascular spaces originating from swollen astrocytes were frequently seen. They were, however, not taken into account because they also regularly occurred in the normal brain after incomplete fixation. The severity and frequency of the different stages of cell change in the cerebral cortex and the proportion of animals with ischemic brain damage are summarized in Fig. 10 and Table 2. Hypoxic-Ischemic Rats, Etomidate-Treated. A high degree of protection was obtained after subcutaneous administration of etomidate (Figs. 7-10, Table 2). Bolus injections of 10 mg/kg given 10 min before carotid FIG. 4. Damage was most prominent in laminae III and VI and could be easily recognized by its spongioid appearance (arrows) (X90). FIG. 5. Mild damage consisting of type II (ischemic) cell change (arrows) and type III (severe) cell change (arrowhead) (X460). FIG. 6. Severe damage consisting mainly of type II (ischemic) cell change (arrows), type III (severe) cell change (arrowheads), type IV (cell loss) (double arrows) and perivascular edema (asterisks) (X460).
VAN
188
FIGS. 7-9. Ipsilateral
cortex
REEMPTS
ET AL.
of hypoxic-ischemic
rats treated
with
etomidate.
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
189
artery ligation and repeated 10 min before nitrogen exposure, or 10 mg/ kg given once at 1 h before nitrogen exposure, led to damage in only one of eight rats and two of eight rats, respectively. This damage was restricted to the ipsilateral hemisphere and consisted exclusively of very moderate microvacuolation or ischemic cell change. A small dose of 2.5 mg/kg given 10 min before carotid artery ligation and again 10 min before nitrogen exposure resulted in only moderate ischemic damage in all but two animals. When this small dose was given once at 1 h before nitrogen exposure, six rats were devoid of lesions, one rat showed mild lesions, and another one had severe lesions. None of the etomidate-treated rats showed lesions in the contralateral neocortex or the left and right hippocampus. Hypoxic-Ischemic rats, Thiopental-Treated. A moderate degree of protection against hypoxic-ischemic damage also was found after subcutaneous thiopental treatment (Fig. 10, Table 2). After the administration of 20 mg/kg thiopental, 10 min before ligation and again 10 min before nitrogen exposure, two of eight rats were devoid of lesions in the ipsilateral hemisphere. The lesions were moderate in five rats and one rat showed severe damage. Damage in the contralateral neocortex was seen in two rats. One rat also had lesions in the ipsilateral hippocampus. DISCUSSION In recent years cerebral hypoxia has been intensively studied ( 10, 17, 2 1,22,28). A large series of animal species has been used to experimentally induce different forms of lowered oxygen availability to the brain and disturbance of neuronal cell function. Oxygenation of the cell can be impaired at least in two ways: disturbance of cerebral blood flow (ischemic or oligemic hypoxia) and disturbance of oxygen supply (hypoxic, anemic, and histotoxic hypoxia; anoxia). The consequences of these insults can be evaluated by different parameters, i.e., biochemical (oxygen and glucose consumption, adenosine triphosphate and phosphocreatine depletion, lactate accumulation), physiological (electroencephalogram, hemodynamics), neurological (behavior, learning), and histological (morphologic and histochemical cell change). Pharmacological studies of the protection against FIG. 7. Survey of the cortical gray matter of a rat which received 10 mg/kg etomidate 1 h before start of the nitrogen exposures. The morphologic picture was fully comparable with that of normal animals. Roman numerals roughly indicate the different cortical layers (x90). FIG. 8. Rat treated with 10 mg/kg etomidate 10 min before ligation of the carotid artery and again 10 min before the nitrogen insult. No cellular (arrows) or vascular (arrowhead) alterations were seen (X460). FIG. 9. Rat treated with 2.5 mg/kg etomidate 10 min before ligation and again 10 min before nitrogen exposure. Next to normal cells (arrows), occasionally type II (ischemic) cell change was seen (arrow-heads). Capillaries (c) appeared unaltered (X460).
190
VAN
9
REEMPTS
ET AL.
Untrrrtcd
3J 91
Elomidrtr
2xlOmglkg
91
Etomidate
lxlOmg/kg
5.c.
-1Omin
S.C. -1h
----
9
-L-n
Etomidata
2x2.5mglkg
S.C. -1Omin
Elomidate
lxZ.Smg/kg
SC.
lhiopentrl
2x20
:
06 ::
1
7
mglkg
-1h
S.C. - 1Omin
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
191
cerebral hypoxic damage include treatment with barbiturates and etomidate (1, 10, 16, 17, 20, 21, 23, 24, 26, 27, 29). Pharmacologic protection against cell damage after hypoxic-ischemic insults is, to our knowledge, seldom morphologically evaluated. This is largely due to the fact that histologic changes appear relatively late in contrast to biochemical and physiologic changes. Moreover, ischemic damage is usually in small foci, difficult to localize, unless more severe hypoxia is imposed. But under such circumstances a high incidence of mortality is encountered in the untreated control animals. Another important factor which hampers morphological assessment is the difficult preservation of brain tissue. Histologic artefacts [i.e., dark cells and hydropic cells (7)] are almost unavoidable in human material; but also brains from large experimental animals are often inadequately fixed, giving rise to a wide variety of morphologic alterations which cannot be attributed to experimental hypoxic conditions, although they appear to be similar. Finally, one has to consider that many anesthetic agents, used during some hypoxic insults, may exert protective effects. The Levine preparation, in which the lesions are evoked in the conscious rat, offers a good outcome for such a morphologic approach. The rat brain is one of the best understood of all mammalian species. It can be fixed easily by perfusion techniques and extensive regions can be investigated. Moreover, the histopathologic changes originating after nitrogen exposure in rats with a ligated carotid artery are well documented (5-7, 14, 15). Neuronal and astrocytic changes are related both to the number of nitrogen exposures and to the survival time and they are largely restricted to the hemisphere ipsilateral to the ligated side. Thanks to these features, this model meets the criteria of reproducibility and practicability, enabling the evaluation of a drug with brain protective properties. The processes which are responsible for the induced injury are complex and far from understood. The neuron relies heavily on a continuous supply of oxygen and energy substrates, for it lacks adequate substrates to function anaerobically. In the Levine model two factors have to be considered. First, there is interruption of the blood flow in the right common carotid artery, an insult which is not harmful in itself, because oxygen and substrates are FIG. 10. Severity and frequency of cellular damage in the cerebral cortex of “Levine” rats. The figure summarizes the degree of damage in the ipsilateral (R) and contralateral (L) cortex of rats (N = 8 for each treatment group) which survived 24 h after unilateral carotidartery ligation and intermittent exposure to nitrogen. The occurrence of four types of cell change (0, type I: microvacuolation; EC& type II: ischemic cell change; m, type III: severe cell change; and I, type IV: cell loss) was scored, respectively, in laminae III, IV + V, and VI accordingly to a three-point scale whereby 1 = occasionally cells involved, 2 = several, and 3 = many cells involved. The scores of these different layers were added for each particular type of change, score 9 indicating thus the maximal degree of damage.
192
VAN
REEMPTS
ET Al.,.
adequately provided via the circle of Willis. Second, the arterial PO2 is repeatedly reduced to minimal values by exposure of the animal to the nitrogen environment. This reduction increases the cerebral blood flow fourfold on the contralateral side but only twofold on the side of the interrupted carotid artery (22). The unilateral “relative” ischemia does not limit substrate supply, but it reduces the supply of oxygen to values that can no longer sustain a normal energy balance (22). In addition these conditions can lead to cardiovascular failure in such a way that secondary ischemia occurs. It is widely accepted that the autoregulation of cerebral blood flow is abolished below a mean arterial pressure of 50 mm Hg (13). It is thus probably the lowering of arterial POz values below limits of 20 to 25 mm Hg (23), combined with a failure of cerebral blood flow autoregulation that is responsible for increased vulnerability of the ipsilateral hemisphere. Under conditions of severe hypoxia it is well known that membrane permeability is increased and that the cellular homeostasis for electrolytes is lost, resulting in the formation of edema. As for the myocardial cell (4), an increased influx of calcium ions also may be deleterious for the structural integrity and function of the neuronal cell. On the other hand, it cannot be excluded that during reanimation, probably as a result of reactive hyperemia, a sudden increase in oxygen supply may initiate deleterious oxidative reactions (22, 24). The results obtained with etomidate and thiopental indicate that both drugs have brain protective properties, either by reducing the severity of damage or by the total prevention of lesions. The effective doses of etomidate were considerably smaller than those of thiopental and most probably are devoid of secondary effects such as cardiac or respiratory depression (18). The protective effects of thiopental could not be enhanced because the animals, when pretreated with higher doses of thiopental, did not survive the hypoxic insult, probably owing to cardiopulmonary insufficiency. The treatment and prevention of cerebral hypoxia is aimed at preserving the balance between O2 demand and O2 supply. This can be obtained by changing the metabolic rate, blood flow, and arterial oxygen content. In some way etomidate may preserve the energy reserves in the neuron. In man it was shown that etomidate is an important cerebral metabolic depressant (19). The fact that the drug is very effective against both electrically and chemically produced convulsions (26, 27) suggests that the energy-consuming hypermetabolism during the ischemic and/or postischemic period is attenuated. Moreover, the results obtained in different models of hypoxiaischemia show a dose-related prolongation of brain function with etomidate (26,29). Etomidate pretreatment was also shown to prevent the behavioral
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
193
and histologic sequelae which develop after resuscitation of rats treated with a lethal intravenous dose of potassium cyanide (2). Hypnosis is not necessarily required to obtain brain protection with etomidate (27, 29). This is evidenced also in the present study: cerebral damage can be avoided even when nitrogen challenge is performed 1 h after S.C. administration of etomidate, when the rats are awake. A similar brain protective action in this model was recently demonstrated with flunarizine, a vasoactive substance that blocks stimulated Ca*+ fluxes through the plasma membrane [(3), J. Van Reempts, manuscript in preparation]. For barbiturates, inhibition of Ca2+ entry at cell membranes has already been reported (9). Although direct Ca2+ -entry blocking properties were not shown for etomidate, it might be that, through its interaction with plasma membrane components, it can counteract the deleterious ion fluxes which occur during and/or after hypoxia. This would explain not only the prevention of the Ca2+-induced hypermetabolism and the further exhaustion of energy reserves, but also the antiedematous effect. An indication for the latter is the absence of spongioid regions in the cortex of all etomidatetreated animals with the exception of one. A vascular action of this drug certainly cannot be excluded. Recent experiments on hemorrhagic shock in dogs (25) and in rats (C. Hermans, unpublished results) showed that etomidate not only rapidly increased mean aortic pressure, but also drastically prolonged the survival time in comparison with control and thiopental-treated animals. By allowing sufficient autoregulation or by providing adequate perfusion in vulnerable territories (5) the drug could guarantee not only a sufficient amount of oxygen, but also a necessary removal of waste products. Although this study cannot provide certainty about the working mechanism of etomidate in brain cell protection, it clearly shows that the drug is a potent antianoxic agent in a certain form of severe hypoxia in rats. The present results together with literature data about etomidate indicate that this drug warrants experimental and clinical research in the prevention of brain ischemia. REFERENCES 1. ANFRED, I., AND 0. SECHER. 1962. Anoxia and barbiturates. Arch. Int. Pharmacodyn. Ther. 89: 61-14. 2. ASHTON, D., J. VAN REEMPTS, AND A. WAUQUIER. 198 1. Behavioural, electroencephalographic and histological study of the protective effect of etomidate against histotoxic dysoxia produced by cyanide. Arch. Int. Pharmacodyn. Ther., in press. 3. BORGERS, M., F. THONE, AND J. M. VAN NUETEN. 1981. The subcellular distribution of calcium and the effects of calcium antagonists as evaluated with a combined oxalatepyroantimonate technique. Acta Histochem. Suppl. 24: 327-332.
194
VAN REEMPTS
ET AL.
4. BORGERS, M. 1981. The role of calcium in the toxicity of the myocardium. Histochem. J. 13: 839-848.
5. BROWN, A. W., AND J. B. BRIERLEY. 1968. The nature, distribution and earliest stages of anoxic-ischaemic nerve cell damage in the rat brain as defined by the optical microscope. Br. J. Exp. Pathol. 49: 87-106. 6. BROWN, A. W., AND J. B. BRIERLEY. 1973. The earliest alterations in the rat neurones and astrocytes after anoxia-ischemia. Acta Neuroparhol. 23: 9-22. 7. BROWN, A. W. 1977. Structural abnormalities in neurones. J. Clin. Pathol. 30 Suppl. (R. COB. Pathol.), 11: 155-169. 8. DE GROOT, J. 1963. The rat forebrain in stereotaxic coordinates. Verh. Kon. Ned. Akad. Wetensch. Afd. Nat. 52, no. 4. N. Hollandsche Uitgeversmaatschappij, Amsterdam. 9. ELROD, S. V., AND S. W. LESLIE. 1980. Acute and chronic effects of barbiturates on depolarization-induced calcium influx into synaptosomes from rat brain regions. J. Pharmacol. Exp. Ther. 212: 131-136. 10. HOSSMANN, K. A. 1979. Experimental basis for the treatment of cerebral ischemia. Pages 253-262 in M. GOLDSTEIN, L. BOLIS, F. FIESCHI, S. GORINA, AND C. H. MILLIKAN, Eds., Advances in Neurology, Vol. 25. Raven Press, New York. 11. JANSSEN, P. A. J., C. J. E. NIEMEGEERS, K. H. L. SCHELLEKENS, AND F. M. LENAERTS. 197 1. Etomidate, R-(+)-ethyl- 1-(a-methylbenzyl) imidazole-5-carboxylate (R 16 659). Arzneimittelforsch 21: 1234-1243. 12. JANSSEN, P. A. J., C. J. E. NIEMEGEERS, AND R. P. H. MARSBOOM. 1975. Etomidate, a potent non-barbiturate hypnotic. Intravenous etomidate in mice, rats, guinea pigs, rabbits and dogs. Arch. Int. Pharmacodyn. Ther. 214: 92-l 32. 13. KOV/~CH, A. G., AND P. SANDOR. 1976. Cerebral blood flow and brain function during hypotension and shock. Annu. Rev. Physiol. 38: 571-596. 14. LEVINE, S. 1960. Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 36: l-17. 15. MCGEE-RUSSELL, S. M., A. W. BROWN, AND J. B. BRIERLEY. 1970. A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain. Brain Res. 20: 193-200. 16. MICHENFELDER, J. D., AND R. A. THEYE. 1973. Cerebral protection by thiopental during hypoxia. Anesthesiology 39: 5 1O-5 17. 17. NEMOTO, E. M. 1978. Pathogenesis of cerebral ischemia-anoxia. Crif. Care Med. 6: 203214.
RENEMAN, R. S., A. M. H. JAGENEAU, R. XHONNEUX, AND P. LADURON. 1975. The cardiovascular pharmacology of etomidate (R 26 490), a new potent and short acting intravenous hypnotic agent. Pages 152-l 56 in A. ARIAS, Ed., Recent Progress in Anaesthesiology and Resuscitation: Proceedings (Eur. Gong. Anaesthesiol., 4th). Exerpta Medica Internat. Congr., Ser. No. 347. North-Holland/Elsevier, Amsterdam/New York. 19. RENOU, A. M., J. VERNHIET, P. MACREZ, P. CONSTANT, J. BILLEREY, M. Y. KHADAROO, AND J. M. CAILLE. 1978. Cerebral blood flow and metabolism during etomidate anaesthesia in man. Br. J. Anaesfhesiol. 50: 1047. 20. ROSSIGNOL, R., AND M. EBIGWEI-IBRU. 1980. Drugs against brain hypoxia. Trends 18.
Pharmacol.
Sci. 1: 287-289.
21. SAFAR, P., A. BLEYAERT, E. M. NEMOTO, J. MOOSSY, AND J. V. SNYDER. 1978. Resuscitation after global brain ischemia-anoxia. Crit. Care Med. 6: 215-227. 22. SIESJ& B. K. 1978. Brain Energy Metabolism. Wiley. New York/Chichester. 23. SMITH, A. L. 1977. Barbiturate protection in cerebral hypoxia. Anesthesiology 47: 285293.
PROTECTION
AGAINST
HYPOXIC
BRAIN DAMAGE
195
24. STEEN, P. A., AND J. D. MICHENFELDER. 1980. Mechanisms of barbiturate protection. Anesthesiology 53: 183-185. 25. WAUQUIER, A., C. HERMANS, W. A, E. VAN DEN BROECK, A. JAGENEAU, AND P. FRANCOIS. 1980. Etomidate vs. thiopental and pentobarbital in haemorrhagic dogs. 7th World Congress of Anaesthesiologists, Hamburg, Abstr. 295. 26. WAUQUIER, A., D. ASHTON, C. J. E. NIEMEGEERS, J. VAN REEMPTS, AND M. BORGERS. 1980. Etomidate: anti-ischemic and anti-anoxic actions in animals. 7th World Congress of Anaesthesiologists, Hamburg, Abstr. 216. 27. WAUQUIER, A., D. ASHTON, G. CLINCKE, C. J. E. NIEMEGEERS, AND P. A. J. JANSSEN. 1980. Etomidat, ein Barbituratfreies Hypnotikum: antikonvulsive, anti-anoxische und hirnprotektive Wirkung im Tierexperiment. Pages 183-203 in A. OPITZANDR. DEGEN, Eds., Anaesthesie bei Zerebralen Krampfanfallen und Intensivtherapie des Status Epilepticus. Verlags. Erlangen. 28. WAUQUIER, A., D. ASHTON, G. CLINCKE, AND J. VAN REEMPTS. 1981. Considerations on models and treatment of brain hypoxia. Pages 95-114 in M. W. VAN HOF AND G. MOHN, Eds., Developments in Neuroscience: Recovery from Brain Damage. Elsevier/ North-Holland Biomedical Press, Amsterdam. 29. WAUQUIER, A., D. ASHTON, G. CLINCKE, AND C. J. E. NIEMEGEERS. 198 1. Antihypoxic effects of etomidate, thiopental and methohexital. Arch. Int. Pharmacodyn. Ther. 249: 330-334.
EXPERIMENTALNEUROLOGY
(1982)
Protective Effects of Etomidate in Hypoxic-lschemic Brain Damage in the Rat. A Morphologic Assessment Jos VAN REEMPTS,
Laboratories
MARCEL BORGERS, JAN VAN CARLO HERMANS’
of Cell Biology
Received
August
and Applied Pharmacology, B-2340 Beerse. Belgium 14, 1981;
revision
received
EYNDHOVEN,
Janssen
November
AND
Pharmaceutics,
10, 1981
Cerebral damage was induced in rats by intermittent exposure to pure nitrogen subsequent to right carotid artery ligation (“Levine preparation”). After a survival period of 24 h all animals developed typical ischemic lesions in the parietal cortex and to a lesser extent in the hippocampus of the hemisphere ipsilateral to the interrupted blood flow. The contralateral hemispheres were almost devoid of damage. The morphologic appearance of the brain tissue of animals pretreated with etomidate at a dose sufficient to induce hypnosis during the nitrogen exposures, resembled that of normoxic animals, suggesting that this drug preserved the structural integrity of the brain tissue. A hypnotic dose of thiopental also reduced neuronal damage to some extent. When exposure to nitrogen started after the rat’s complete recovery from hypnosis, etomidate still significantly suppressed the formation of lesions. These results indicate that etomidate, in addition to its hypnotic and anticonvulsant activities, is very effective in preventing cerebral damage induced by a severe hypoxic-ischemic insult and that the hypnotic effect does not seem to be essential in assuring this protection. There are several possible mechanisms by which this drug exerts its protective effects.
INTRODUCTION Little or no histologic data are available on pharmacological protection against brain cell damage. One major reason for this is the paucity of suitable models in which reproducible brain damage can be induced in the ’ Correspondence should be Janssen Pharmaceutics, B-2340 of Dr. A. Wauquier, Professor Mr. L. Wouters for statistical their technical assistance, and
addressed to Mr. Van Beerse, Belgium. The R. Reneman, and Mr. evaluation, to Mr. L. to Mrs. D. Verkuringen
Reempts, Laboratory of Cell Biology, authors appreciate the valuable advice T. Jageneau. They are also grateful to Leijssen and Mrs. M. Van de Ven for for typing the manuscript.
181 0014-4886/82/040181-15$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
182
VAN
REEMPTS
ET AL.
conscious rat. The hypoxic-ischemic rat model, previously described by Levine (14) was shown to be a reliable model for this purpose. In this “Levine preparation” one hemisphere of the forebrain is made more vulnerable to hypoxia by the ligation of a common carotid artery. Intermittent exposure of such animals to pure nitrogen induces progressive damage to the hemisphere whose blood supply has been altered. A comprehensive histologic description of the nature and distribution of early and late stages of nerve cell damage in this model was given by Brown and Brierley (5), making the Levine preparation especially suitable for the present morphologic study. Etomidate, R-( +) ethyl- 1-( 1-phenylethyl)- 1H-imidazole-5carboxylate, is a potent, short-acting, and safe nonbarbiturate hypnotic ( 11, 12) with prominent anticonvulsant and brain protective activities (26, 27, 29). The aim of this study was to add histologic evidence to these previously reported effects. MATERIALS
AND
METHODS
A total of 52 nonfasted male Wistar rats of approximately 250 g was used in this study. Carotid Artery Ligation. The right common carotid artery was dissected out under ether anesthesia. Blood flow in this artery was interrupted by cutting the vessel between two ligatures. Nitrogen Exposure. After a recuperation period of 3 to 4 h, each animal was exposed to pure nitrogen in a tightly closed Perspex chamber continuously flushed with nitrogen at a flow of 9 liters/min. Each exposure lasted 1 min and was followed by artificial ventilation using a rubber bulb, standardized to three periods of 10 inflations. The exposures were repeated nine times within a standard period of 40 min. The onset of convulsions and apnea was recorded. After the last nitrogen exposure all rats were housed in groups for 24 h. During that period water and food pellets were freely available. Treatment. The “Levine” rats were divided into six groups as shown in Table 1. Apart from these groups, four rats with only a ligated artery served as control. They were not exposed to nitrogen and remained untreated. Fixation. At the end of the 24-h survival period the animals were anesthetized with ether. After injection of heparin (0.15 ml; 5000 IU/ml) into the tail vein, the chest was opened, and the descending aorta clamped. Intracardiac perfusion was carried out through the left ventricle during 5 min with Karnovsky’s fixative (2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer 0.1 M, pH 7.4) at room temperature. After decapitation, the brain was left in the skull for at least 1 h, while immersed in
PROTECTION
AGAINST
HYPOXIC TABLE
183
BRAIN DAMAGE
1
Treatment Schedule of “Levine” Rats Exposed Intermittently to Pure Nitrogen after Ligation of the Right Common Carotid Artery Time of injection
Group”
Treatment
Dose S.C. (w/kg)
Before carotid ligation
I II III IV V VI
None Etomidate* Etomidate Etomidate Etomidate Thiopental’
10 10 2.5 2.5 20
-10 min -10 min -10 min
Before nitrogen exposure -10 -1 -10 -1 -10
min h min h min
a N = 8 for each group. * Etomidate; R 16 659, Janssen Pharmaceutics. ’ Thiopental; Abbott.
the same fixative. Using a stereotaxic apparatus, a 2-mm transverse slice was cut corresponding to the plane A2.2-A4.2 of the atlas of De Groot (8). After an overnight stay in the same fixative at room temperature, 200pm sections were cut with an Oxford Vibratome. Sections were postfixed in 1% 0~0~ buffered with Verona1 acetate, 0.05 M, pH 7.4 and impregnated in 0.5% uranium acetate. The parietal cortex and the hippocampus were excised, dehydrated in a graded series of ethanol, and routinely embedded in LX 112 epoxy resin (Ladd Ind.). Histologic Evaluation. Toluidine blue-stained sections (2-pm thickness) from selected regions of the right and left cortex and hippocampus were completely screened at the highest light microscope magnification. The occurrence of the four types of ischemic cell change as described by Brown and Brierley (5) was evaluated. Type I changes (microvacuolation), type II changes (ischemic cell change with or without incrustations), type III changes (severe cell change), and type IV changes (cell loss) were listed according to a three-point scale whereby 1 = occasionally cells involved, 2 = several, and 3 = many cells involved. The scores were given separately for lamina III, laminae IV-V, and lamina VI of the cortex and for the CAl, CA3, and CA4 layers of the hippocampus. Dark neurons or hydropic astrocytes, mainly due to incomplete fixation, were not taken into account. To obtain a global picture of cortical damage, the scores of the different layers were added for each particular type of change, a score of 9 thus indicating the maximum degree of damage (Fig. 10).
184
VAN REEMPTS
ET AL.
FIGS. l-3. Ipsilateral cortex of control rats with interrupted carotid flow and not exposed to nitrogen.
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
185
RESULTS All rats survived the hypoxic-ischemic insult. Only in a few rats was the perfusion fixation incomplete in some foci of the brain. These rats however, were not eliminated because the fixation artefacts, which were limited to the formation of dark cells, hydropic cells, and cells with a slightly different nuclear chromatin pattern, could easily be distinguished from the changes induced by hypoxic ischemia (Fig. 3). Ligated Control Rats. Four rats in which the common carotid artery was ligated, but which were not exposed to nitrogen were completely devoid of lesions 24 h later (Figs. l-3). The histologic appearance of both hemispheres was similar and comparable to that of normal rats, indicating an adequate fixation even in the hemisphere to which carotid flow was interrupted. Hypoxic-Ischemic Rats, Untreated. After 35 to 45 s of nitrogen exposure, all hypoxic-ischemic animals developed convulsions followed by apnea. Gasping started within 1 min. Reanimation became more difficult with increasing number of nitrogen exposures, so that additional periods of artificial ventilation were required. Twenty-four hours after exposure to the nitrogen all animals showed lesions varying in severity and extent. The lesions were most prominent in the ipsilateral neocortex (laminae III and VI). They were found only occasionally in the ipsilateral hippocampus. Except for two rats, mild lesions also were found in the contralateral neocortex. Four types of cell changes were encountered (Figs. 4-6). In type I changes (microvacuolation), the cells exhibited a dark cytoplasm filled with small vacuoles. The nucleus was densely stained with an almost normal chromatin pattern. These cells were occasionally found. Type II changes (ischemic cell change mostly with incrustations) were seen in all untreated rats. These cells were considerably shrunken, their nuclei were pyknotic and the cytoplasm and cell processes were very dense and clumped. Type III changes (severe cell change) were characterized by swelling of the nuclei, loss of the cytoplasmic density and occurrence of large membranous vacuolation in and around the cell body. Type IV changes (cell loss) were FIG. 1. Survey of the different cortical layers (I-VI) to show the absence of damage in the neuronal as well as the vascular compartments (X90). FIG. 2. Detail of lamina III. Neuronal cells (arrow) and astrocytes (arrow-head) appeared completely normal. Capillaries (c) were widely distended and perivascular spaces could not be recognized (X460). FIG. 3. Cortical area showing incomplete fixation. Changes consisted mainly of dark neurons (D) and hydropic astrocytes (H) which could easily be distinguished from hypoxic-ischemic changes (X570).
186
VAN
FIGS. 4-6.
Ipsilateral
REEMPTS
cortex
ET AL.
of hypoxic-ischemic
untreated
rats.
PROTECTION
AGAINST
HYPOXIC
TABLE
187
BRAIN DAMAGE
2
Number of Animals Showing the Various Types of Ischemic Brain Damage in the Ipsilateral Cortex Treatment” Etomidatc 2x 10 Wk& 8x., -10 min
Etomidate 1 x 10 w/W SC. -1 h
Etomidatc 2 x 2.5 me/b? s.c., -10 min
Etomidate I x 2.5 w/4’ s.c., -I h
Thiopental 2 x 20 w/k& s.c., -10 min
2 “.S.
2 n.s.
scare
NOM
21 Pd
"
n.s.
5~ 11 (ischemic cell change)
21
8
1
2
P
-
0.001
0.007
"3.
0.007
n.s.
Type III (severe cell change)
21 P
6 -
0 0.007
0 0.007
1 0.041
I 0.041
3 “3
Type IV (all loss)
bl P
4
0
0
0
I
2 "S.
Type of cell change
Type1 (microvacuolation)
0
I
tl.8.
I “A
n.s. 6
n.s.
2
"3.
Ncxr. Smre L I, ‘N = 8 for each group. b Bolus injections given 10 min before carotid artery ligation and again IO min &fore ’ Bolus injection given once at 1 h before nitrogen exposure. ‘P = Fisher two-tailed exact probability test; KS. = not significant.
Its.
nitrogen
6
exposure.
seen in a large part of the brains of rats that also showed type III damage. The latter two types of cell change involved both neurons and astrocytes and were responsible for the typical spongioid state of large regions of the brain. Perivascular spaces originating from swollen astrocytes were frequently seen. They were, however, not taken into account because they also regularly occurred in the normal brain after incomplete fixation. The severity and frequency of the different stages of cell change in the cerebral cortex and the proportion of animals with ischemic brain damage are summarized in Fig. 10 and Table 2. Hypoxic-Ischemic Rats, Etomidate-Treated. A high degree of protection was obtained after subcutaneous administration of etomidate (Figs. 7-10, Table 2). Bolus injections of 10 mg/kg given 10 min before carotid FIG. 4. Damage was most prominent in laminae III and VI and could be easily recognized by its spongioid appearance (arrows) (X90). FIG. 5. Mild damage consisting of type II (ischemic) cell change (arrows) and type III (severe) cell change (arrowhead) (X460). FIG. 6. Severe damage consisting mainly of type II (ischemic) cell change (arrows), type III (severe) cell change (arrowheads), type IV (cell loss) (double arrows) and perivascular edema (asterisks) (X460).
VAN
188
FIGS. 7-9. Ipsilateral
cortex
REEMPTS
ET AL.
of hypoxic-ischemic
rats treated
with
etomidate.
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
189
artery ligation and repeated 10 min before nitrogen exposure, or 10 mg/ kg given once at 1 h before nitrogen exposure, led to damage in only one of eight rats and two of eight rats, respectively. This damage was restricted to the ipsilateral hemisphere and consisted exclusively of very moderate microvacuolation or ischemic cell change. A small dose of 2.5 mg/kg given 10 min before carotid artery ligation and again 10 min before nitrogen exposure resulted in only moderate ischemic damage in all but two animals. When this small dose was given once at 1 h before nitrogen exposure, six rats were devoid of lesions, one rat showed mild lesions, and another one had severe lesions. None of the etomidate-treated rats showed lesions in the contralateral neocortex or the left and right hippocampus. Hypoxic-Ischemic rats, Thiopental-Treated. A moderate degree of protection against hypoxic-ischemic damage also was found after subcutaneous thiopental treatment (Fig. 10, Table 2). After the administration of 20 mg/kg thiopental, 10 min before ligation and again 10 min before nitrogen exposure, two of eight rats were devoid of lesions in the ipsilateral hemisphere. The lesions were moderate in five rats and one rat showed severe damage. Damage in the contralateral neocortex was seen in two rats. One rat also had lesions in the ipsilateral hippocampus. DISCUSSION In recent years cerebral hypoxia has been intensively studied ( 10, 17, 2 1,22,28). A large series of animal species has been used to experimentally induce different forms of lowered oxygen availability to the brain and disturbance of neuronal cell function. Oxygenation of the cell can be impaired at least in two ways: disturbance of cerebral blood flow (ischemic or oligemic hypoxia) and disturbance of oxygen supply (hypoxic, anemic, and histotoxic hypoxia; anoxia). The consequences of these insults can be evaluated by different parameters, i.e., biochemical (oxygen and glucose consumption, adenosine triphosphate and phosphocreatine depletion, lactate accumulation), physiological (electroencephalogram, hemodynamics), neurological (behavior, learning), and histological (morphologic and histochemical cell change). Pharmacological studies of the protection against FIG. 7. Survey of the cortical gray matter of a rat which received 10 mg/kg etomidate 1 h before start of the nitrogen exposures. The morphologic picture was fully comparable with that of normal animals. Roman numerals roughly indicate the different cortical layers (x90). FIG. 8. Rat treated with 10 mg/kg etomidate 10 min before ligation of the carotid artery and again 10 min before the nitrogen insult. No cellular (arrows) or vascular (arrowhead) alterations were seen (X460). FIG. 9. Rat treated with 2.5 mg/kg etomidate 10 min before ligation and again 10 min before nitrogen exposure. Next to normal cells (arrows), occasionally type II (ischemic) cell change was seen (arrow-heads). Capillaries (c) appeared unaltered (X460).
190
VAN
9
REEMPTS
ET AL.
Untrrrtcd
3J 91
Elomidrtr
2xlOmglkg
91
Etomidate
lxlOmg/kg
5.c.
-1Omin
S.C. -1h
----
9
-L-n
Etomidata
2x2.5mglkg
S.C. -1Omin
Elomidate
lxZ.Smg/kg
SC.
lhiopentrl
2x20
:
06 ::
1
7
mglkg
-1h
S.C. - 1Omin
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
191
cerebral hypoxic damage include treatment with barbiturates and etomidate (1, 10, 16, 17, 20, 21, 23, 24, 26, 27, 29). Pharmacologic protection against cell damage after hypoxic-ischemic insults is, to our knowledge, seldom morphologically evaluated. This is largely due to the fact that histologic changes appear relatively late in contrast to biochemical and physiologic changes. Moreover, ischemic damage is usually in small foci, difficult to localize, unless more severe hypoxia is imposed. But under such circumstances a high incidence of mortality is encountered in the untreated control animals. Another important factor which hampers morphological assessment is the difficult preservation of brain tissue. Histologic artefacts [i.e., dark cells and hydropic cells (7)] are almost unavoidable in human material; but also brains from large experimental animals are often inadequately fixed, giving rise to a wide variety of morphologic alterations which cannot be attributed to experimental hypoxic conditions, although they appear to be similar. Finally, one has to consider that many anesthetic agents, used during some hypoxic insults, may exert protective effects. The Levine preparation, in which the lesions are evoked in the conscious rat, offers a good outcome for such a morphologic approach. The rat brain is one of the best understood of all mammalian species. It can be fixed easily by perfusion techniques and extensive regions can be investigated. Moreover, the histopathologic changes originating after nitrogen exposure in rats with a ligated carotid artery are well documented (5-7, 14, 15). Neuronal and astrocytic changes are related both to the number of nitrogen exposures and to the survival time and they are largely restricted to the hemisphere ipsilateral to the ligated side. Thanks to these features, this model meets the criteria of reproducibility and practicability, enabling the evaluation of a drug with brain protective properties. The processes which are responsible for the induced injury are complex and far from understood. The neuron relies heavily on a continuous supply of oxygen and energy substrates, for it lacks adequate substrates to function anaerobically. In the Levine model two factors have to be considered. First, there is interruption of the blood flow in the right common carotid artery, an insult which is not harmful in itself, because oxygen and substrates are FIG. 10. Severity and frequency of cellular damage in the cerebral cortex of “Levine” rats. The figure summarizes the degree of damage in the ipsilateral (R) and contralateral (L) cortex of rats (N = 8 for each treatment group) which survived 24 h after unilateral carotidartery ligation and intermittent exposure to nitrogen. The occurrence of four types of cell change (0, type I: microvacuolation; EC& type II: ischemic cell change; m, type III: severe cell change; and I, type IV: cell loss) was scored, respectively, in laminae III, IV + V, and VI accordingly to a three-point scale whereby 1 = occasionally cells involved, 2 = several, and 3 = many cells involved. The scores of these different layers were added for each particular type of change, score 9 indicating thus the maximal degree of damage.
192
VAN
REEMPTS
ET Al.,.
adequately provided via the circle of Willis. Second, the arterial PO2 is repeatedly reduced to minimal values by exposure of the animal to the nitrogen environment. This reduction increases the cerebral blood flow fourfold on the contralateral side but only twofold on the side of the interrupted carotid artery (22). The unilateral “relative” ischemia does not limit substrate supply, but it reduces the supply of oxygen to values that can no longer sustain a normal energy balance (22). In addition these conditions can lead to cardiovascular failure in such a way that secondary ischemia occurs. It is widely accepted that the autoregulation of cerebral blood flow is abolished below a mean arterial pressure of 50 mm Hg (13). It is thus probably the lowering of arterial POz values below limits of 20 to 25 mm Hg (23), combined with a failure of cerebral blood flow autoregulation that is responsible for increased vulnerability of the ipsilateral hemisphere. Under conditions of severe hypoxia it is well known that membrane permeability is increased and that the cellular homeostasis for electrolytes is lost, resulting in the formation of edema. As for the myocardial cell (4), an increased influx of calcium ions also may be deleterious for the structural integrity and function of the neuronal cell. On the other hand, it cannot be excluded that during reanimation, probably as a result of reactive hyperemia, a sudden increase in oxygen supply may initiate deleterious oxidative reactions (22, 24). The results obtained with etomidate and thiopental indicate that both drugs have brain protective properties, either by reducing the severity of damage or by the total prevention of lesions. The effective doses of etomidate were considerably smaller than those of thiopental and most probably are devoid of secondary effects such as cardiac or respiratory depression (18). The protective effects of thiopental could not be enhanced because the animals, when pretreated with higher doses of thiopental, did not survive the hypoxic insult, probably owing to cardiopulmonary insufficiency. The treatment and prevention of cerebral hypoxia is aimed at preserving the balance between O2 demand and O2 supply. This can be obtained by changing the metabolic rate, blood flow, and arterial oxygen content. In some way etomidate may preserve the energy reserves in the neuron. In man it was shown that etomidate is an important cerebral metabolic depressant (19). The fact that the drug is very effective against both electrically and chemically produced convulsions (26, 27) suggests that the energy-consuming hypermetabolism during the ischemic and/or postischemic period is attenuated. Moreover, the results obtained in different models of hypoxiaischemia show a dose-related prolongation of brain function with etomidate (26,29). Etomidate pretreatment was also shown to prevent the behavioral
PROTECTION
AGAINST
HYPOXIC
BRAIN
DAMAGE
193
and histologic sequelae which develop after resuscitation of rats treated with a lethal intravenous dose of potassium cyanide (2). Hypnosis is not necessarily required to obtain brain protection with etomidate (27, 29). This is evidenced also in the present study: cerebral damage can be avoided even when nitrogen challenge is performed 1 h after S.C. administration of etomidate, when the rats are awake. A similar brain protective action in this model was recently demonstrated with flunarizine, a vasoactive substance that blocks stimulated Ca*+ fluxes through the plasma membrane [(3), J. Van Reempts, manuscript in preparation]. For barbiturates, inhibition of Ca2+ entry at cell membranes has already been reported (9). Although direct Ca2+ -entry blocking properties were not shown for etomidate, it might be that, through its interaction with plasma membrane components, it can counteract the deleterious ion fluxes which occur during and/or after hypoxia. This would explain not only the prevention of the Ca2+-induced hypermetabolism and the further exhaustion of energy reserves, but also the antiedematous effect. An indication for the latter is the absence of spongioid regions in the cortex of all etomidatetreated animals with the exception of one. A vascular action of this drug certainly cannot be excluded. Recent experiments on hemorrhagic shock in dogs (25) and in rats (C. Hermans, unpublished results) showed that etomidate not only rapidly increased mean aortic pressure, but also drastically prolonged the survival time in comparison with control and thiopental-treated animals. By allowing sufficient autoregulation or by providing adequate perfusion in vulnerable territories (5) the drug could guarantee not only a sufficient amount of oxygen, but also a necessary removal of waste products. Although this study cannot provide certainty about the working mechanism of etomidate in brain cell protection, it clearly shows that the drug is a potent antianoxic agent in a certain form of severe hypoxia in rats. The present results together with literature data about etomidate indicate that this drug warrants experimental and clinical research in the prevention of brain ischemia. REFERENCES 1. ANFRED, I., AND 0. SECHER. 1962. Anoxia and barbiturates. Arch. Int. Pharmacodyn. Ther. 89: 61-14. 2. ASHTON, D., J. VAN REEMPTS, AND A. WAUQUIER. 198 1. Behavioural, electroencephalographic and histological study of the protective effect of etomidate against histotoxic dysoxia produced by cyanide. Arch. Int. Pharmacodyn. Ther., in press. 3. BORGERS, M., F. THONE, AND J. M. VAN NUETEN. 1981. The subcellular distribution of calcium and the effects of calcium antagonists as evaluated with a combined oxalatepyroantimonate technique. Acta Histochem. Suppl. 24: 327-332.
194
VAN REEMPTS
ET AL.
4. BORGERS, M. 1981. The role of calcium in the toxicity of the myocardium. Histochem. J. 13: 839-848.
5. BROWN, A. W., AND J. B. BRIERLEY. 1968. The nature, distribution and earliest stages of anoxic-ischaemic nerve cell damage in the rat brain as defined by the optical microscope. Br. J. Exp. Pathol. 49: 87-106. 6. BROWN, A. W., AND J. B. BRIERLEY. 1973. The earliest alterations in the rat neurones and astrocytes after anoxia-ischemia. Acta Neuroparhol. 23: 9-22. 7. BROWN, A. W. 1977. Structural abnormalities in neurones. J. Clin. Pathol. 30 Suppl. (R. COB. Pathol.), 11: 155-169. 8. DE GROOT, J. 1963. The rat forebrain in stereotaxic coordinates. Verh. Kon. Ned. Akad. Wetensch. Afd. Nat. 52, no. 4. N. Hollandsche Uitgeversmaatschappij, Amsterdam. 9. ELROD, S. V., AND S. W. LESLIE. 1980. Acute and chronic effects of barbiturates on depolarization-induced calcium influx into synaptosomes from rat brain regions. J. Pharmacol. Exp. Ther. 212: 131-136. 10. HOSSMANN, K. A. 1979. Experimental basis for the treatment of cerebral ischemia. Pages 253-262 in M. GOLDSTEIN, L. BOLIS, F. FIESCHI, S. GORINA, AND C. H. MILLIKAN, Eds., Advances in Neurology, Vol. 25. Raven Press, New York. 11. JANSSEN, P. A. J., C. J. E. NIEMEGEERS, K. H. L. SCHELLEKENS, AND F. M. LENAERTS. 197 1. Etomidate, R-(+)-ethyl- 1-(a-methylbenzyl) imidazole-5-carboxylate (R 16 659). Arzneimittelforsch 21: 1234-1243. 12. JANSSEN, P. A. J., C. J. E. NIEMEGEERS, AND R. P. H. MARSBOOM. 1975. Etomidate, a potent non-barbiturate hypnotic. Intravenous etomidate in mice, rats, guinea pigs, rabbits and dogs. Arch. Int. Pharmacodyn. Ther. 214: 92-l 32. 13. KOV/~CH, A. G., AND P. SANDOR. 1976. Cerebral blood flow and brain function during hypotension and shock. Annu. Rev. Physiol. 38: 571-596. 14. LEVINE, S. 1960. Anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 36: l-17. 15. MCGEE-RUSSELL, S. M., A. W. BROWN, AND J. B. BRIERLEY. 1970. A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain. Brain Res. 20: 193-200. 16. MICHENFELDER, J. D., AND R. A. THEYE. 1973. Cerebral protection by thiopental during hypoxia. Anesthesiology 39: 5 1O-5 17. 17. NEMOTO, E. M. 1978. Pathogenesis of cerebral ischemia-anoxia. Crif. Care Med. 6: 203214.
RENEMAN, R. S., A. M. H. JAGENEAU, R. XHONNEUX, AND P. LADURON. 1975. The cardiovascular pharmacology of etomidate (R 26 490), a new potent and short acting intravenous hypnotic agent. Pages 152-l 56 in A. ARIAS, Ed., Recent Progress in Anaesthesiology and Resuscitation: Proceedings (Eur. Gong. Anaesthesiol., 4th). Exerpta Medica Internat. Congr., Ser. No. 347. North-Holland/Elsevier, Amsterdam/New York. 19. RENOU, A. M., J. VERNHIET, P. MACREZ, P. CONSTANT, J. BILLEREY, M. Y. KHADAROO, AND J. M. CAILLE. 1978. Cerebral blood flow and metabolism during etomidate anaesthesia in man. Br. J. Anaesfhesiol. 50: 1047. 20. ROSSIGNOL, R., AND M. EBIGWEI-IBRU. 1980. Drugs against brain hypoxia. Trends 18.
Pharmacol.
Sci. 1: 287-289.
21. SAFAR, P., A. BLEYAERT, E. M. NEMOTO, J. MOOSSY, AND J. V. SNYDER. 1978. Resuscitation after global brain ischemia-anoxia. Crit. Care Med. 6: 215-227. 22. SIESJ& B. K. 1978. Brain Energy Metabolism. Wiley. New York/Chichester. 23. SMITH, A. L. 1977. Barbiturate protection in cerebral hypoxia. Anesthesiology 47: 285293.
PROTECTION
AGAINST
HYPOXIC
BRAIN DAMAGE
195
24. STEEN, P. A., AND J. D. MICHENFELDER. 1980. Mechanisms of barbiturate protection. Anesthesiology 53: 183-185. 25. WAUQUIER, A., C. HERMANS, W. A, E. VAN DEN BROECK, A. JAGENEAU, AND P. FRANCOIS. 1980. Etomidate vs. thiopental and pentobarbital in haemorrhagic dogs. 7th World Congress of Anaesthesiologists, Hamburg, Abstr. 295. 26. WAUQUIER, A., D. ASHTON, C. J. E. NIEMEGEERS, J. VAN REEMPTS, AND M. BORGERS. 1980. Etomidate: anti-ischemic and anti-anoxic actions in animals. 7th World Congress of Anaesthesiologists, Hamburg, Abstr. 216. 27. WAUQUIER, A., D. ASHTON, G. CLINCKE, C. J. E. NIEMEGEERS, AND P. A. J. JANSSEN. 1980. Etomidat, ein Barbituratfreies Hypnotikum: antikonvulsive, anti-anoxische und hirnprotektive Wirkung im Tierexperiment. Pages 183-203 in A. OPITZANDR. DEGEN, Eds., Anaesthesie bei Zerebralen Krampfanfallen und Intensivtherapie des Status Epilepticus. Verlags. Erlangen. 28. WAUQUIER, A., D. ASHTON, G. CLINCKE, AND J. VAN REEMPTS. 1981. Considerations on models and treatment of brain hypoxia. Pages 95-114 in M. W. VAN HOF AND G. MOHN, Eds., Developments in Neuroscience: Recovery from Brain Damage. Elsevier/ North-Holland Biomedical Press, Amsterdam. 29. WAUQUIER, A., D. ASHTON, G. CLINCKE, AND C. J. E. NIEMEGEERS. 198 1. Antihypoxic effects of etomidate, thiopental and methohexital. Arch. Int. Pharmacodyn. Ther. 249: 330-334.