- Email: [email protected]
Modifications of the permeability of the blood-brain barrier and local cerebral metabolism in pentobarbital- and ketamine-anaesthetized rats
0028-3908189 $3.00+ 0.00 Copyright Q 1989Pergamon Press plc
h’europharmaeologyVol. 28,No. 9, pp. 997-1002.1989
Printed in Great Britain. All rights reserved
MODIFICATIONS OF THE PERMEABILITY OF THE BLOOD-BRAIN BARRIER AND LOCAL CEREBRAL METABOLISM IN PENTOBARBITALAND KETAMINE-ANAESTHETIZED RATS A. SAIJA,’ P. PRINCI,~ R. DE PASQUALE’*and G. COSTAR ‘Department Farmaco-Biologico, School of Pharmacy, and ‘Centro Interdipartimentale di Informazione Farmaco-Tossicologica, University of Messina and ‘Institute of Pharmacology, School of Pharmacy, University of Camerino, Italy (Accepted 20 March 1989)
Summary-The state of deep surgical anaesthesia, induced by intraperitoneal injection of pentobarbital sodium (54 mg/kg) or ketamine hydrochloride (150 mg/kg) in the rat, was accompanied by a significant reduction in the permeability of the blood-brain barrier evaluated by calculating a unidirectional blood-tobrain constant (Ki) for the circulating tracer [“‘C]a-aminoisobutyric acid. Pentobarbital-induced anaesthesia was also characterized by a widespread and marked depression of local cerebral glucose utilization; on the contrary, when rats were anaesthetized with ketamine, cerebral glucose utilization increased in the striatum and hippocampus and decreased in the cerebellum and brain-stem. It is suggested, as a hypothesis, that two different mechanisms, depending on the kind of the anaesthetic drug used, may be involved in the changes in the permeability of the blood-brain barrier, observed in anaesthetized animals: (a) a neurogenic component; (b) a direct interaction of the anaesthetic with elements of the microvasculature. Kqv words-blood-brain
barrier permeability, LCGU, pentobarbital, ketamine, anaesthesia, rat.
The blood-brain barrier arises from epithelial-like tight junctions which connect adjoining capillary endothelium together in the microvasculature of the brain (Betz and Goldstein, 1984). The concept of the blood-brain barrier is gradually evolving from one of a passive and relatively immutable structure, to that of a dynamic interface between the blood and the brain, alterable by pharmacological and neurogenie means (Pardridge, 1987). There is substantial evidence that neuronal circuits within the central nervous system modify the tone of non-neuronal structures, influencing various aspects of the function of cerebral microvessels. A tight coupling between brain function and cerebral perfusion has long since been demonstrated (Lou, Edvinsson and MacKenzie, 1987); nevertheless, there is little evidence for a correlation between cerebral functional activity and permeability of the blood-brain barrier. The goal of the present study was to determine whether the state of deep surgical anaesthesia (that is a drug-induced absence of perception of all sensations) is accompanied by regional modifications of the permeability of the blood-brain barrier in the rat. In these experiments, two different anaesthetic drugs were used, pentobarbital, which depresses the cerebral metabolic rate (Nagai, Narumi, Miyamoto, Saji and Nagawa, 1983), and ketamine, which, unlike most other centrally-acting anaesthetics, has been shown to increase cerebral glucose
and blood flow (Cavazzuti, Porro, Biral, Benassi and Barbieri, 1987). For this reason, in order to investigate the existence of a relationship between changes of cerebral functional activity and alterations of the permeability of the blood-brain barrier, the effects of pentobarbital- and ketamine-induced anaesthesia on the rates of local cerebral glucose utilization, which is useful as an index of regional brain activity, were also determined. utilization
METHODS Theory and calculations
Modifications of the permeability of the bloodbrain barrier were determined using a new tracer technique that employs a small molecular weight radiolabelled aminoacid, [‘4C]a-aminoisobutyric acid ([14C]AIB), to identify and quantify small changes in permeability (Gross, Teasdale, Graham, Angerson and Harper, 1982; Picozzi, Todd and Crockard, 1985; Saija, Princi, De Pasquale and Costa, 1987; Tyson, Teasdale, Graham and McCulloch, 1982). The model used for the estimation of the vascular permeability of the brain was developed by Ohno, Pettigrew and Rapoport (1978). A blood-to-brain unidirectional transfer constant (Ki) for [‘4C]AIB may be calculated from the following relationship: Ki
=
Ci(T) PT
Cp dt
*To whom correspondence should be addressed. 991
where Ci( r} is the parenchymal tracer concentration in brain at the time T (which is the duration of the experiment) and Cp is the tracer concentration in arterial plasma. Because &Yis related to the permeability-surface area product (PS) and blood-flow (F), by PS = F ln(1 - W/F) and PS
The experiments were carried out on male Wistar rats (23%240g b.w.). Animals were maintained under normal controlled lighting and temperature conditions and allowed free access to food and water until used.
All rats underwent surgical preparation on the day of the experiment. When the study was carried out in conscious animals (drug-free), the rats were lightly anaesthetized with ether, short polyethylene catheters were inserted in the left femoral vein for administration of tracer and in the left femoral artery for sampling of blood. Rats were then immobilized from the waist down by the means of a loose-fitting plaster cast and were allowed to recover from the anesthesia for 3-4 hr. before injection of tracer. When the experiments were carried out in anaesthetized animals, the rats were injected intrape~toneally (i.p.f with pentobarbital sodium (54 mg/kg) or ketamine hydrochloride (150 mg/kg). Surgical preparation was identical to that for the conscious rats; after the completion of operative procedures, the animals were allowed a steady state period of 15min before the experiment was begun. Local cerebral glucose utilization was determined in a half of each group of animals (conscious, pentobarbital- and ketamine-anaestheti~d~; values of Ki for [14C]AIB were calculated in the other half.
in plasma was longer in anaesthetiz~, than in conscious rats (tliZe of [‘4C]AIB in plasma: 9.32 + 0.51 min in conscious rats, 15.03 + 0.45 min in ketamineanaesthetiz~ rats and l&74* 0.92min in pentobarbital-anaesthetized rats); for this reason a 20 min experimental period was selected for conscious rats and a 30 min experimental period for pentobarbitaland ketamine-anaesthetized animals. At the end of the experimental period all rats were sacrified by the rapid intravenous injection of 1 ml saturated KC1 solution, and the brain was rapidly removed and dissected on dry ice (Paxinos and Watson, 1982). The choroid plexus was carefully removed from the lateral ventricles and around the cerebellum. Solubilisation of the samples was accomplished by adding I ml of Sotuene 100 (Packard) and incubating the vials at 60°C overnight. Each vial was then filled with 4ml of Hionic-Fluor (Packard). Samples of blood were centrifuged; then, 30~1 of plasma were diluted in 0.5 ml distilled water and counted in 6 ml of HionicFluor. Beta-counting was performed by a Packard PL scintillation counter. Single sample quenching was monitored by the external standard method. Sample counts were corrected for background and quenching.
All animals were injected intravenously with a bolus (60 pCi/kg) of 2-deoxy-o-(l-I’4C])glucose ([‘“C]DG). Small samples of blood (80-100 ~1) were collected in polyethylene tubes at predetermined intervals during the experimental period (45min in all animals), for assay of [14C]DG and concentrations of glucose. At the end of the experimental period the animals were sacrified by the rapid jntravenous injection of I ml saturated KC1 solution. Dissection of the brain and assay of the concentration of tracer in specimens of plasma and tissue were performed as described above. Concentrations of glucose in plasma were determined by the glucose ossidase assay.
Another series of experiments was performed to assess the effects of pentobarbital- and ketamineinduced anaesthesia on various physiological parameters. Three groups of 6 rats (conscious, pentobarbitaland k~tamine-anaesthetized animals) underwent the same surgical procedures as for measurements of Ki for [‘4CJAIB and cerebral glucose utilization (cf. Surgical preparation). Blood pressure and arterial blood pH, p0, and pCOZ were measured before (0 min) and at different times (10,20,30,45 minf after the injection of a bolus f0.3ml/rat i.v.) of saline solution (NaCI 0.9%) instead of f’“C]DG or [‘4C]AIB: All animals (conscious and anaesthetized) were the first point (time 0) corresponded to the beginning (injection of tracer) of [14C]AIB and [“C]DG experiinjected intravenously (i.v.) with a bolus (15-20 pCi/ rat) of [14C]AIB. Small samples of blood (80-100 ~1) ments. Mean arterial blood pressure (MAP) was monitored by connecting the arterial catheter to a strain were collected in polyethylene tubes at predetermined intervals. A preliminary series of experiments showed gauge transducer and both systolic and diastolic that the decay time of the concentration of [14C]AIB pressures were displayed on channels of a poligrapb
Anaesthetics and blood-brain (Basile, Italy). Arterial blood samples were ~thd~wn
for dete~ination of pH, pOZ and pC0, (pH-biood gas analyzer, AVL AG, Biomedical Instruments, Switzerland). Statistical analysis
Results are expressed as mean f SE. Differences in Ki values for [14C]AIB, rates of glucose utilization and
physiological parameters, between control and anaesthetized groups, were tested for statistical significance by Student’s “t”-test for unpaired data. Statistical significance was accepted where P c 0.05. Drugs used
[14C]AIB (59 mCi/mmol) and [‘%]DG (59 mCi/ mmol) were purchased from Amersham (England); pentobarbital sodium was purchased from Serva (Heidelberg); ketamine hydrochloride (Ketalar) was purchased from Parke-Davis (Italy).
RESULTS ~ethodologicai comment
A critical component in the [i4C]AIB technique is the cont~bution of intravascular tracer to the final con~nt~tion of tracer in brain tissue. Accurate estimates of vascular volume are difficuft, because vascular volume cannot be calculated in the same animals used for the experiments with [14C]AIB, In addition, an excessive or a prolonged experimental time may increase the brain-to-blood back diffusion of the tracer. The experimental time of 20 or 30min was selected to strike an optimal compromise, allowing both a small final intravascular concentration of tracer and minimal back diffusion of tracer from brain to blood. The Ki values for all regions of the brain examined were consistent with those described by other authors (Picozzi et al., 1985; Gross et al., 1982) and previously obtained in this laboratory (Saija, Princi, De Pasquale and Costa, 1988a; 1988b). When determinations of cerebral glucose utilization were made, an attempt was made to take samples from areas of homogenous gray matter and to avoid contamination by adjacent white matter. In structures such as the hippocampus, however, some substructural heterogeneity within the sample was unavoidable. Because homogeneity of tissue is crucial for the determination of cerebral glucose utilization and the kinetic constants of the model have different values for gray and white matter, some of the vaiues may not have reflected exactly the true mean metabolic rate. The advantages of the present method were its relative simplicity (which permits it to be readily applied to a larger number of animals) and the possibility to estimate Ki values for [14C]AIB and local rates of glucose utilization, using a same method of dissection. Similar studies of modifications of cerebral glucose utilization in dissected samples of brain were undertaken successfully by other authors
999
barrier permeability
(Ginsberg, Busto, Booyhe and Campbell, 1981). Finally, values for cerebral giucose utilization calculated in the present experiments appeared to be in accordance with those reported in the literature and obtained by autoradiographs (Cavazzuti et al., 1987; Ingvar, Abdul-Rahaman and Siesjo, 1980). Effect of pentobarbitai on the permeability of the blood-brain barrier and cerebral glucose utilization
The administration of an anaesthetic dose of pentobarbital (54 mg/kg i.p.) decreased significantly the local permeability of the blood-brain barrier in all areas of the brain examined (Fig. 1); these changes were associated with a parallel marked depression of cerebral glucose utilization which was widespread throughout the brain (Fig. 1). Effect of ketamine on the permeability of the blood-brain barrier and on cerebral glucose utilization
Ketamine decreased Ki values for [14C]AIB in all areas of the brain examined (Fig. 1). The same anaesthetic dose of the drug (150 mg/kg, i.p.) increased significantly cerebral glucose utilization in the striatum and hippocampus, with a simultaneous decrement of metabolic rate in the cerebellum and brain-stem (Fig. 1). Effect of pentobarbitat and ketamine on physiological parameters
Blood pressure was decreased by pentobarbital anaesthesia, in comparison with values measured in conscious rats (Table 1). Conversely, a rise in blood pressure was observed in ketamine-anaesthetized animals (Table 1). Pentobarbital and ketamine tended to increase the blood pC0, and reduce the blood ~0~; as a result, the pH of the blood shifted slightly to acidic side from pH 7.4 (Table 1). DISCUSSION
The present experiments showed that the state of deep surgical anaesthesia, when induced by two different anaesthetics, such as pentobarbital and ketamine, was accompanied by a significant decrease in the permeability of the blood-brain barrier in several regions of the brain. There are several known mechanisms which alter the characteristics of the blood-brain barrier, including osmotic stress, hypertension, trauma, inflammation, anoxia or seizures (Stewart, Hayakawa and Carlen, 1988). None of these mechanisms can be invoked as an expIanation for the decreased ~~eability of the blood-brain barrier, observed in pentobarbital- or ketamine-anaesthetized animals. Pentobarbital and ketamine elicit, respectively, a decrease and an increment of blood pressure in the rat, in comparison with unanaesthetized animals, according also to results reported in literature (Carruba, Bondiolotti, Picotti, Catteruccia and Da Prada, 1987; Wixson, White, Hughes, Lang and Marshall, 1987); but, hypotensions (also severe) was
A.
1000
Ki
for
FC
14C-AIB
PC
OC
(ml/g/min
ST
LCGU (pmol/l
HI
SAIJA et
* 10B3)
HY
CB
BS
CB
BS
MG/KG
I.P.
OOg/min)
60
FC
PC
UC
q CUNSCIO~S RATS q PENTUBARBITAl f$d KETAMXNE
ST
HI
SUDIUM
HYDRIICHLCIRIDE
HY
54
150
MG/KG
I.P.
al.
thesta .: cannot lead to the decrement in the permeabthty of the blood-brain barrier, which was shown in anaesthetized rats, because, on the contrary, anoxia (at least 40 min of complete ischemia) damages the endothelial cells of the blood-brain barrier (Petito, Pulsinelli, Jacobson and Plum, 1982). Anyway, in the present experiments, throughout the experimental period, anaesthetized rats (also those injected with pentobarbital. which slows cerebral blood flow and reduces oxygenation of the brain) continued to have a good colour and robust respiration; this was an additional proof that they did not suffer from profound anoxia. The existence of a relationship between afterations of the ~~eability of the blood-brain barrier and changes of cerebral blood flow induced by anaesthetic drugs, could also be excluded, because, in this mathematical model, the Kr’ for [14C]AIB was independent of cerebral blood flow values (cf Methods). Pentobarbital anaesthesia is accompanied by widespread metabolic depression of the brain besides changes in the characteristics of the blood-brain barrier. This is in agreement with results reported by Ingvar ef al. (1980), that phenobarbital-induced surgical anaesthesia depressed the metabolic rate of glucose to 31-60% of the values obtained with nitrous oxide. The present results seem to suggest that the dramatically depressed metabolic rate (index of marked modifications in the activity of cerebral neuronal circuits) and the reduced permeability of the blood-brain barrier were, to some extent, correlated. In support of this hypothesis are the previous observations that administration of an external stimulus (exposure to a high intensity light) increased the permeability of the blood-brain barrier (structures of which control homeostasis in the brain and, so, information processing) in one area of the brain
Table I. Influence of anaesthesia induced by ~nto~rbital sodium (54 mgjkg Lp.) or ketamine hydrochloride f 150 mg/kg i.p.) on various physiological parameters at different times. The values arc mean f SE of 6 determinations Time (min) PH
Fig. 1. Influence of pentobarbital- or ketamine-induced anaesthesia on Ki for [%]AIB and cerebral glucose utilization (LCGU) in the rat, as compared with conscious animals in several areas of the brain. (FC = Frontal Cortex; PC = Temporoparietal Cortex; OC = Occipital Cortex; ST = Striatum; HI = Hippocampus; HY = Hypothalamus; CB = Cerebellum; BS = Brain-Stem.) The values are means & SE of 6 determinations.
PO, mmHg
PC% mmHg
not shown to alter the characteristics of the bloodbrain barrier (Domer, Boertje and Sampson, 1985) and only a very large and rapid increase in blood pressure increases the permeability of the bloodbrain barrier (Ellison, Povlishock and Hayes, 1985). The small changes of p02 and pC0, that are observed in the course of pentobarbitaf and ketamine anaes-
MAP mmHg
0 IO 20 30 45 0 IO 20 30 45 0 IO 20 30 45 0 10 20 30 45
Conscious rats 7.41 + 0.007 7.40 + 0.009 7.41 i 0.008 7.39 * 0.007 7.39 + 0.005 92.7 k 4.3 I 92.9 i 4.42 93.5 * 3.91 94.6 & 4.24 96.1 + 3.85 35.61 3.12 35.9 2 2.97 35.3 i 3.25 34.9 + 2.77 34.7 f 3.34 125.8 f 5.76 128.3 + 4.93 120.7 + 5.12 120.5 k4.51 117.4 f 5.20
*P 4 0.05vs conscious rats. MAP = mean arterial pressure.
Pentobarbital 7.33 + 0.008* 7.34 L 0.010* 7.36 4 0.01 I 7.37 ?J 0.008 7.38 +0.012 X4.2+3.15 84.3 i 3.36 89.3 i 2.g3 90.0 + 2.97 94.4 2 3.22 43.1 2 2.73 43. I + 2.75 40.8 * 3.19 38. I + 2.73 36.5 F 2.51 107.1 + 3.72 110.7 f 3.55 112.5~4.10 115.3 + 4.27 I 15.4 + 2.96
Ketamine 7.34+o.oII* 7.35 j: 0.009* 7.37 I: 0.008 7.38 rt 0.010 7.38 $: 0.009 85.4 jz 2.52 87.8 2 3.44 90. I i 2.95 92.t 13.17 95.2 $2.82 41.7~2.16 41.7 k2.16 39.Ok3.13 32.2 4 3.51 36.8 + 3.35 142.2 4 6.76 140.7 4 5.92 135.4 k 5.43 130.5 +_3.97 128.9 k 4.35
Anaesthetics and blood-brain
(occipital cortex) that is thought to elaborate the information administered (Saija et al., 1988a); in addition, modifications of vascular permeability of the brain may also be induced by stress, probably as a consequence of an altered release of neurotramitters (Sharma and Dey, 1981). Unlike barbiturates, that exert direct vasodilator actions on cerebral arterial smooth muscle, ketamine, at anaesthetic doses, was found to exert spasmogenic actions on cerebral arterioles, venules and arteries; distinct phencyclidine-like receptors which subserve contraction appear to exist on large, as well as microscopic, cerebral blood vessels (Altura and Altura, 1984). So. it could be hypothesized that ketamine alters the transport of [14C]AIB across the bloodbrain barrier as a consequence of its spasmogenic action at the level of cerebral microcirculation. In conclusion, the present study provides evidence that the state of deep surgical anaesthesia, induced by two different centrally-acting anaesthetics (pentobarbital and ketamine), was accompanied by a decrease of local permeability of the blood-brain barrier in several regions of the brain; however, different mechanisms must be invoked to explain the alterations in permeability of the blood-brain barrier observed in pentobarbital- or ketamine-anaesthetized rats. Also, great care must be taken to interpret the data; however, the existence of a neurogenic component in the control of the permeability of the blood-brain barrier can be proposed when anaesthesia is induced by drugs, since pentobarbital widely and markedly depresses the functional activity of neuronal circuits in the brain. On the other hand, it is important not to loose sight of the importance of the effects, which may be exerted by other anaesthetics, such as ketamine, directly on cerebral microvessels.
barrier permeability
1001
Ginsberg M. D., Busto R., Booyhe T. E. and Campbell J. A. (1981) A radioisotopic method for the simultaneous quantitation of regional cerebral blood flow and glucose utilization in small dissected samples: validation studies and values in the nitrous oxide-anesthetized rat. Brain Res. 230: l65--179.
Gross P. M., Teasdale G. M., Graham D. I., Angerson W. J. and Harper A. M. (1982) Intra-arterial histamine increases blood-brain transport in rats. Am. J. Phvsiol. 243: H307-H317. Ingvar M., Abdul-Rahaman A. and Siesjo B. K. (1980) Local cerebral glucose consumption in the artificially ventilated rat: influence of nitrous oxide analgesia and of phenobarbital anaesthesia. Acfa physiol. stand. 109: 177-185. Lou H. C., Edvinsson L. and MacKenzie E. T. (1987) The concept of coupling blood flow to brain function: revision required? Ann. Neural. 22: 289-297. McCulloch J. (1982) Mapping functional alterations in the CNS with [‘%]deoxyglucose. In: Handbook of Psychopharmacology (Iversen L. L., Iversen S. D. and Snyder S. H., Eds), Vol. 15, pp. 321409. Plenum, New York. Nagai Y., Narumi S.. Miyamoto M., Saji Y. and Nagawa Y. (1983) A TRH analog (DN-1417): effect on local cerebral glucose utilization in conscious and pentobarbitalized rats determined by the autoradiographic 2-deoxy-[‘Qglucose method. Jap. J. Pharmac. 33: 635-645. Ohno K., Pettigrew K. and Rapoport S. (1978) Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat. Am. J. Physiol. 235: 299-307. Pardridge W. M. (1987) The gate keeper: How molecules are screened for admission to the brain. Sciences 27: 5G55. Paxinos G. and Watson C. (1982) The Rat Brain in Stereoraxic Coordinates. Academic Press, New York. Petit0 C. K., Pulsinelli W. A., Jacobson G. and Plum F. (1982) Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction. J. Neuropath. exp. Neural. 41: 423436. Picozzi P., Todd N. and Crockard A. (1985) Regional blood-brain barrier permeability changes after restoration of blood flow in postischemic gerbil brains: a quantitative study. J. cereb. Blood FloursMetab. 5: l&16. Saija A.. Princi P., De Pasquale R. and Costa G. (1987) Regional blood-brain barrier permeability changes in response to ACTH plasma levels modifications. Bull. molec. Biol. Med. 12: 97-109.
REFERENCES Altura B. T. and Altura B. M. (1984) Effects of barbiturates, phencyclidine, ketamine and analogues on cerebral circulation and cerebrovascular muscle. Microcirc. Em&helium Lymphatics 1: 169-184. Betz A. L. and Goldstein G. W. (1984) Brain capillaries. Structure and Function. In: Handbook of Neurochemistry (Lajtha A., Ed.), 2nd edn, pp. 465484. Plenum, London. Carruba M. 0.. Bondiolotti G.. Picotti G. B.. Catteruccia N. and Da Prada M. (1987) Effects of diethyl ether, halothane. ketamine and urethane on sympathetic activity in the rat. Eur. J. Pharmac. 134: 15-24. Cavazzuti M., Porro C. A., Biral G. P.. Benassi C. and Barbieri C. G. (1987) K&amine effects on local cerebral blood flow and metabolism in the rat. J. cereb. Blood Flow Metab. 7: 806-81 I. Domer F. R., Boertje S. B. and Sampson J. B. (1985)
Permeability of the blood-brain barrier is not changed by vasoactive doses of arachidonic acid and prostaglandins in rats. Res. Commun them. Path. Pharmac. 48: 27-37. Ellison M. D., Povlishock J. T. and Hayes R. L. (1985) Examination of the blood-brain barrier transfer of aaminoisobutyric acid and horseradish peroxidase: regional alterations in blood-brain barrier function following acute hypertension. J. cereb. Blood Now Metab. 6: 471480.
Saija A., Princi P., De Pasquale R. and Costa G. (1988a) High intensity light exposure increases blood-brain barrier transport in rats. Pharmac. Res. Commun. 20: 553-559. Saija A., Princi P., De Pasquale R. and Costa G. (1988b) Circadian modifications of blood-brain barrier (BBB) permeability in the rat. Pharmac. Res. Commun. 20: 249-259. Sharma H. S. and Dey P. K. Dey (1981) Impairment of blood-brain barrier (BBB) in rat by immobilization stress: role of serotonin (5-HT). Indian J. Physiol. Pharmac. 25: 111-122.
Sokoloff L. (1980) The [“‘C]deoxyglucose
method for the quantitative determination of local cerebral glucose utilization: theoretical and practical consideration. In: Cerebral Merabolism and Neural Function (Passonneau J. W.. Hawkins R. A., Lust W. D. and Welsh F. A., Eds), pp. 319-330. Williams and Wilkins, Baltimore. Sokoloff L. (1983) Measurements of local glucose utilization and its use in localization of functional activity in the Central Nervous System of animals and man. Rec. Prog. Hormone Res. 39: 75-126. Sokoloff L.. Reivich M., Kennedy C., Des Rosiers M. H., Patlack C. S., Pettigrew K. D., Sakurada 0. and Shinohara M. (1977) The [“‘Cldeoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28: 897-916. Stewart P. A., Hayakawa E. M. and Carlen P. L. (1988)
1002
A. SAIJAet
Ethanol and ~tobarbi~l in combination increase bloodbrain barrier permeability to horseradish peroxidase. Brain Rex 443: 12-20.
Tyson G., Teasdale G., Graham D. and McCuiloch J. (1982) Cerebrovascular permeability following MCA occlusion in the rat. J. Neurosurg. 57: 186-196.
af.
Wixson S. K., White W. J. Hughes H. C. Jr, Lang C. M. and Marshall W. K. (1987) The effects of pentobarbital, fentanyl-droperidol, ketamine-xylazine and ketaminediazepam on arterial blood pH, blood gases, mean arterial blood pressure and heart rate in adult male rats. Lab. Anim. Sci. 37: 736742.
h’europharmaeologyVol. 28,No. 9, pp. 997-1002.1989
Printed in Great Britain. All rights reserved
MODIFICATIONS OF THE PERMEABILITY OF THE BLOOD-BRAIN BARRIER AND LOCAL CEREBRAL METABOLISM IN PENTOBARBITALAND KETAMINE-ANAESTHETIZED RATS A. SAIJA,’ P. PRINCI,~ R. DE PASQUALE’*and G. COSTAR ‘Department Farmaco-Biologico, School of Pharmacy, and ‘Centro Interdipartimentale di Informazione Farmaco-Tossicologica, University of Messina and ‘Institute of Pharmacology, School of Pharmacy, University of Camerino, Italy (Accepted 20 March 1989)
Summary-The state of deep surgical anaesthesia, induced by intraperitoneal injection of pentobarbital sodium (54 mg/kg) or ketamine hydrochloride (150 mg/kg) in the rat, was accompanied by a significant reduction in the permeability of the blood-brain barrier evaluated by calculating a unidirectional blood-tobrain constant (Ki) for the circulating tracer [“‘C]a-aminoisobutyric acid. Pentobarbital-induced anaesthesia was also characterized by a widespread and marked depression of local cerebral glucose utilization; on the contrary, when rats were anaesthetized with ketamine, cerebral glucose utilization increased in the striatum and hippocampus and decreased in the cerebellum and brain-stem. It is suggested, as a hypothesis, that two different mechanisms, depending on the kind of the anaesthetic drug used, may be involved in the changes in the permeability of the blood-brain barrier, observed in anaesthetized animals: (a) a neurogenic component; (b) a direct interaction of the anaesthetic with elements of the microvasculature. Kqv words-blood-brain
barrier permeability, LCGU, pentobarbital, ketamine, anaesthesia, rat.
The blood-brain barrier arises from epithelial-like tight junctions which connect adjoining capillary endothelium together in the microvasculature of the brain (Betz and Goldstein, 1984). The concept of the blood-brain barrier is gradually evolving from one of a passive and relatively immutable structure, to that of a dynamic interface between the blood and the brain, alterable by pharmacological and neurogenie means (Pardridge, 1987). There is substantial evidence that neuronal circuits within the central nervous system modify the tone of non-neuronal structures, influencing various aspects of the function of cerebral microvessels. A tight coupling between brain function and cerebral perfusion has long since been demonstrated (Lou, Edvinsson and MacKenzie, 1987); nevertheless, there is little evidence for a correlation between cerebral functional activity and permeability of the blood-brain barrier. The goal of the present study was to determine whether the state of deep surgical anaesthesia (that is a drug-induced absence of perception of all sensations) is accompanied by regional modifications of the permeability of the blood-brain barrier in the rat. In these experiments, two different anaesthetic drugs were used, pentobarbital, which depresses the cerebral metabolic rate (Nagai, Narumi, Miyamoto, Saji and Nagawa, 1983), and ketamine, which, unlike most other centrally-acting anaesthetics, has been shown to increase cerebral glucose
and blood flow (Cavazzuti, Porro, Biral, Benassi and Barbieri, 1987). For this reason, in order to investigate the existence of a relationship between changes of cerebral functional activity and alterations of the permeability of the blood-brain barrier, the effects of pentobarbital- and ketamine-induced anaesthesia on the rates of local cerebral glucose utilization, which is useful as an index of regional brain activity, were also determined. utilization
METHODS Theory and calculations
Modifications of the permeability of the bloodbrain barrier were determined using a new tracer technique that employs a small molecular weight radiolabelled aminoacid, [‘4C]a-aminoisobutyric acid ([14C]AIB), to identify and quantify small changes in permeability (Gross, Teasdale, Graham, Angerson and Harper, 1982; Picozzi, Todd and Crockard, 1985; Saija, Princi, De Pasquale and Costa, 1987; Tyson, Teasdale, Graham and McCulloch, 1982). The model used for the estimation of the vascular permeability of the brain was developed by Ohno, Pettigrew and Rapoport (1978). A blood-to-brain unidirectional transfer constant (Ki) for [‘4C]AIB may be calculated from the following relationship: Ki
=
Ci(T) PT
Cp dt
*To whom correspondence should be addressed. 991
where Ci( r} is the parenchymal tracer concentration in brain at the time T (which is the duration of the experiment) and Cp is the tracer concentration in arterial plasma. Because &Yis related to the permeability-surface area product (PS) and blood-flow (F), by PS = F ln(1 - W/F) and PS
The experiments were carried out on male Wistar rats (23%240g b.w.). Animals were maintained under normal controlled lighting and temperature conditions and allowed free access to food and water until used.
All rats underwent surgical preparation on the day of the experiment. When the study was carried out in conscious animals (drug-free), the rats were lightly anaesthetized with ether, short polyethylene catheters were inserted in the left femoral vein for administration of tracer and in the left femoral artery for sampling of blood. Rats were then immobilized from the waist down by the means of a loose-fitting plaster cast and were allowed to recover from the anesthesia for 3-4 hr. before injection of tracer. When the experiments were carried out in anaesthetized animals, the rats were injected intrape~toneally (i.p.f with pentobarbital sodium (54 mg/kg) or ketamine hydrochloride (150 mg/kg). Surgical preparation was identical to that for the conscious rats; after the completion of operative procedures, the animals were allowed a steady state period of 15min before the experiment was begun. Local cerebral glucose utilization was determined in a half of each group of animals (conscious, pentobarbital- and ketamine-anaestheti~d~; values of Ki for [14C]AIB were calculated in the other half.
in plasma was longer in anaesthetiz~, than in conscious rats (tliZe of [‘4C]AIB in plasma: 9.32 + 0.51 min in conscious rats, 15.03 + 0.45 min in ketamineanaesthetiz~ rats and l&74* 0.92min in pentobarbital-anaesthetized rats); for this reason a 20 min experimental period was selected for conscious rats and a 30 min experimental period for pentobarbitaland ketamine-anaesthetized animals. At the end of the experimental period all rats were sacrified by the rapid intravenous injection of 1 ml saturated KC1 solution, and the brain was rapidly removed and dissected on dry ice (Paxinos and Watson, 1982). The choroid plexus was carefully removed from the lateral ventricles and around the cerebellum. Solubilisation of the samples was accomplished by adding I ml of Sotuene 100 (Packard) and incubating the vials at 60°C overnight. Each vial was then filled with 4ml of Hionic-Fluor (Packard). Samples of blood were centrifuged; then, 30~1 of plasma were diluted in 0.5 ml distilled water and counted in 6 ml of HionicFluor. Beta-counting was performed by a Packard PL scintillation counter. Single sample quenching was monitored by the external standard method. Sample counts were corrected for background and quenching.
All animals were injected intravenously with a bolus (60 pCi/kg) of 2-deoxy-o-(l-I’4C])glucose ([‘“C]DG). Small samples of blood (80-100 ~1) were collected in polyethylene tubes at predetermined intervals during the experimental period (45min in all animals), for assay of [14C]DG and concentrations of glucose. At the end of the experimental period the animals were sacrified by the rapid jntravenous injection of I ml saturated KC1 solution. Dissection of the brain and assay of the concentration of tracer in specimens of plasma and tissue were performed as described above. Concentrations of glucose in plasma were determined by the glucose ossidase assay.
Another series of experiments was performed to assess the effects of pentobarbital- and ketamineinduced anaesthesia on various physiological parameters. Three groups of 6 rats (conscious, pentobarbitaland k~tamine-anaesthetized animals) underwent the same surgical procedures as for measurements of Ki for [‘4CJAIB and cerebral glucose utilization (cf. Surgical preparation). Blood pressure and arterial blood pH, p0, and pCOZ were measured before (0 min) and at different times (10,20,30,45 minf after the injection of a bolus f0.3ml/rat i.v.) of saline solution (NaCI 0.9%) instead of f’“C]DG or [‘4C]AIB: All animals (conscious and anaesthetized) were the first point (time 0) corresponded to the beginning (injection of tracer) of [14C]AIB and [“C]DG experiinjected intravenously (i.v.) with a bolus (15-20 pCi/ rat) of [14C]AIB. Small samples of blood (80-100 ~1) ments. Mean arterial blood pressure (MAP) was monitored by connecting the arterial catheter to a strain were collected in polyethylene tubes at predetermined intervals. A preliminary series of experiments showed gauge transducer and both systolic and diastolic that the decay time of the concentration of [14C]AIB pressures were displayed on channels of a poligrapb
Anaesthetics and blood-brain (Basile, Italy). Arterial blood samples were ~thd~wn
for dete~ination of pH, pOZ and pC0, (pH-biood gas analyzer, AVL AG, Biomedical Instruments, Switzerland). Statistical analysis
Results are expressed as mean f SE. Differences in Ki values for [14C]AIB, rates of glucose utilization and
physiological parameters, between control and anaesthetized groups, were tested for statistical significance by Student’s “t”-test for unpaired data. Statistical significance was accepted where P c 0.05. Drugs used
[14C]AIB (59 mCi/mmol) and [‘%]DG (59 mCi/ mmol) were purchased from Amersham (England); pentobarbital sodium was purchased from Serva (Heidelberg); ketamine hydrochloride (Ketalar) was purchased from Parke-Davis (Italy).
RESULTS ~ethodologicai comment
A critical component in the [i4C]AIB technique is the cont~bution of intravascular tracer to the final con~nt~tion of tracer in brain tissue. Accurate estimates of vascular volume are difficuft, because vascular volume cannot be calculated in the same animals used for the experiments with [14C]AIB, In addition, an excessive or a prolonged experimental time may increase the brain-to-blood back diffusion of the tracer. The experimental time of 20 or 30min was selected to strike an optimal compromise, allowing both a small final intravascular concentration of tracer and minimal back diffusion of tracer from brain to blood. The Ki values for all regions of the brain examined were consistent with those described by other authors (Picozzi et al., 1985; Gross et al., 1982) and previously obtained in this laboratory (Saija, Princi, De Pasquale and Costa, 1988a; 1988b). When determinations of cerebral glucose utilization were made, an attempt was made to take samples from areas of homogenous gray matter and to avoid contamination by adjacent white matter. In structures such as the hippocampus, however, some substructural heterogeneity within the sample was unavoidable. Because homogeneity of tissue is crucial for the determination of cerebral glucose utilization and the kinetic constants of the model have different values for gray and white matter, some of the vaiues may not have reflected exactly the true mean metabolic rate. The advantages of the present method were its relative simplicity (which permits it to be readily applied to a larger number of animals) and the possibility to estimate Ki values for [14C]AIB and local rates of glucose utilization, using a same method of dissection. Similar studies of modifications of cerebral glucose utilization in dissected samples of brain were undertaken successfully by other authors
999
barrier permeability
(Ginsberg, Busto, Booyhe and Campbell, 1981). Finally, values for cerebral giucose utilization calculated in the present experiments appeared to be in accordance with those reported in the literature and obtained by autoradiographs (Cavazzuti et al., 1987; Ingvar, Abdul-Rahaman and Siesjo, 1980). Effect of pentobarbitai on the permeability of the blood-brain barrier and cerebral glucose utilization
The administration of an anaesthetic dose of pentobarbital (54 mg/kg i.p.) decreased significantly the local permeability of the blood-brain barrier in all areas of the brain examined (Fig. 1); these changes were associated with a parallel marked depression of cerebral glucose utilization which was widespread throughout the brain (Fig. 1). Effect of ketamine on the permeability of the blood-brain barrier and on cerebral glucose utilization
Ketamine decreased Ki values for [14C]AIB in all areas of the brain examined (Fig. 1). The same anaesthetic dose of the drug (150 mg/kg, i.p.) increased significantly cerebral glucose utilization in the striatum and hippocampus, with a simultaneous decrement of metabolic rate in the cerebellum and brain-stem (Fig. 1). Effect of pentobarbitat and ketamine on physiological parameters
Blood pressure was decreased by pentobarbital anaesthesia, in comparison with values measured in conscious rats (Table 1). Conversely, a rise in blood pressure was observed in ketamine-anaesthetized animals (Table 1). Pentobarbital and ketamine tended to increase the blood pC0, and reduce the blood ~0~; as a result, the pH of the blood shifted slightly to acidic side from pH 7.4 (Table 1). DISCUSSION
The present experiments showed that the state of deep surgical anaesthesia, when induced by two different anaesthetics, such as pentobarbital and ketamine, was accompanied by a significant decrease in the permeability of the blood-brain barrier in several regions of the brain. There are several known mechanisms which alter the characteristics of the blood-brain barrier, including osmotic stress, hypertension, trauma, inflammation, anoxia or seizures (Stewart, Hayakawa and Carlen, 1988). None of these mechanisms can be invoked as an expIanation for the decreased ~~eability of the blood-brain barrier, observed in pentobarbital- or ketamine-anaesthetized animals. Pentobarbital and ketamine elicit, respectively, a decrease and an increment of blood pressure in the rat, in comparison with unanaesthetized animals, according also to results reported in literature (Carruba, Bondiolotti, Picotti, Catteruccia and Da Prada, 1987; Wixson, White, Hughes, Lang and Marshall, 1987); but, hypotensions (also severe) was
A.
1000
Ki
for
FC
14C-AIB
PC
OC
(ml/g/min
ST
LCGU (pmol/l
HI
SAIJA et
* 10B3)
HY
CB
BS
CB
BS
MG/KG
I.P.
OOg/min)
60
FC
PC
UC
q CUNSCIO~S RATS q PENTUBARBITAl f$d KETAMXNE
ST
HI
SUDIUM
HYDRIICHLCIRIDE
HY
54
150
MG/KG
I.P.
al.
thesta .: cannot lead to the decrement in the permeabthty of the blood-brain barrier, which was shown in anaesthetized rats, because, on the contrary, anoxia (at least 40 min of complete ischemia) damages the endothelial cells of the blood-brain barrier (Petito, Pulsinelli, Jacobson and Plum, 1982). Anyway, in the present experiments, throughout the experimental period, anaesthetized rats (also those injected with pentobarbital. which slows cerebral blood flow and reduces oxygenation of the brain) continued to have a good colour and robust respiration; this was an additional proof that they did not suffer from profound anoxia. The existence of a relationship between afterations of the ~~eability of the blood-brain barrier and changes of cerebral blood flow induced by anaesthetic drugs, could also be excluded, because, in this mathematical model, the Kr’ for [14C]AIB was independent of cerebral blood flow values (cf Methods). Pentobarbital anaesthesia is accompanied by widespread metabolic depression of the brain besides changes in the characteristics of the blood-brain barrier. This is in agreement with results reported by Ingvar ef al. (1980), that phenobarbital-induced surgical anaesthesia depressed the metabolic rate of glucose to 31-60% of the values obtained with nitrous oxide. The present results seem to suggest that the dramatically depressed metabolic rate (index of marked modifications in the activity of cerebral neuronal circuits) and the reduced permeability of the blood-brain barrier were, to some extent, correlated. In support of this hypothesis are the previous observations that administration of an external stimulus (exposure to a high intensity light) increased the permeability of the blood-brain barrier (structures of which control homeostasis in the brain and, so, information processing) in one area of the brain
Table I. Influence of anaesthesia induced by ~nto~rbital sodium (54 mgjkg Lp.) or ketamine hydrochloride f 150 mg/kg i.p.) on various physiological parameters at different times. The values arc mean f SE of 6 determinations Time (min) PH
Fig. 1. Influence of pentobarbital- or ketamine-induced anaesthesia on Ki for [%]AIB and cerebral glucose utilization (LCGU) in the rat, as compared with conscious animals in several areas of the brain. (FC = Frontal Cortex; PC = Temporoparietal Cortex; OC = Occipital Cortex; ST = Striatum; HI = Hippocampus; HY = Hypothalamus; CB = Cerebellum; BS = Brain-Stem.) The values are means & SE of 6 determinations.
PO, mmHg
PC% mmHg
not shown to alter the characteristics of the bloodbrain barrier (Domer, Boertje and Sampson, 1985) and only a very large and rapid increase in blood pressure increases the permeability of the bloodbrain barrier (Ellison, Povlishock and Hayes, 1985). The small changes of p02 and pC0, that are observed in the course of pentobarbitaf and ketamine anaes-
MAP mmHg
0 IO 20 30 45 0 IO 20 30 45 0 IO 20 30 45 0 10 20 30 45
Conscious rats 7.41 + 0.007 7.40 + 0.009 7.41 i 0.008 7.39 * 0.007 7.39 + 0.005 92.7 k 4.3 I 92.9 i 4.42 93.5 * 3.91 94.6 & 4.24 96.1 + 3.85 35.61 3.12 35.9 2 2.97 35.3 i 3.25 34.9 + 2.77 34.7 f 3.34 125.8 f 5.76 128.3 + 4.93 120.7 + 5.12 120.5 k4.51 117.4 f 5.20
*P 4 0.05vs conscious rats. MAP = mean arterial pressure.
Pentobarbital 7.33 + 0.008* 7.34 L 0.010* 7.36 4 0.01 I 7.37 ?J 0.008 7.38 +0.012 X4.2+3.15 84.3 i 3.36 89.3 i 2.g3 90.0 + 2.97 94.4 2 3.22 43.1 2 2.73 43. I + 2.75 40.8 * 3.19 38. I + 2.73 36.5 F 2.51 107.1 + 3.72 110.7 f 3.55 112.5~4.10 115.3 + 4.27 I 15.4 + 2.96
Ketamine 7.34+o.oII* 7.35 j: 0.009* 7.37 I: 0.008 7.38 rt 0.010 7.38 $: 0.009 85.4 jz 2.52 87.8 2 3.44 90. I i 2.95 92.t 13.17 95.2 $2.82 41.7~2.16 41.7 k2.16 39.Ok3.13 32.2 4 3.51 36.8 + 3.35 142.2 4 6.76 140.7 4 5.92 135.4 k 5.43 130.5 +_3.97 128.9 k 4.35
Anaesthetics and blood-brain
(occipital cortex) that is thought to elaborate the information administered (Saija et al., 1988a); in addition, modifications of vascular permeability of the brain may also be induced by stress, probably as a consequence of an altered release of neurotramitters (Sharma and Dey, 1981). Unlike barbiturates, that exert direct vasodilator actions on cerebral arterial smooth muscle, ketamine, at anaesthetic doses, was found to exert spasmogenic actions on cerebral arterioles, venules and arteries; distinct phencyclidine-like receptors which subserve contraction appear to exist on large, as well as microscopic, cerebral blood vessels (Altura and Altura, 1984). So. it could be hypothesized that ketamine alters the transport of [14C]AIB across the bloodbrain barrier as a consequence of its spasmogenic action at the level of cerebral microcirculation. In conclusion, the present study provides evidence that the state of deep surgical anaesthesia, induced by two different centrally-acting anaesthetics (pentobarbital and ketamine), was accompanied by a decrease of local permeability of the blood-brain barrier in several regions of the brain; however, different mechanisms must be invoked to explain the alterations in permeability of the blood-brain barrier observed in pentobarbital- or ketamine-anaesthetized rats. Also, great care must be taken to interpret the data; however, the existence of a neurogenic component in the control of the permeability of the blood-brain barrier can be proposed when anaesthesia is induced by drugs, since pentobarbital widely and markedly depresses the functional activity of neuronal circuits in the brain. On the other hand, it is important not to loose sight of the importance of the effects, which may be exerted by other anaesthetics, such as ketamine, directly on cerebral microvessels.
barrier permeability
1001
Ginsberg M. D., Busto R., Booyhe T. E. and Campbell J. A. (1981) A radioisotopic method for the simultaneous quantitation of regional cerebral blood flow and glucose utilization in small dissected samples: validation studies and values in the nitrous oxide-anesthetized rat. Brain Res. 230: l65--179.
Gross P. M., Teasdale G. M., Graham D. I., Angerson W. J. and Harper A. M. (1982) Intra-arterial histamine increases blood-brain transport in rats. Am. J. Phvsiol. 243: H307-H317. Ingvar M., Abdul-Rahaman A. and Siesjo B. K. (1980) Local cerebral glucose consumption in the artificially ventilated rat: influence of nitrous oxide analgesia and of phenobarbital anaesthesia. Acfa physiol. stand. 109: 177-185. Lou H. C., Edvinsson L. and MacKenzie E. T. (1987) The concept of coupling blood flow to brain function: revision required? Ann. Neural. 22: 289-297. McCulloch J. (1982) Mapping functional alterations in the CNS with [‘%]deoxyglucose. In: Handbook of Psychopharmacology (Iversen L. L., Iversen S. D. and Snyder S. H., Eds), Vol. 15, pp. 321409. Plenum, New York. Nagai Y., Narumi S.. Miyamoto M., Saji Y. and Nagawa Y. (1983) A TRH analog (DN-1417): effect on local cerebral glucose utilization in conscious and pentobarbitalized rats determined by the autoradiographic 2-deoxy-[‘Qglucose method. Jap. J. Pharmac. 33: 635-645. Ohno K., Pettigrew K. and Rapoport S. (1978) Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat. Am. J. Physiol. 235: 299-307. Pardridge W. M. (1987) The gate keeper: How molecules are screened for admission to the brain. Sciences 27: 5G55. Paxinos G. and Watson C. (1982) The Rat Brain in Stereoraxic Coordinates. Academic Press, New York. Petit0 C. K., Pulsinelli W. A., Jacobson G. and Plum F. (1982) Edema and vascular permeability in cerebral ischemia: comparison between ischemic neuronal damage and infarction. J. Neuropath. exp. Neural. 41: 423436. Picozzi P., Todd N. and Crockard A. (1985) Regional blood-brain barrier permeability changes after restoration of blood flow in postischemic gerbil brains: a quantitative study. J. cereb. Blood FloursMetab. 5: l&16. Saija A.. Princi P., De Pasquale R. and Costa G. (1987) Regional blood-brain barrier permeability changes in response to ACTH plasma levels modifications. Bull. molec. Biol. Med. 12: 97-109.
REFERENCES Altura B. T. and Altura B. M. (1984) Effects of barbiturates, phencyclidine, ketamine and analogues on cerebral circulation and cerebrovascular muscle. Microcirc. Em&helium Lymphatics 1: 169-184. Betz A. L. and Goldstein G. W. (1984) Brain capillaries. Structure and Function. In: Handbook of Neurochemistry (Lajtha A., Ed.), 2nd edn, pp. 465484. Plenum, London. Carruba M. 0.. Bondiolotti G.. Picotti G. B.. Catteruccia N. and Da Prada M. (1987) Effects of diethyl ether, halothane. ketamine and urethane on sympathetic activity in the rat. Eur. J. Pharmac. 134: 15-24. Cavazzuti M., Porro C. A., Biral G. P.. Benassi C. and Barbieri C. G. (1987) K&amine effects on local cerebral blood flow and metabolism in the rat. J. cereb. Blood Flow Metab. 7: 806-81 I. Domer F. R., Boertje S. B. and Sampson J. B. (1985)
Permeability of the blood-brain barrier is not changed by vasoactive doses of arachidonic acid and prostaglandins in rats. Res. Commun them. Path. Pharmac. 48: 27-37. Ellison M. D., Povlishock J. T. and Hayes R. L. (1985) Examination of the blood-brain barrier transfer of aaminoisobutyric acid and horseradish peroxidase: regional alterations in blood-brain barrier function following acute hypertension. J. cereb. Blood Now Metab. 6: 471480.
Saija A., Princi P., De Pasquale R. and Costa G. (1988a) High intensity light exposure increases blood-brain barrier transport in rats. Pharmac. Res. Commun. 20: 553-559. Saija A., Princi P., De Pasquale R. and Costa G. (1988b) Circadian modifications of blood-brain barrier (BBB) permeability in the rat. Pharmac. Res. Commun. 20: 249-259. Sharma H. S. and Dey P. K. Dey (1981) Impairment of blood-brain barrier (BBB) in rat by immobilization stress: role of serotonin (5-HT). Indian J. Physiol. Pharmac. 25: 111-122.
Sokoloff L. (1980) The [“‘C]deoxyglucose
method for the quantitative determination of local cerebral glucose utilization: theoretical and practical consideration. In: Cerebral Merabolism and Neural Function (Passonneau J. W.. Hawkins R. A., Lust W. D. and Welsh F. A., Eds), pp. 319-330. Williams and Wilkins, Baltimore. Sokoloff L. (1983) Measurements of local glucose utilization and its use in localization of functional activity in the Central Nervous System of animals and man. Rec. Prog. Hormone Res. 39: 75-126. Sokoloff L.. Reivich M., Kennedy C., Des Rosiers M. H., Patlack C. S., Pettigrew K. D., Sakurada 0. and Shinohara M. (1977) The [“‘Cldeoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28: 897-916. Stewart P. A., Hayakawa E. M. and Carlen P. L. (1988)
1002
A. SAIJAet
Ethanol and ~tobarbi~l in combination increase bloodbrain barrier permeability to horseradish peroxidase. Brain Rex 443: 12-20.
Tyson G., Teasdale G., Graham D. and McCuiloch J. (1982) Cerebrovascular permeability following MCA occlusion in the rat. J. Neurosurg. 57: 186-196.
af.
Wixson S. K., White W. J. Hughes H. C. Jr, Lang C. M. and Marshall W. K. (1987) The effects of pentobarbital, fentanyl-droperidol, ketamine-xylazine and ketaminediazepam on arterial blood pH, blood gases, mean arterial blood pressure and heart rate in adult male rats. Lab. Anim. Sci. 37: 736742.