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Influence of profound hypothermia on the blood-brain barrier permeability during acute arterial hypertension
INFLUENCE BLOOD-BRAIN
OF PROFOUND HYPOTHERMIA ON THE BARRIER PERMEABILITY DURING ACUTE ARTERIAL HYPERTENSION
BARiA GZTAS, MEHMET KAYA and SAFiNAZ CAMURCU Ilepurtment
of Physiology, Istanbul Faculty of Medicine, Uniter-sit.y Capa, Istanbul 34390, Turkey
of Istunhrtl,
SUMMARY In hypothermic rats with acute hypertension induced by intravenous injection of adrenalin, regional changes in blood-brain barrier permeability to macromolecules were investigated using Evans blue as indication. Evans blue albumin extravasation was determined as a macroscopic finding and a quantitative estimation with a spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. Five groups of rats were studied: Group I: normothermia+acute hypertension; Group II: hypothermia+acute hypertension; Group III: control hypothermia; Group IV: normothermia+hypotension; Group V: control normothermia. The rats were anaesthetized with diethyl-ether. Body temperature was lowered by submerging anaesthetized animals in an ice water bath. The colonic temperature was reduced to 20+1 “C. During adrenaline-induced acute hypertension the mean arterial blood pressure increased in both normothermic and hypothermic animals. Blood-brain barrier lesions were present in 40% of normothermic rats, and 60% of hypothermic rats after adrenaline-induced hypertension. Mean value for Evans blue dye in the whole brain was found to be 0.530f0.202 mg% in the normothermic rats and 0.752f0.256 mg% in the hypothermic rats during adrenaline-induced hypertension. This difference between normothermic and hypothermic rats was found to be statistically significant (PcO.01). Our results showed that the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic rats after adrenaline-induced acute hypertension. KFY WORDS:blood-brain
barrier, hypothermia, Evans blue.
INTRODUCTION Profound induced hypothermia ( 15 ’ to 20 “C) combined with total circulatory arrest has been in use for several years to correct cardiac defects in neonates [ I]. 1043-6618/92/050075-10/$03.00/O
0 1992 The Italian Pharmacological Society
76
Pharmacological
Research,
Vol. 26, No. 1, 1992
On the other hand, profound hypothermia and circulatory arrest is used most commonly to facilitate surgical repair of giant intracranial aneurysms that would otherwise be inoperable [2]. Under conditions of deep hypothermia, cerebral pressure flow autoregulation is lost. Cerebral glucose utilization is decreased during induced hypothermia, but significant changes in the level of lactic acid are produced by the hypothermic brain [3]. Hypothermia also reduces cerebral oxygen consumption. The activity of the brain, as measured by the electroencephalogram, is consistent with the decreased cerebral biochemical findings [3]. A greater lowering of the temperature (18 “C) may lead to disturbed functions of the central nervous system [4]. An increased permeability of the blood-brain barrier was one of the factors discussed as being responsible for these disturbances [5,6]. In contrast to other organs, the microvascular endothelial cells in the brain are sealed together by continuous tight junctions; they do not contain fenestrations or transendothelial channels, and they have little transcellular vesicular transport [7]. The blood-brain barrier regulates the uptake rate of substances from the blood into the brain parenchyma and protects the brain from certain exogenous or endogenousfactors deriving from the blood stream [5, 81. This feature constitutes the morphological basis for the blood-brain barrier. The biochemical components of the barrier are to be seen in the degradation of certain substances by enzymes localized in the endothelial membranes [6, 81. Despite the use of these biological extremes of temperature, their effects on blood-brain barrier permeability are unknown. Light microscopic and biochemical tracer investigations have provided contradictory results on whether hypothermia increases or decreases the permeability of the blood-brain barrier [6,9-l I]. On the other hand, it has been shown in many studies that acute hypertenion can increase the blood-brain barrier permeability to protein in human beings and experimental animals. Despite extensive clinical observations on hypothermia, there has been no experimental study on the selective regional blood-brain barrier breakdown during hypothermia plus adrenaline-induced hypertension. We wish to find whether there is a difference in blood-brain barrier permeability during acute hypertension between normothermic and hypothermic conditions and the present experiments were therefore planned in order to answer the following questions: (1) Does deep hypothermia influence the blood-brain barrier permeability? (2) Does hypothermia have a protective or aggravating effect on blood-brain barrier permeability during adrenaline-induced hypertension? (3) Do hypothermia or hypotension effect blood-brain barrier dysfunction in hypothermic conditions?
MATERIAL
AND METHODS
A total of 131 adult male Wistar rats (weight 240-290 g) were used in this study. They were initially anaesthetized with diethyl ether. A femoral artery was cannulated for recording of mean arterial blood pressure. Mean arterial blood pressure was recorded by connecting the arterial catheter to a stain gauge
Phu~rnclt,oloXic,ul Research, Vol. 26. No. 1, 1992
transducer (Ugo-Basil). The femoral vein injections. Evans blue (4 ml/kg) was given blood-brain barrier function.
77
was cannulated for intravenous intravenously as an indicator of
Induction of hypothermia Body temperature was lowered by submerging anaesthetized animals in an ice water bath (l-2 “C) up to their necks. Rectal temperature was measured with a digital thermometer inserted 34 cm into the rectum. The colonic temperature was reduced to 20+1 “C under these conditions; the brain temperature was 4-S “C higher than that measured at the rectum [ 121. After the desired temperature was reached (lo-15 min, depending on the weight of the animal), the rats were taken out of the restraining table, and their fur dried. After a rectal temperature of 20 “C was attained, the temperature of heat exchange was controlled to maintain the temperature for the experimental duration. Five groups of rats were studied: Group I: normothermia+acute hypertension; Group II: hypothermia+acute hypertension; Group III: hypothermic control; Group IV: normothermia+hypotension; Group V: normothermic control. In the first and second group of rats after recording initial blood pressure, 4 ml/kg of 2% Evans blue was injected i.v. and after 5 min adrenaline (40 pug/kg) was rapidly injected in the normothermic and hypothermic animals (Groups I and II). Groups III and V served as controls for the permeability of blood-brain barrier and received Evans blue only. In Group IV animals Evans blue was injected and after 5 min 1 mg/kg nitroprusside was administered i.v. At the end of experiments, i.e. approximately 30 min after Evans blue or drug injection under diethyl-ether anaesthesia, all the rats were killed by perfusion through the heart with saline followed by 10% freshly prepared formaldehyde for 5 min to fix the brain in situ and to avoid artificial staining of the brain during removal. Then, brains were removed and, after macroscopic inspection of the brain surface and photographic registration of any Evans blue albumin extravasation, were cut by hand in 2-3-mm thick coronal blocks and presence and distribution of Evans blue leakage also recorded photographically. Barrier opening was graded as follows from observations and photographs of the intensity and extent of Evans blue staining: grade O=no staining of brain: grade l+=faint and localized staining; grade 2+=moderate blue staining; grade _?+=extensive, dark staining [ 131. A quantitative estimation of dye in the brain was also carried out according to the method of Harada et al. [14] in a separate group of animals in the same experimental conditions. Briefly, the brain was removed and bisected at the midline. Each half cerebrum and cerebellum were placed in tared tubes that were immediately reweighed. They were homogenized with 5 ml of phosphate-buffered saline containing a 5 ml% solution of 1 N NaOH. The homogenized brain was centrifuged (10 000 rpm for 10 min) and a spectrophotometric analysis at 620 nm was performed to measure the amount of resolved dye. Macroscopic findings described above correlated well with this quantitative estimation with the spectrophotometer. Data are expressed as mean&n, and statistical analysis was by Student’s r-test. Adrenalin and Evans blue were obtained from the Sigma Chemical Company.
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RESULTS The degree of blood-brain barrier breakdown and mean arterial blood pressure before and after drug administration are presented in Table I. There was a considerable decrease in mean arterial blood pressure during induction of hypothermia. When rectal temperature was decreased from 37fl “C to 20+1 “C, mean arterial blood pressure decreased from 113+11 mmHg to 61+9 mmHg (PC 0.01). We can say that hypothermia in rats results in an approximately 40% decrease of the mean arterial blood pressure in all hypothermic animals. In all rats a single rapid intravenous administration of 40 ,ug/kg adrenaline resulted in an immediate increase in mean arterial blood pressure (Fig. 1). The initial mean femoral arterial blood pressure was 94+11 mmHg in normothermic animals and 66 1-16 mmHg in hypothermic rats. These pressures rapidly increased to 172k 16 mmHg in normothermic and 18 l+ 13 mmHg in hypothermic animals after the adrenaline injections (Table I). The duration of increased blood pressure was 2.2-t 0.27 min in normothermic rats and 4.lfl min in hypothermic rats during adrenaline induced hypertension (Groups I, II). The duration difference between normothermic and hypothermic animals was found to be statistically significant (PcO.01). There was also a considerable decrease in mean arterial blood pressure after i.v. administration of 1 mg/kg nitroprusside (Group IV). The initial mean arterial blood pressure 114+9 mmHg and this decreased to 56&10 mmHg after drug injections (Table I).
Blood-brain barrier permeability changes The regional distribution of blood-brain barrier breakdown and its frequency are summarized in Table II. No Evans blue albumin extravasation was seen in the brains from normothermic control rats except in the pineal body, pituitary gland and choroid plexus-regions in which capillaries are known to be leaky. Evans blue dye was 0.279kO.48 mg% whole brain in this group (Group V). In the first group, no macroscopically evident Evans blue albumin complex was observed in the brain in 1.5 out of 25 rats. Minimal extravasation of Evans blue albumin was observed in five rats (grade l+) and moderate extravasation of Evans blue albumin in the remaining five rats (grade 2+) (Group I). In these animals distinct Evans blue leakage was observed in the cerebral cortex. These rats had small circumscribed areas of Evans blue albumin extravasation with a diameter of l-4 mm in the occipital, temporal and frontal cortex. Two of the rats had small spots of extravasation in the cerebellum. In hypothermic plus adrenaline-induced rats, blood-brain barrier leakage occurred similarly to that in normothermic rats but the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic ones. The magnitudes of the blood-brain barrier permeability differences we observed between normothermic and hypothermic conditions during adrenaline-induced acute hypertension were surprisingly large. The mean value for Evans blue dye was found to be 0.530+ 0.202 mg% whole brain in normothermic and 0.752f0.256 mg% whole brain in hypothermic animals after adrenaline-induced seizures (Fig. 2). This difference between normothermic and hypothermic animals was found to be significant (P< 0.01).
98k13
II
*In comparison to initial value PCO.01. n=number of animals.
control
Normothermic (Group V)
S6flO*
114f9
9
Normothermic+hypotension (Group IV)
61f9*
113+11
13
Hypothermic control (Group III)
66+16*
-
94fll
-
After hypothermia
barrier
-
181+13*
172+16*
Maximal
blood pressure (mmHg)
Initial
Mean arterial
105+12
23
Hypothermia+adrenaline (Group II)
n 25
groups
Normothermia+adrenaline (Group I)
Experimental
Table I Mean arterial blood pressure (MABP) and degree of blood-brain groups
76
78
AP
9
-
11
6
9
15
0
-
3
3
7
5
It
1
4
5
2t
3
3t
Degree of BBB Breakdown
4.1*1*
2.2kO.27
Duration (min)
(BBB) breakdown during experimental
Pharmacological Research, Vol. 26, No. I, I992
80
150 2 B z 2
100 -
50-
f 0
::
, 6
H I 12
9
1 15
I 18
1 21
24
1 30
27
33
TIME(MIN)
Fig. 1. Mean arterial blood pressure (MABP) record from a hypothermic animal treated with adrenaline. During the hypothermia (H) blood pressure decreased and administration adrenaline (A) resulted in an immediate increase. EB: Evans blue.
intravenous
Table II Frequency and intensity of regional distribution of adrenaline-induced blood-brain barrier breakdown in normothermic and hypothermic animals Brain
regions
Frontal cortex Occipital cortex Temporal cortex Parieto-occipital cortex
Hypothermia+Adrenaline Grade of staining
Normothermia+Adrenaline Grade of staining 0
1t
2t
3+
0
1-t
2t
3”r
17* I.5 22
5 5 3
3 5
-
11 9 IO
6 7 7
4 4 4
2 3 2
15
5
9
7
4
3
-
5
*Number of animals. (0-3-t): grade of Evans blue albumin extravasation (see text).
In the third group, induced hypothermia only, no macroscopically evident Evans blue albumin complex was observed in the brain in nine out of 13 animals. Minimal extravasation of Evans blue albumin was occurred in the three rats. The blood-brain barrier breakdown to Evans blue was found to be less intense (Grade l+) in hippocampus and hypothalamus in these animals. Only one animal showed moderate Evans blue albumin extravasation (Grade 2+). The cerebellum also exhibited blood-brain barrier breakdown. Evans blue dye was 0.507+0.180 mg% whole brain in this group. In the fourth group, which received only nitroprusside, minimal extravasation of Evans blue albumin was observed in the brain in six out
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Plrarrrrur~ologl~~aiResearch. Vol. 26, No. I, I992
1.2 r
m
Normothcrmio
m
Hypothrrmio
Fig. 2. Evans blue (mg% whole brain) content in the control, hypotension and adrenalineinduced hypertension in normothermic and hypothermic rats. Each column representsthe mean of IO rats. Mean&D. *P
of nine rats. The neuroanatomic distribution and extent of Evans blue penetration was similar to that in the hypothermic group (Group III). Evans blue dye was 0.408+0.160 mg% whole tirain in this group. There was no significant difference between hypothermic control and normothermic hypotensive groups (Groups III, IV) but there was a statistically significant difference between normothermic control and hypothermic control animals (P ~0.01).
DISCUSSION Evans blue albumin extravasation was determined as a macroscopic finding. However, quantitative estimation with a spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. Macroscopic findings correlated well with this quantitative estimation with spectrophotometer. Kajiwara et al. [ 151 have also confirmed that macroscopic findings correlated well with the spectrophotometer. The question of whether severe hypothermia lowers or increases the permeability of the blood-brain barrier has not yet been unequivocally decided [5, 6, lo]. Schindelmeiser et al. [6] studied the blood-brain barrier in animals using lanthanum nitrate as a tracer after profound hypothermia. From their study, it appeared that the blood-brain barrier is partially disturbed at temperatures of 7 “C and below [6]. In earlier studies, Lourie ef al., Miller et al. and Baldwin studied the brain using
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Pharmac~okqic~al Research, Vol. 26. No. I. 1992
different blood-brain barrier tracers such as trypan blue and sodium fluorescein respectively [I 1, 16, 171. They found that cerebral vascular permeability was increased during profound hypothermia. In contrast to the above results, Krantis found that hypothermia induced a reduction in the permeation of radiolabeled tracer substances across the blood-brain barrier [lo]. The partly contradictory results may be explained by different methods of cooling, different tracers, temperature ranges and evaluation methods [6]. In the present study, minimal alteration of blood-brain barrier permeability was observed in some hypothermic rats. Although the indicator used in this study, Evans blue, may not allow the discrimination of small changes in permeability possible with the other agents, blood-brain barrier breakdown observed during hypothermia was in areas of the brain similar to findings in normothermic hypotensive animals. Therefore we can speculate that hypotension rather than hypothermia increases the blood-brain barrier permeability during hypothermia. When rectal temperature was decreased from 37 “C to 20+1 “C, mean arterial blood pressure decreased significantly. Ishikawa et al. [18] have shown that nitroprusside- or trimethaphan-induced hypotension disturbed blood-brain barrier permeability in dogs anaesthetized with halothane. Acute hypertension is known to be associated with disturbances of the blood-brain barrier [ 19-231. In these conditions there is a loss of autoregulation of cerebral blood flow [22]. In the present investigations adrenaline produced blood pressure elevations similar in amplitude in normothermic and hypothermic animals but the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic rats in these experimental conditions. We were surprised at the magnitude of blood-brain barrier permeability differences we observed between normothermic and hypothermic rats in these experimental conditions. Hypothermia, in contrast to what was previously suggested, aggravates the permeability of the blood-brain barrier in hypertension. The detailed mechanism of the vulnerability of hypothermia on the blood-brain barrier is not known, but the results suggest some possible reason for the provacative effect of hypothermia on blood-brain barrier permeability during acute hypertension. The first reason why the extravasation of Evans blue albumin is most pronounced in the brains of hypothermic rats may be the duration of the mean arterial blood pressure during acute hypertension. Although there was no significant difference betwen normothermic and hypothermic groups in maximal blood pressure attained, a significant difference was found in the duration (Table I). Besides the rate of increase in pressure, the duration of this increased pressure was the important factor in the disruption of the blood-brain barrier during hypertension and convulsions [24, 251. Generally, the animals with shorter duration of maximal mean arterial blood pressure have less intense blood-brain barrier permeability, in contrast to the animals with higher duration of maximal mean arterial blood pressure and increase in blood-brain barrier permeability (251. Therefore, mean arterial blood pressure duration observed in hypothermic animals injected with adrenaline seems to be the parameter which determines the degree of blood-brain barrier permeability because the duration of increased blood pressure was 2.2f0.27 min in normothermic rats and 4. I l+l min in hypothermic rats
during adrenaline-induced hypertension. The importance of duration of high pressure on blood-brain barrier dysfunction was also observed in our previous studies with adrenaline and generalized convulsions in normothermic animals [20, 25, 261. Several other phenomena contribute to alterations of the blood-brain barrier breakdown in acute hypertension during hypothermia. These include an increase in the viscosity, a two-fold increase at 20 “C, alterations in cerebral vessel resistance, and a redistribution of cerebral blood flow. Each of these have an important role in determining the blood-brain barrier permeability [3]. Second, hypothermia alters the distribution of blood flow to organ systems. Cerebral glucose utilization and blood flow to the brain was significantly decreased during induced hypothermia, but significant change in the levels of lactic acid are produced by the hypothermic brain [3, 271. The influx of potassium into astrocytes could also be reduced. Both lactic acid and increased potassium ion concentrations are known potent vasodilators of cerebral vessels. Cerebral vasodilatation, which increases the tension in the vessel wall, enhances the blood-brain barrier permeability [20, 281 because the cerebrovascular tone is of main importance, i.e. dilatation enhances and vasoconstriction decreases Evans blue albumin leakage. Neither alterations in vascular tone nor in neurogenic activity can fully explain the differences in vulnerability of blood-brain barrier between normothermic and hypothermic animals during our experimental conditions. It is likely that alteration in the cerebral endothelial cell membrane is more important than we realise. That hypothermic rats are more prone to develop permeability disturbances than normothermic rats during acute hypertension may be of clinical interest. However, the neurological consequences of these barrier disturbances are at present uncertain. The extravasation of protein into the brain is particularly indicative ot the vasogenic type of cerebral oedema [29]. As a result we can say that hypothermic rats are more prone to develop acute arterial permeability disturbances than normothermic rats during hypertension.
REFERENCES I. Greeley WJ, Ungerleider RS, Smith R, Reves JG, Durhan NC. The effects of deep hypothermic cardiopulmonary bypass and total circulatory arrest on cerebral blood flow in infants and children. J Thorax Cardiovasc Surg 1989; 97: 737115. 2. Cyatte D, Elefteriades J, Kim B. Profound hypothermia and circulatory arrest for aneurysm surgery. J Neurosurg 1989; 70: 489-9 I. 3. Black PR, Devanter SV, Lawrance HC. Effects of hypothermia on systemic and organ system metabolism and function. J Surg Res 1976; 20: 49-63. 4. Molina JE, Einzig S, Mastri AR, et al. Brain damage in profound hypothermia. .I 7’l1or.ac, C’urdiovasc
Surg 1984; 87: 596-604.
5. Bradbury M. The concept of a blood-brain barrier. New York: Wiley, 1979. 6. Schindelmeiser J, Bergmann M, Lehmenkiihler A, Kersting V. Tracer permeability of rat cortical blood vessels during regional hypothermia. Acfa Neuroparhol 1987; 73: 349-65.
7. Betz AL. Transport of ions across the blood-brain barrier. Fed Proc 1986; 45: 2050-4. 8. Bradbury M. The blood-brain 1985; 57: 213-22.
barrier transport across the cerebral endothelium.
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Pharmacological Research,Vol. 26, No. I. 1992
9. Klatzo I, Piraux A, Laskowski EJ. The relationship between edema, blood-brain barrier and tissue elements in a local brain injury. J Neuropathol Exp New-01 1958; 17: 548-64. 10. Krantis A. Hypothermia-induced reduction in the permeation of radio-labelled tracer substances across the blood-brain barrier. Acta Neuropathol (Berl) 1983; 60: 61-9. 11. Miller DR, Hallaba MAS, Steegmann AT. Effect of profound hypothermia with circulatory arrest in dogs special references to changes in cerebrovascular permeability. Ann Surg 1965; 161: 272-85. 12. Emirbekow EZ, Sershen H, Lajtha A. Lack of effects of hypothermia on cerebral amino acid uptake in vivo. Brain Res 1977; 125: 187-9 1. 13. Rapoport SI, Frederick WR, Ohno K, Pettigrew KD. Quantitative aspect of reversible osmotic opening of the blood-brain barrier. Am J Physiol 1980; 238: R421-3 1. 14. Harada AM, Takeuchi M, Fukato T, Katagiri KA. Simple method for the quantitative extraction of dye extravasated into the skin. J Pharm Pharmacol 1971; 23: 218-9. 15. Kajiwara K, Ito H, Fukumoto T. Lymphocyte infiltration into normal rat brain following hyperosmotic blood-brain barrier opening. J Neuroimmunol 1980; 27: 133-40. 16. Baldwin M, Galinda A, Farrier R. Cerebral reaction to sodium fluorescein during profound hypothermia. Neurology 1962; 12: 193-201. 17. Lourie H, Weinstein WJ, O’Leary JL. The effect of hypothermia upon vital staining of the brain. J Nerv Ment Dis 1960; 130: l-5. 18. Ishikawa T, Funatsu N, Okamoto K, Takeshita H, McDowall G. Blood-brain barrier function following drug-induced hypotension in the dog. Anesthesiology 1983; 59: 526-31. 19. Bolwing TG, Hertz MM, Westergaard E. Acute hypertension causing blood-brain barrier breakdown during epileptic seizures. Acta New-01 Stand 1977; 56: 33542. 20. Gztav B, Sandalci U, Eren A, Gokhan N. The influence of adrenalin hypertension on the permeability of blood-brain barrier. IRCS Med Sci 1984; 12: 722-5. 21. Gztag B. Hypertension induced protein leakage in the brain in acute ethanol administered rats. Res Commun Substances of Abuse 1989; 10: 189-92. 22. Suzuki R, Nitsch C, Fujiwara K, Klatzo I. Regional changes in cerebral blood flow and blood-brain barrier permeability during epileptiform seizures and in acute hypertension in rabbits. J Cereb Blood Flow Metab 1984; 4: 96-100. 23. Westergaard E. The blood-brain barrier to horseradish peroxidase under normal and experimental conditions. Acta Neuropath (Berl) 1977; 39: 181-7. 24. Johansson B. Pharmacological modification of hypertension blood-brain barrier opening. Acta Pharmacol Toxic01 1981; 48: 242-7. 25. Gztag B, Kaya M. The effect of acute hypertension on blood-brain barrier permeability to albumin during experimentally induced epileptic seizures. Pharmacol Res 1991; 23: 41-6. 26. Gzta? B, Sandalct U. Blood-brain barrier permeability after pentylenetetrazol and electrically induced seizures. ZRCS Med Sci 1984; 12: 488-9. 27. Michenfelder JD, Theys RA. Hypothermia: Effects on canine brain and whole body metabolism. Anesthesiology 1968; 29: 1107-12. 28. Johansson B. Blood-brain barrier dysfunction in acute arterial hypertension after papaverine-induced vasodilatation. Acta Neurol Stand 1974; SO: 573-80. 29. Klatzo I, Neuropathological aspects of brain edema. J Neuropathol Exp New-01 1967; 24: l-13.
OF PROFOUND HYPOTHERMIA ON THE BARRIER PERMEABILITY DURING ACUTE ARTERIAL HYPERTENSION
BARiA GZTAS, MEHMET KAYA and SAFiNAZ CAMURCU Ilepurtment
of Physiology, Istanbul Faculty of Medicine, Uniter-sit.y Capa, Istanbul 34390, Turkey
of Istunhrtl,
SUMMARY In hypothermic rats with acute hypertension induced by intravenous injection of adrenalin, regional changes in blood-brain barrier permeability to macromolecules were investigated using Evans blue as indication. Evans blue albumin extravasation was determined as a macroscopic finding and a quantitative estimation with a spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. Five groups of rats were studied: Group I: normothermia+acute hypertension; Group II: hypothermia+acute hypertension; Group III: control hypothermia; Group IV: normothermia+hypotension; Group V: control normothermia. The rats were anaesthetized with diethyl-ether. Body temperature was lowered by submerging anaesthetized animals in an ice water bath. The colonic temperature was reduced to 20+1 “C. During adrenaline-induced acute hypertension the mean arterial blood pressure increased in both normothermic and hypothermic animals. Blood-brain barrier lesions were present in 40% of normothermic rats, and 60% of hypothermic rats after adrenaline-induced hypertension. Mean value for Evans blue dye in the whole brain was found to be 0.530f0.202 mg% in the normothermic rats and 0.752f0.256 mg% in the hypothermic rats during adrenaline-induced hypertension. This difference between normothermic and hypothermic rats was found to be statistically significant (PcO.01). Our results showed that the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic rats after adrenaline-induced acute hypertension. KFY WORDS:blood-brain
barrier, hypothermia, Evans blue.
INTRODUCTION Profound induced hypothermia ( 15 ’ to 20 “C) combined with total circulatory arrest has been in use for several years to correct cardiac defects in neonates [ I]. 1043-6618/92/050075-10/$03.00/O
0 1992 The Italian Pharmacological Society
76
Pharmacological
Research,
Vol. 26, No. 1, 1992
On the other hand, profound hypothermia and circulatory arrest is used most commonly to facilitate surgical repair of giant intracranial aneurysms that would otherwise be inoperable [2]. Under conditions of deep hypothermia, cerebral pressure flow autoregulation is lost. Cerebral glucose utilization is decreased during induced hypothermia, but significant changes in the level of lactic acid are produced by the hypothermic brain [3]. Hypothermia also reduces cerebral oxygen consumption. The activity of the brain, as measured by the electroencephalogram, is consistent with the decreased cerebral biochemical findings [3]. A greater lowering of the temperature (18 “C) may lead to disturbed functions of the central nervous system [4]. An increased permeability of the blood-brain barrier was one of the factors discussed as being responsible for these disturbances [5,6]. In contrast to other organs, the microvascular endothelial cells in the brain are sealed together by continuous tight junctions; they do not contain fenestrations or transendothelial channels, and they have little transcellular vesicular transport [7]. The blood-brain barrier regulates the uptake rate of substances from the blood into the brain parenchyma and protects the brain from certain exogenous or endogenousfactors deriving from the blood stream [5, 81. This feature constitutes the morphological basis for the blood-brain barrier. The biochemical components of the barrier are to be seen in the degradation of certain substances by enzymes localized in the endothelial membranes [6, 81. Despite the use of these biological extremes of temperature, their effects on blood-brain barrier permeability are unknown. Light microscopic and biochemical tracer investigations have provided contradictory results on whether hypothermia increases or decreases the permeability of the blood-brain barrier [6,9-l I]. On the other hand, it has been shown in many studies that acute hypertenion can increase the blood-brain barrier permeability to protein in human beings and experimental animals. Despite extensive clinical observations on hypothermia, there has been no experimental study on the selective regional blood-brain barrier breakdown during hypothermia plus adrenaline-induced hypertension. We wish to find whether there is a difference in blood-brain barrier permeability during acute hypertension between normothermic and hypothermic conditions and the present experiments were therefore planned in order to answer the following questions: (1) Does deep hypothermia influence the blood-brain barrier permeability? (2) Does hypothermia have a protective or aggravating effect on blood-brain barrier permeability during adrenaline-induced hypertension? (3) Do hypothermia or hypotension effect blood-brain barrier dysfunction in hypothermic conditions?
MATERIAL
AND METHODS
A total of 131 adult male Wistar rats (weight 240-290 g) were used in this study. They were initially anaesthetized with diethyl ether. A femoral artery was cannulated for recording of mean arterial blood pressure. Mean arterial blood pressure was recorded by connecting the arterial catheter to a stain gauge
Phu~rnclt,oloXic,ul Research, Vol. 26. No. 1, 1992
transducer (Ugo-Basil). The femoral vein injections. Evans blue (4 ml/kg) was given blood-brain barrier function.
77
was cannulated for intravenous intravenously as an indicator of
Induction of hypothermia Body temperature was lowered by submerging anaesthetized animals in an ice water bath (l-2 “C) up to their necks. Rectal temperature was measured with a digital thermometer inserted 34 cm into the rectum. The colonic temperature was reduced to 20+1 “C under these conditions; the brain temperature was 4-S “C higher than that measured at the rectum [ 121. After the desired temperature was reached (lo-15 min, depending on the weight of the animal), the rats were taken out of the restraining table, and their fur dried. After a rectal temperature of 20 “C was attained, the temperature of heat exchange was controlled to maintain the temperature for the experimental duration. Five groups of rats were studied: Group I: normothermia+acute hypertension; Group II: hypothermia+acute hypertension; Group III: hypothermic control; Group IV: normothermia+hypotension; Group V: normothermic control. In the first and second group of rats after recording initial blood pressure, 4 ml/kg of 2% Evans blue was injected i.v. and after 5 min adrenaline (40 pug/kg) was rapidly injected in the normothermic and hypothermic animals (Groups I and II). Groups III and V served as controls for the permeability of blood-brain barrier and received Evans blue only. In Group IV animals Evans blue was injected and after 5 min 1 mg/kg nitroprusside was administered i.v. At the end of experiments, i.e. approximately 30 min after Evans blue or drug injection under diethyl-ether anaesthesia, all the rats were killed by perfusion through the heart with saline followed by 10% freshly prepared formaldehyde for 5 min to fix the brain in situ and to avoid artificial staining of the brain during removal. Then, brains were removed and, after macroscopic inspection of the brain surface and photographic registration of any Evans blue albumin extravasation, were cut by hand in 2-3-mm thick coronal blocks and presence and distribution of Evans blue leakage also recorded photographically. Barrier opening was graded as follows from observations and photographs of the intensity and extent of Evans blue staining: grade O=no staining of brain: grade l+=faint and localized staining; grade 2+=moderate blue staining; grade _?+=extensive, dark staining [ 131. A quantitative estimation of dye in the brain was also carried out according to the method of Harada et al. [14] in a separate group of animals in the same experimental conditions. Briefly, the brain was removed and bisected at the midline. Each half cerebrum and cerebellum were placed in tared tubes that were immediately reweighed. They were homogenized with 5 ml of phosphate-buffered saline containing a 5 ml% solution of 1 N NaOH. The homogenized brain was centrifuged (10 000 rpm for 10 min) and a spectrophotometric analysis at 620 nm was performed to measure the amount of resolved dye. Macroscopic findings described above correlated well with this quantitative estimation with the spectrophotometer. Data are expressed as mean&n, and statistical analysis was by Student’s r-test. Adrenalin and Evans blue were obtained from the Sigma Chemical Company.
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Research.
Vol. 26, No. 1. I992
RESULTS The degree of blood-brain barrier breakdown and mean arterial blood pressure before and after drug administration are presented in Table I. There was a considerable decrease in mean arterial blood pressure during induction of hypothermia. When rectal temperature was decreased from 37fl “C to 20+1 “C, mean arterial blood pressure decreased from 113+11 mmHg to 61+9 mmHg (PC 0.01). We can say that hypothermia in rats results in an approximately 40% decrease of the mean arterial blood pressure in all hypothermic animals. In all rats a single rapid intravenous administration of 40 ,ug/kg adrenaline resulted in an immediate increase in mean arterial blood pressure (Fig. 1). The initial mean femoral arterial blood pressure was 94+11 mmHg in normothermic animals and 66 1-16 mmHg in hypothermic rats. These pressures rapidly increased to 172k 16 mmHg in normothermic and 18 l+ 13 mmHg in hypothermic animals after the adrenaline injections (Table I). The duration of increased blood pressure was 2.2-t 0.27 min in normothermic rats and 4.lfl min in hypothermic rats during adrenaline induced hypertension (Groups I, II). The duration difference between normothermic and hypothermic animals was found to be statistically significant (PcO.01). There was also a considerable decrease in mean arterial blood pressure after i.v. administration of 1 mg/kg nitroprusside (Group IV). The initial mean arterial blood pressure 114+9 mmHg and this decreased to 56&10 mmHg after drug injections (Table I).
Blood-brain barrier permeability changes The regional distribution of blood-brain barrier breakdown and its frequency are summarized in Table II. No Evans blue albumin extravasation was seen in the brains from normothermic control rats except in the pineal body, pituitary gland and choroid plexus-regions in which capillaries are known to be leaky. Evans blue dye was 0.279kO.48 mg% whole brain in this group (Group V). In the first group, no macroscopically evident Evans blue albumin complex was observed in the brain in 1.5 out of 25 rats. Minimal extravasation of Evans blue albumin was observed in five rats (grade l+) and moderate extravasation of Evans blue albumin in the remaining five rats (grade 2+) (Group I). In these animals distinct Evans blue leakage was observed in the cerebral cortex. These rats had small circumscribed areas of Evans blue albumin extravasation with a diameter of l-4 mm in the occipital, temporal and frontal cortex. Two of the rats had small spots of extravasation in the cerebellum. In hypothermic plus adrenaline-induced rats, blood-brain barrier leakage occurred similarly to that in normothermic rats but the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic ones. The magnitudes of the blood-brain barrier permeability differences we observed between normothermic and hypothermic conditions during adrenaline-induced acute hypertension were surprisingly large. The mean value for Evans blue dye was found to be 0.530+ 0.202 mg% whole brain in normothermic and 0.752f0.256 mg% whole brain in hypothermic animals after adrenaline-induced seizures (Fig. 2). This difference between normothermic and hypothermic animals was found to be significant (P< 0.01).
98k13
II
*In comparison to initial value PCO.01. n=number of animals.
control
Normothermic (Group V)
S6flO*
114f9
9
Normothermic+hypotension (Group IV)
61f9*
113+11
13
Hypothermic control (Group III)
66+16*
-
94fll
-
After hypothermia
barrier
-
181+13*
172+16*
Maximal
blood pressure (mmHg)
Initial
Mean arterial
105+12
23
Hypothermia+adrenaline (Group II)
n 25
groups
Normothermia+adrenaline (Group I)
Experimental
Table I Mean arterial blood pressure (MABP) and degree of blood-brain groups
76
78
AP
9
-
11
6
9
15
0
-
3
3
7
5
It
1
4
5
2t
3
3t
Degree of BBB Breakdown
4.1*1*
2.2kO.27
Duration (min)
(BBB) breakdown during experimental
Pharmacological Research, Vol. 26, No. I, I992
80
150 2 B z 2
100 -
50-
f 0
::
, 6
H I 12
9
1 15
I 18
1 21
24
1 30
27
33
TIME(MIN)
Fig. 1. Mean arterial blood pressure (MABP) record from a hypothermic animal treated with adrenaline. During the hypothermia (H) blood pressure decreased and administration adrenaline (A) resulted in an immediate increase. EB: Evans blue.
intravenous
Table II Frequency and intensity of regional distribution of adrenaline-induced blood-brain barrier breakdown in normothermic and hypothermic animals Brain
regions
Frontal cortex Occipital cortex Temporal cortex Parieto-occipital cortex
Hypothermia+Adrenaline Grade of staining
Normothermia+Adrenaline Grade of staining 0
1t
2t
3+
0
1-t
2t
3”r
17* I.5 22
5 5 3
3 5
-
11 9 IO
6 7 7
4 4 4
2 3 2
15
5
9
7
4
3
-
5
*Number of animals. (0-3-t): grade of Evans blue albumin extravasation (see text).
In the third group, induced hypothermia only, no macroscopically evident Evans blue albumin complex was observed in the brain in nine out of 13 animals. Minimal extravasation of Evans blue albumin was occurred in the three rats. The blood-brain barrier breakdown to Evans blue was found to be less intense (Grade l+) in hippocampus and hypothalamus in these animals. Only one animal showed moderate Evans blue albumin extravasation (Grade 2+). The cerebellum also exhibited blood-brain barrier breakdown. Evans blue dye was 0.507+0.180 mg% whole brain in this group. In the fourth group, which received only nitroprusside, minimal extravasation of Evans blue albumin was observed in the brain in six out
XI
Plrarrrrur~ologl~~aiResearch. Vol. 26, No. I, I992
1.2 r
m
Normothcrmio
m
Hypothrrmio
Fig. 2. Evans blue (mg% whole brain) content in the control, hypotension and adrenalineinduced hypertension in normothermic and hypothermic rats. Each column representsthe mean of IO rats. Mean&D. *P
of nine rats. The neuroanatomic distribution and extent of Evans blue penetration was similar to that in the hypothermic group (Group III). Evans blue dye was 0.408+0.160 mg% whole tirain in this group. There was no significant difference between hypothermic control and normothermic hypotensive groups (Groups III, IV) but there was a statistically significant difference between normothermic control and hypothermic control animals (P ~0.01).
DISCUSSION Evans blue albumin extravasation was determined as a macroscopic finding. However, quantitative estimation with a spectrophotometer using homogenized brain to release the dye was also performed to evaluate the macroscopic findings. Macroscopic findings correlated well with this quantitative estimation with spectrophotometer. Kajiwara et al. [ 151 have also confirmed that macroscopic findings correlated well with the spectrophotometer. The question of whether severe hypothermia lowers or increases the permeability of the blood-brain barrier has not yet been unequivocally decided [5, 6, lo]. Schindelmeiser et al. [6] studied the blood-brain barrier in animals using lanthanum nitrate as a tracer after profound hypothermia. From their study, it appeared that the blood-brain barrier is partially disturbed at temperatures of 7 “C and below [6]. In earlier studies, Lourie ef al., Miller et al. and Baldwin studied the brain using
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Pharmac~okqic~al Research, Vol. 26. No. I. 1992
different blood-brain barrier tracers such as trypan blue and sodium fluorescein respectively [I 1, 16, 171. They found that cerebral vascular permeability was increased during profound hypothermia. In contrast to the above results, Krantis found that hypothermia induced a reduction in the permeation of radiolabeled tracer substances across the blood-brain barrier [lo]. The partly contradictory results may be explained by different methods of cooling, different tracers, temperature ranges and evaluation methods [6]. In the present study, minimal alteration of blood-brain barrier permeability was observed in some hypothermic rats. Although the indicator used in this study, Evans blue, may not allow the discrimination of small changes in permeability possible with the other agents, blood-brain barrier breakdown observed during hypothermia was in areas of the brain similar to findings in normothermic hypotensive animals. Therefore we can speculate that hypotension rather than hypothermia increases the blood-brain barrier permeability during hypothermia. When rectal temperature was decreased from 37 “C to 20+1 “C, mean arterial blood pressure decreased significantly. Ishikawa et al. [18] have shown that nitroprusside- or trimethaphan-induced hypotension disturbed blood-brain barrier permeability in dogs anaesthetized with halothane. Acute hypertension is known to be associated with disturbances of the blood-brain barrier [ 19-231. In these conditions there is a loss of autoregulation of cerebral blood flow [22]. In the present investigations adrenaline produced blood pressure elevations similar in amplitude in normothermic and hypothermic animals but the extravasation of Evans blue albumin was most pronounced in the brains of hypothermic rats compared to normothermic rats in these experimental conditions. We were surprised at the magnitude of blood-brain barrier permeability differences we observed between normothermic and hypothermic rats in these experimental conditions. Hypothermia, in contrast to what was previously suggested, aggravates the permeability of the blood-brain barrier in hypertension. The detailed mechanism of the vulnerability of hypothermia on the blood-brain barrier is not known, but the results suggest some possible reason for the provacative effect of hypothermia on blood-brain barrier permeability during acute hypertension. The first reason why the extravasation of Evans blue albumin is most pronounced in the brains of hypothermic rats may be the duration of the mean arterial blood pressure during acute hypertension. Although there was no significant difference betwen normothermic and hypothermic groups in maximal blood pressure attained, a significant difference was found in the duration (Table I). Besides the rate of increase in pressure, the duration of this increased pressure was the important factor in the disruption of the blood-brain barrier during hypertension and convulsions [24, 251. Generally, the animals with shorter duration of maximal mean arterial blood pressure have less intense blood-brain barrier permeability, in contrast to the animals with higher duration of maximal mean arterial blood pressure and increase in blood-brain barrier permeability (251. Therefore, mean arterial blood pressure duration observed in hypothermic animals injected with adrenaline seems to be the parameter which determines the degree of blood-brain barrier permeability because the duration of increased blood pressure was 2.2f0.27 min in normothermic rats and 4. I l+l min in hypothermic rats
during adrenaline-induced hypertension. The importance of duration of high pressure on blood-brain barrier dysfunction was also observed in our previous studies with adrenaline and generalized convulsions in normothermic animals [20, 25, 261. Several other phenomena contribute to alterations of the blood-brain barrier breakdown in acute hypertension during hypothermia. These include an increase in the viscosity, a two-fold increase at 20 “C, alterations in cerebral vessel resistance, and a redistribution of cerebral blood flow. Each of these have an important role in determining the blood-brain barrier permeability [3]. Second, hypothermia alters the distribution of blood flow to organ systems. Cerebral glucose utilization and blood flow to the brain was significantly decreased during induced hypothermia, but significant change in the levels of lactic acid are produced by the hypothermic brain [3, 271. The influx of potassium into astrocytes could also be reduced. Both lactic acid and increased potassium ion concentrations are known potent vasodilators of cerebral vessels. Cerebral vasodilatation, which increases the tension in the vessel wall, enhances the blood-brain barrier permeability [20, 281 because the cerebrovascular tone is of main importance, i.e. dilatation enhances and vasoconstriction decreases Evans blue albumin leakage. Neither alterations in vascular tone nor in neurogenic activity can fully explain the differences in vulnerability of blood-brain barrier between normothermic and hypothermic animals during our experimental conditions. It is likely that alteration in the cerebral endothelial cell membrane is more important than we realise. That hypothermic rats are more prone to develop permeability disturbances than normothermic rats during acute hypertension may be of clinical interest. However, the neurological consequences of these barrier disturbances are at present uncertain. The extravasation of protein into the brain is particularly indicative ot the vasogenic type of cerebral oedema [29]. As a result we can say that hypothermic rats are more prone to develop acute arterial permeability disturbances than normothermic rats during hypertension.
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