Iniern. J. Neuroscience, 1999, Val. 99, pp. 105--112 Reprints available directly from the publisher Photocopying permitted by license only

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THE INFLUENCE OF NIFEDIPINE

PERMEABILITY DURING ON BLOOD-BRAIN BARRIER

BICUCULLINE-INDUCED SEIZURES

BARIA OZTAS

Department of Physiology, Istanbul Faculty of Medicine, University of Istanbul (7apa/Istanbu1/34390/Turkey

(Received in finalform I 2 January 1999)

The influence of the calcium channel blocker nifedipine plus bicuculline-induced seizures on the permeability of the blood-brain barrier to protein was studied in rats. Evans-blue was used as a blood-brain barrier tracer. Four groups of rats were studied. Group I: control, Group 11: Nifedipine, Group 111: bicuculline-induced seizure, Group IV: Nifedipine + seizure. The mean value for Evans-blue dye in the brain was found to be 0.23 f 0.03 mg/g in control animals and 0.32 f 0.06 mg/g in the group of all rats during nifedipine-induced hypotension. This difference between control and hypotensive animals was not statistically significant ( p < 0.5). Mean value for Evans-blue dye In the brain was found to be 1.4 f 0.3 mg/g in bicuculline-induced seizure, and 0.73 f 0.2mg/g in the group of nifedipine plus bicuculline-induced seizures. This differ- ence between Group 111 and Group I V was found statistically significant ( p < .01). The cal- cium channel blocker nifedipine significantly prevents the blood brain barrier disruption during bicuculline-induced seizure.

Keywords: Blood-brain barrier; Evans-blue; calcium channel blocker; seizure

1. INTRODUCTION

The blood-brain barrier minimizes the entry of water soluble molecules, proteins and ions into brain tissue. The anatomical basis for restricted pas- sage of large molecules and water soluble molecules has been attributed to the tight interendothelial junctions and the relative lack of pinocytotic activ- ity which characterize the blood vessels of the brain (Davson and Segal, 1995). Breakdown of the blood-brain barrier occurs in many pathological conditions of the central nervous system including epileptic seizures

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I06 B. OZTAS

(Greenwood, 1991; Oztag and Camurcu, 1989; Oztag and Kaya, 1994). Several workers have studied the effect of seizures on the blood-brain barrier permeability (Davson and Segal, 1995; Oztag and Sandalci, 1984; Oztag et al., 1992). An increased permeability to protein of the blood-brain barrier has also been demonstrated after electrically induced seizures (Bolwing et al., 1977a; Oztag and Camurcu, 1989). The uptake into brain tissue of various marker substances such as Evans-blue, Horseradish peroxidase, lantanum, or radioactively labelled albumin has been used as an index of increased cerebrovascular permeability during experimentally-induced seizures.

On the other hand, calcium is known to play an important role in main- taining the tightness of the blood-brain barrier (Nagashima et a/., 1994) and calcium channel blokers are increasingly used in cerebral diseases such as focal brain ischemia (Cohan, 1990; Pazzili et al., 1989). Calcium plays an essential role in regulating membrane exitability, hormone and neurotrans- mitter secretion, and enzyme activation. Cerebral blood flow autoregulation is provided by the contractility of vascular smooth muscles which depend on calcium, particularly in the cerebral vessels. The rationale of calcium antago- nism is the prevention of vasospasm by blocking the entry of calcium into vascular smooth muscles and thus impairing their contractility (Cohan, 1990; Scholz, 1997). The protein leakage into the brain can be modified by drugs changing the cerebrovascular tone, as well as by drugs acting on the cerebral endothelial cell membrane, possibly by inhibiting pinocytosis (Joo’, 1994). Calcium antagonists could change both calcium dependent pinocy- totic activity in capillary endothelial cells and the vascular tone. In this study, we examined the effect of pretreatment with a calcium channel blocker, nifedipine, on the increased cerebrosvascular permeability to protein that develops in bicuculline induced seizures.

2. MATERIALS AND METHODS

The experiments were performed on 45 male Wistar Albino rats, having free access to food and water, and weighing between 210-240 g.

2.1. Experimental Design

The rats were anaesthetized with diethylether. A catheter was introduced into the right femoral artery. This was connected to a pressure transduced to allow blood pressure recordings before, during and after drug administra- tion. A femoral vein catheter was inserted to inject the drug and a single dose of Evans-blue (4ml of 2% solution in saline/kg).

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BICUCULLINE-INDUCED SEIZURES 107

Evans-blue was used as an indicator of blood-brain barrier function. Evans-blue binds to serum albumin in vivo and is easily detected macro- scopically providing a convenient gross evaluation of leakage. Rats were pretreated with nifedipine (20 ug/kg) which was dissolved in the commercial solvent. The substance was handled in sodium light to prevent degradation of the drug. Ten min later, convulsion was induced by an intravenous injec- tion of bicuculline (1.2 ug/kg). Mean arterial blood pressure was recorded continuously during the experiments (Ugo-Basil). Four group rats were stud- ied: Group I: Control, Group 11: Nifedipine, Group 111: Bicuculline-induced seizure, Group IV: Nifedipine + bicuculline-induced seizure. Approximately 20 minutes after bicuculline or nifedipine injections, all rats were killed by perfusion of saline via the left heart ventricle to remove blood and tracers from cerebral blood flow, under diethyl-ether anesthesia.

2.2. Estimation of the Degree of Blood-Brain Barrier Opening

The degree of blood brain opening in terms of extravasation of Evans-blue albumin was graded on the basis of the gross appearance of the brain accord- ing to the method by Rapoport et al. (1980) as follows: Grade 0, no staining on the surface of on the coronal section; grade 1 t, just noticeable staining; grade 2 +, moderate blue staining; and grade 3 +, dark blue staining on the surface or on the coronal sections. A quantitative estimation of dye in the brain was also carried out earlier (Oztag and Kiiqiik, 1995). 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. The homogenized brain was centrifuged (10.000 rpm for 10min) and a spectro- photometrical analysis at 620 nm of wavelength was performed to measure the amount of resolved dye.

Macroscopical findings described above correlated well with this quanti- tative estimation with spectrophotometer.

2.3. Statistical Analysis

Data were expressed as mean f SD, and statistical analyse was performed by Students t-test. p values less than .05 were considered to be significant.

3. RESULTS

The degree of blood-brain barrier breakdown and mean arterial blood pressure before and after drug administration are presented in Table I .

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108 B. OZTAS

TABLE I breakdown for the various experimental groups

Experimental MABP(mmHg) Degree of BBB break down groups n Initial After nifedipine Maximal 0 I + 2+ 3+

Mean arterial blood pressure (MABP) and degree of blood-brain barrier (BBB)

Control 8 1 0 8 f 9 - - 8 - - ~

(Group I) Nifedipine 12 1 0 2 f 1 0 57+10* ~ -

(Group 11) (Immediately) Seizure 13 1 0 4 f l l ~ 178*12* 4 3 3 3 (Group 111) Nifedipine 12 108 f 3 8 4 1 1 1 * 1 2 0 5 1 3 5 5 2 - + Seizure (10min later) (Group IV)

10 2 ~

*In comparison to initial value, p < .01

There was a considerable decrease in mean arterial blood pressure after nifedi- pine injection. The mean decrease in mean arterial blood pressure induced by intravenous nifedipine was 45mmHg in anestetized rats. Prior to the administration of bicuculline, the pressure 24 mmHg lower than the initial mean arterial blood pressure. The initial mean arterial blood pressure was 102 f 10mmHg in control animals in 57 f 10mmHg after nidefipine injec- tion ( p < .Ol) . We can say that injection of nifedipine in rats results in an approximately 45% abrupt decrease of the mean arterial blood pressure in all animals. In all animals a single rapid intravenous administration of 1.2 mg/kg bicuculline resulted in an immediate increase in mean arterial blood pressure. This rapid increase in mean arterial blood pressure was 74 mmHg in control animals (Group 111) and 36 mmHg in nifedipine treated animals (Group IV).

3.1. Blood-Brain Barrier Permeability Changes

Our experiments were carried out with two different methods to investigate the integrity of the blood brain barrier i.e., Evans-blue dye was measured quantitatively and qualitatively. No Evans-blue albumin extravazation was seen in the brains of control rats except in the pineal body, pituitary gland and choroid plexus, i.e., regions in which capillaries are known to be leaky. In the second group, which underwent nifedipine-induced hypotension, no macroscopically evident Evans-blue albumin complex was observed in the brain in ten out of twelve rats. Nevertheless, the values from control group and from the nifedipine-treated animals can be compared because they were obtained under identical conditions, except for the administration of nifedipine. A slightly higher Evans-blue content was found in the brain regions of rats given nifedipine than in the corresponding control rats, but

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BICUCULLINE-INDUCED SEIZURES 109

the values were not significantly different. The mean value for Evans-blue dye in the brain was found to be 0.23 f 0.03 mg/g control animals and 0.32 f 0.06 mg/g in the group of all rats during nifedipine-induced hypo- tension. This difference between control and hypotensive animals was not statistically significant (p < 0.5). The most severe protein leakage was seen in the thalamus, hypothalamus, hypocampus, globus pallidus, nucleus caudatus and midbrain in bicuculline induced seizure (Group 111). In Group IV. 5 brains showed no Evans-blue albumin extravasation. The other five brains showed a few small areas of Evans-blue albumin leakage (grade I f ) , and 4 brains showed moderate blue staining (grade 2+). No macroscopic hemorrhages could be observed on the surface of the brains in both experimental groups.

The mean value for Evans-blue dye in the brain was 1.4*0.3mg/g in the third Group and 0.73 f 0.2mg/g in the fourth group. This difference

1.5 E v A N S B U E

m g

/ g 0.6

-

L 1

0

CONTROL

NIFEDIPINE

n S E I Z U R E

* NIFEDIPINE+SEIZURE

--+-- FIGURE 1

**p < .O1 seizure versus nifedipine + seizure. Evans-blue content in the experimental groups. p < .01 control versus seizure;

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110 B. 6ZTAS

between Group I11 and Group IV was statistically significant ( p < .Ol). The extravasation of Evans-blue albumin was most pronounced in the brains of control rats + bicuculline compared to nifedipine-induced hypotension + bicucullin-induced seizures Figure 1.

4. DISCUSSION

This is the first study to examine the effect of nifedipine on the blood-brain barrier permeability during bicuculline-induced seizure. Other studies have implicated on important role for calcium and the flux of calcium across cerebral endothelium in disruption of the blood-brain barrier during acute hypertension (Nag, 1991; Sakaki et al., 1991). Nag (1991) showed that treatment of rats with calcium antagonist flunarizine could inhibit the per- meability of the blood-brain barrier to Evans-blue dye during norepinephrine- induced acute hypertension. Similar results were obtained in the present study. This study demonstrates that pretreatment of epileptic rats with nifedipine results in a significant reduction in protein transfer in brain tissue.

But in contrast to our data, Edvinsson et al. (1983) have suggested that nimodipine potentiates disruption of the blood-brain barrier during acute hypertension. Hollerhage et al. (1988) have also shown that animals pre- treated with nimodipine showed more Evans-blue dye extravasation then animals pretreated with the vehicle. Thus, the precise role of calcium and calcium antagonists in modulation of the breakdown of the blood-brain barrier during acute hypertension remains controversial (Mayhan and Didion, 1995). Epileptic rats showed a significant increase in protein transfer in whole brain ( p < .Ol). Increased blood-brain barrier permeability in bi- cuculline induced seizure has been well documented using Evans-blue as tracer. (Suzuki et a/., 1984) Blood-brain barrier permeability to Evans-blue was confined to central and basal parts of the brain that was similar to previous reports (Pazzili et al., 1989; Suzuki et al., 1984). The calcium entry blocker used it in this study was nifedipine which is potent in reversing cerebral vasospasm, as defined angiographically, following experimental subarachnoid haemorrhage (Allen et al., 1979).

The detailed mechanism of the protective mechanism of nifedipine on the blood-brain barrier permeability during bicuculline-induced seizure is not clear, but may be related to mean arterial blood pressure. It is known that epileptic seizures can open the blood-brain barrier and that the barrier break- down is related to hemodynamic events rather than to the seizures per se. No blood-brain barrier breakdown takes place if the concomitant mean arterial

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BICUCULLINE-INDUCED SEIZURES 1 1 1

blood pressure increase is prevented (Blowing ct ul., 1977). Pretreatment with nifedipine can not increase mean arterial blood pressure more than 36 mmHg during bicuculline-induced seizure. But increase in mean arterial blood pressure due to seizures was 74 mmHg in bicuculline-induced seizure (Tab. I).

On the other hand, Braun and Freed (1990) have shown that nifedipine caused a significant dose-dependent decrease in total seizure time. Rather than preventing seizures, nifedipine decreased the duration of seizures epi- sodes, so that the mice recovered more rapidly from each individual seizure. Oztag and Kaya (1991) have shown that animals with shorter duration of maximal mean arterial blood pressure and no alteration in blood-brain barrier permeability have less intense seizure. Several studies suggest that some forms of seizures can be blocked by calcium channel inhibitors, such as flunarizine and nifedipine. Calcium channel antagonists have been re- ported as useful drugs in the management of patients with intractable epilepsy (Braun and Freed, 1990; Larkin et ul., 1988; Nag, 1991; Sonder and Trevisol-Bittencourt, 1990). The present data suggest that nifedipine might be useful as a treatment of dysfunctional blood-brain barrier permeability during status epilepticus.

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