Blood-brain barrier changes with kainic acid-induced limbic seizures

EXPERIMENTAL

NEUROLOGY

79,422-433

(1983)

Blood-Brain Barrier Changes with Kainic Acid-Induced Limbic Seizures DAVID K. ZUCKER, G. FREDERICK

WOOTEN,

AND ERIC W. LOTHMAN’

Departments of Neurology, Division of Clinical Neuropharmacology, and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 Received May 1 I, 1982; revision received August 13, 1982 Bats were treated with kainic acid (KA) i.v. to produce increasingly severe limbic seizures that were monitored with a behavioral rating scale. At various times after the induction of seizures, the animals’ blood-brain barriers (EBB) were studied with (Y[“C]aminoisobutyric acid ([14C]AIBA) autoradiography. Using optical density ratios, a coefficient was devised to assessthe functional integrity of the B-BB in discrete anatomic regions and to quantitatively compare these measurements among different groups of experimental animals. In animals that exhibited only mild seizures, the EBB was not different from controls. Animals with severe limbic seizures, however, showed alterations. For as long as 2 h alter delivery of KA, the B-BB appeared normal, from 2 to 24 h, the permeability to [‘%]AIBA was markedly increased throughout the brain, especially in limbic regions; from 24 h to 7 days the EBB returned to normal except for a small residual change in limbic structures. These findings were confirmed with Evans blue dye studies of the B-BB. A correlation between focal accentuation of B-BB alterations and neuropathologic changes was found. These experiments indicated that recurrent limbic seizures may lead to a breakdown in the EBB independent of systemic metabolic derangements. Marked focal metabolic and electrical changes, however, occurred in several limbic structures several hours before the blood-brain barrier was altered. INTRODUCTION

Kainic acid (KA) is a potent neurotoxin (17). When injected into the central nervous system, it destroys neuronal cell bodies while relatively sparAbbreviations: KA-kainic acid, B-BB-blood-brain barrier, [‘4C]AIBA-[‘4C]a-aminoisobutyric acid. ’ This work was supported in part by U.S. Public Health Service grant NS 14834 and a grant from the Institute of Medical Education and Research of the City of St. Louis. The current address of Dr. Zucker is Department of Psychiatry, University of Chicago Pritzker Medical School. Address reprint quests to Dr. Lothman, Department of Neurology, Washington Univ. School of Medicine, St. Louis, MO 63110. 422 0014-4886/83/020422-12$03.00/O Copyright 0 1983 by Academic Ress, Inc. All rights of reproduction in any form reserved.

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ing axons and terminals (5, 6, 26). Kainic acid also possesses a strong excitatory action which has proved useful for studying the pathophysiologic mechanisms of epilepsy and associated phenomena (18). Administered systemically, KA causes distinctive changes in behavior and restricted electroencephalographic, metabolic, and histologic alterations (15, 16,20,24). These alterations seem to be the consequences of the ability of KA to selectively induce seizures in the hippocampus and related limbic structures (15, 18). Blood-brain barrier (EBB) permeability changes have been described with generalized seizures produced by electrical stimulation or convulsants (7, 9, 10-14, 22). The causative events remain undefined and represent a deficit in understanding basic mechanisms of epilepsy. It is also unknown whether or not similar changes in the B-BB occur with more restricted seizures such as those induced by KA. Furthermore, it is possible that alterations in the B-BB may a&ct the distribution of blood-borne tracers used in experimental studies of seizures-related metabolic phenomena (e.g., [‘4C]deoxyglucose). This would make a characterization of the changes an important methodologic consideration. For these reasons, a study of the effect of KA-induced seizures on the EBB was undertaken in unanesthetized rats. A refinement of the c+aminoisobutyric acid (AIBA) technique, based on optical density measurements of [14C]AIBA autoradiographs (1, 2), was devised for quantitative comparison. The changes found with this method were correlated with behavioral observations and neuropathology. METHODS Thirty-nine unanesthetized, male albino rats, weighing 280 to 350 g, were studied. All injections were made into the tail vein. Kainic acid (Sigma, St. Louis), dissolved in normal saline (PH adjusted to 7.4 with sodium hydroxide), was injected during 15 s as a 0.28 to 0.35-ml bolus. The [ 14C]AIBA autoradiographs were obtained from brains of 33 animals. Thirty-one animals had received KA at various times before the [14C]AIBA injections (Table I), and two animals received injections of saline without KA. Ten minutes prior to killing, the animals were injected during 10 s with 10 &i [ 14C]AIBA (New England Nuclear, 5 1.6 mCi/mmol) in 0.3 ml. Autoradiographs were prepared according to established methods (3). Briefly, tier pentobarbital anesthesia, animals were perfusion-fixed with cacodylate-buffered paraformaldehyde. Brains were then removed, frozen in liquid Freon, placed in Lip shaw M-l Embedding Matrix, and cut at -2O’C in duplicate 20-pm-thick sections that were mounted on cardboard and exposed 5 to 6 weeks to Kodak SB5 film. Optical densities of various anatomic regions were measured with a Leitz MPV microscopic photometer with adjustable aperture. To normalize the results among different brains, the optical density of each measured region

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TABLE 1 Summary of Behavioral Scores, Blood-Brain Barrier (B-BB) Patterns and Neuropathology in Animals Receiving Kainic Acid (RA) and Studied with a-[‘4C]aminoisobmyric Acid ([i4C]AIBA) KA dose

1 m/kg

4 w/b

12 mg/kg

Time interval

Behavioral score

EBB pattern”

2h 1 &Y I days 7 days

Intact Intact”

2hr 1 &Y I days 7 days 7 days

Intact Intact” Intact Intacta Intact

Neuropathology

Intact

45 min 2h 2h 1 day 1 day 3 days 3 days 3 days 3 days -I days 7 days

4 5 5 4 5 4 4 5 6 2 5

Intact” IntacF Intac9 Intacr Intact Intact” Intact” Intact* II” Intact Intact”

0 0 0 0 0 0 0 0 N* 0 0

45 min 45 min 2h 2h 2h 1 &Y 1 &Y 3 days 7 days 7 days 7 days

5 5 6 6 6 6 6 6 6 6 6

Intact” Intact” I’ I” I0 I” II” II” II” II” II”

0 V' V V V VJ N N N N N

LIIndicates autoradiographs on which B-BB coefficients were obtained (see Table 2). “Intact” denotes no change from control pattern. * N indicates necrosis. cV indicates vacuolar changes.

was divided by the optical density of the third ventricular choroid plexus of that brain, a region where no B-BB exists (8). The resultant ratios were called B-BB permeability coefficients. For a given structure, there were no statistical

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differences between the right and left sides and, therefore, left and right side measurements were pooled. After obtaining autoradiographs, tissue sections were stained with cresyl violet and examined by light microscopy for identification of anatomic structures on the autoradiographs as well as for neuropathology. Permeability of the B-BB (23) was studied in six animals with Evans blue 45 min after injection of KA (12 mg/kg, N = 2) and 2 h (12 mg/kg, N = 2 or 14 mg/kg, N = 2). These animals were injected 10 min prior to killing with 2.0 ml 2% Evan’s blue solution. They were anesthesized with pentobarbital and perfused with buffered fixative until the effluent was clear. The brains were removed, placed 5 min in iced saline, and cut into 2-mmthick coronal sections. Regions of increased permeability were detected by visual examination under low power magnification (2 to 4 X). Behavioral changes were rated on the following scale based on criteria described elsewhere ( 15): 0 1 2 3 4

Normal staring Increased exploratory activity, “wet dog shakes” Automatisms: chewing, head bob, sniffing Mild limbic convulsions: head and facial twitches, increased salivation, pupillary dilation, isolated forelimb jerks 5 Major limbic convulsions: rearing with bilateral synchronized forelimb clonus + loss of balance 6 Recurrent major limbic convulsions lasting longer than 5 min RESULTS

The purpose of this report is to describe the types of B-BB changes that accompany KA-induced seizures. The behavioral responses observed matched those of a previous study (15). Of note here are the observations that the threshold for mild limbic seizures was 4 mg/kg, 7 mg/kg produced major limbic seizures in approximately 50% of the cases, 12 mg/kg caused recurrent major limbic seizures (“limbic status”) about 90% of the time, and behavioral responses were maximal about 2 h after injection. The neuropathologic consequences of KA have also been described elsewhere (4,24,27,28). Because of the detailed descriptions provided in those communications, the behavioral and neuropathologic changes will be considered here only as they correlate with the B-BB changes. With visual inspection of the autoradiographs obtained in these experiments, three EBB patterns were detected-the normal (or “intact”) and two abnormal types with increased B-BB permeability (Fig. 1). The B-BB pattern present depended on the dose and elapsed time since injection of

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CONTROL

KA

INTACT

r”;

u

FIG. 1. Blood-brain barrier (B-BB) and neuropathologic changes associated with kainic acid (KA)-induced seizures. Each row contains the a-[14C]aminoisobutyric acid ([‘%]AIBA) autoradiograph (left) and cresyl violet-stained section used to produce that image (right). Top rowcontrol from animal receiving no KA. Note localization of tracer in the leptomeninges, choroid plexus, and arcuate regions ofthe hypothalamus. Second row-intact B-BB from animal receiving

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KA, and abnormal autoradiographs correlated with intense behavioral seizures and neuropathology (Table 1). An example of a [i4C]AIBA autoradiograph obtained from an animal receiving no KA is shown in the top row of Fig. 1. The [i4C]AIBA was localized in the leptomeninges, choroid plexus, ventricular lining, pineal body, and hypothalamus. Similar B-BB autoradiographs were obtained in 21 animals receiving IL4 (second row of Fig. 1 and Table 1). This pattern was seen at all times examined after injections of ~4 mg/kg; in all rats except one after 7 mg/kg (the exception was a pattern II at 3 days, see below); and 45 min after the 12 mg/kg dose. The general behavioral score of this group of animals was less than 4 although occasional isolated mild or major limbic convulsions raised some scores to 4 or 5. None of the animals in this group received a score of 6 and no neuropathologic alterations were detected in them. The patterns of increased B-BB permeability to [i4C]AIBA were detected only in animals with more severe seizures and were accompanied by neuropathologic changes. Pattern I (third row in Fig. 1) was seen in four of five rats after injections of 12 mg/kg KA in the 2-h to l-day intervals (Table 1). This pattern was characterized by a diffise increase in B-BB permeability throughout the brain, most marked over the septum, hippocampal complex, and hypothalamus. In addition, a rim of increased activity was seen around the piriform cortex, amygdaloid nuclei, and entorhinal cortex, occasionally extending into the basal ganglia, regions involved in severe limbic seizures ( 15,16). All brains showing this pattern came from animals with a behavioral score of 6. The neuropathology in these brains (Figs. 1 and 2, Table 1) consisted of acute necrosis and hemorrhage in limbic structures (particularly in the piriform cortex, amygdaloid region, and entorhinal cortex) and diffise vacuolar changes in limbic and extralimbic structures (24, 28). Pattern II autoradiographs (bottom line in Fig. 1) were characterized by a return toward the normal EBB pattern. This was seen in six rats, from 1 day to 1 week after injections of 7 or 12 mg/kg KA (Table 1). All these animals had initially experienced severe seizures with a behavioral score of 6 but within 24 h seizures had ceased. Thereafter, these animals were more aggressive and excitable than naive rats. Neuropathologic findings consisted of gliosis and early cystic changes around necrotic regions in the limbic 12 mg/kg KA 45 min earlier. Histologically, the tissue appeared normal. Third row-pattern I EBB changes from animal receiving 12 mg/kg KA, 24 h earlier. Neuropathology consisted of vacuolar changes and early necrosis in the dorsal hippocampus, midline thalamus, amygdaloid nuclei, and piriform cortex. Bottom row-pattern II obtained 3 days after injection of 7 mg& KA. Necrosis was present in the piriform cortex and amygdala and there was neuronal loss on the dorsal hippocampus.

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TABLE 2 Blood-Brain Barrier Permeability Coefficients Experimental Control (N=2) Piriform cortex Amygdaloid nuclei Entorhinal cortex

Motor cortex striate cortex Septum Dorsal hippocampus CA1 CA3 Dentate gyms Hypothalamus Thalamus Corpus collosum

“Intact” (N= 13)

Pattern I (N=4)

Pattern II (N= 6)

0.04 0.04 0.06 0.06 0.06 0.09

0.03 0.03 0.03 0.03 0.03 0.03

+ * + * + +

0.01 0.01 0.02 0.02 0.02 0.01

0.52 0.51 0.50 0.53 0.48 0.94

* f + * f f

0.10** o&l** 0.04** 0.13** 0.08** 0.18**

0.08 0.12 0.12 0.07 0.08 0.10

k * + * + f

0.07 0.06*+ 0.12 0.05 0.06 0.08*

0.07 0.03 0.08 0.08 0.06 0.03

0.04 0.05 0.06 0.05 0.03 0.03

+ f + f + +

0.03 0.03 0.02 0.03 0.01 0.01

0.48 0.58 0.69 0.73 0.60 0.60

f k f f + +

0.06** 0.15** 0.15*+ 0.08** 0.18** 0.18**

0.08 0.10 0.11 0.09 0.07 0.08

+ 0.09 zk 0.07 f 0.09 zk 0.05 f 0.05 e 0.06

* P c 0.05, Student’s t test.

** P < 0.001.

structures and anterior midline and lateral thalamic nuclei (24, 28). Ventricular dilation (hydrocephalus ex vacua) was present with some of the longer survival times. Using EBB permeability coefficients, quantitative analyses were conducted on all control, pattern I, pattern II autoradiographs, and 13 of the “intact” autoradiographs (Table 2). Several representative limbic and nonlimbic structures which normally manifest a B-BB were examined. For the control group all regions selected for study had low B-BB coefficients, indicating little passage of [14C]AIBA from the blood into the brain. The group of animals that had received KA and displayed an intact B-BB on the autoradiographs had coefficients that were not different from controls. For the pattern I autoradiograms, the coefficients of all regions studied were markedly elevated by 10 to 30-fold. In the case of pattern II autoradiographs, the B-BB

Fro. 2. Representativeneuropathologic changes induced by KA. A-the dorsal hippocampus showed vacuolar changes and early necrosis in regio inferior. B-the dorsal hippocampus showed neuronal loss in regio inferior and regio superior. C-necrosis of the amygdala and piriform cortex. A is from pattern I, and B and C are from pattern II animals of Fig. I. Calibration bar 2 mm for A and B, 1.5 mm for C.

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coefficients were not significantly different from controls except in the amygdaloid region and septal nuclei. There was a trend toward continued increase in other regions, however, although they did not attain statistical significance. In six rats the pattern of deposition of Evan’s blue dye was similar to that of [i4C]AIBA 45 min and 2 h after IL4 administration. DISCUSSION The existence of a barrier that either totally or partially excludes various compounds in the blood from entering brain parenchyma is well established (6, 21). Anatomically, the B-BB has been localized to the endothelial cells lining cerebral blood vessels. Movement of different substrates through this barrier is dependent on certain properties of the barrier (e.g., enzyme activities and the carrier-mediated transport system) as well as the physical and chemical properties of the blood-borne substances. Important factors include molecular size, degree of ionization, lipid solubility, and protein binding of the substance (6, 21). With these considerations in mind, various methods have been used to study the B-BB. Those techniques have been limited both with respect to quantification and anatomic resolution. In the present study, we used a nonmetabolized amino acid, a-aminoisobutyric acid, which has been shown to have minimal transport across the intact B-BB (19). In addition, AIBA has been shown to possess other qualities requisite for studying the B-BB. It has limited diffusion from its site of entry into the brain because it is accumulated by viable brain cells and there is also limited diffusion from brain back to blood (1, 2). As a further refinement of the autoradiographic technique which provides anatomical resolution of the B-BB in separate regions, we developed the concept of a B-BB permeability coefficient for individual structures. This coefficient allows for quantification of regional changes in the B-BB and permits statistical comparisons. To confirm the changes found with [14C]AIBA studies, several animals were studied with Evan’s blue dye. We saw autoradiographic evidence of B-BB changes only when the animals had experienced severe seizure activity and the corresponding brain sections showed neuropathology. Considerable data have been gathered to support the idea that neuropathologic changes produced by KA are a consequence of its epileptogenic effect (4, 15, 16, 18, 28). The B-BB alterations noted in those experiments lagged behind the onset of electrophysiologic and behavioral seizures (15, 16); the dose of KA required to produce B-BB changes was above the threshold for seizures and, for a given amount of KA, B-BB changes were present only in animals with a behavioral score of 6. Our observations suggest that the B-BB changes are also the consequence of

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severe, recurrent seizures. The presence of focal B-BB changes at later times (pattern II) coincides with the development of focal necrosis (4,24,28) and can be explained by the loss of structural components of the B-BB. The diffuse increase in EBB permeability (pattern I) that appears earlier is more difficult to explain. The transition from pattern I to pattern II, however, indicates that at least part of the acute B-BB changes are reversible and may reflect a functional disturbance. The possible causal relationship between seizures and B-BB changes is also suggested by previous studies of such changes after electrically (11, 22) or pharmacologically (7, 9, 10, 12- 14) induced seizures. Changes have been seen after 3 to 5 min of seizures (12, 14) or a single electrically induced seizure (22). In those prior studies of generalized seizures, changes were most frequently reported in the thalamus, hypothalamus, and cerebral cortex (7, 11, 12, 14, 22). In contrast, the limbic system was most severely involved in the current study of localized seizures. The preferential excitation of the limbic system by KA (15) would seem to be the most likely explanation for these differences in the locus of B-BB changes. The direct mechanism for the B-BB changes is unknown. We have examined systemic blood pressure and blood chemistries and found that in the time frame in which B-BB changes were found in this study, systemic metabolic derangements were not present which could account for the permeability changes (15). Cerebral blood flow, however, may increase in KAinduced seizures, especially in limbic structures (unpublished results). Although such regional hemodynamic changes may have a role in altering the EBB, the contributions of changes in flow to B-BB permeability remain to be determined. It has been suggested also by some authors (7, 11) that the changes are dependent on neuronal activity and/or local metabolic alterations. The close relationship between neuropathologic, electroencephalographic, and B-BB changes with KA-induced convulsions supports their point of view. Others (10, 22), however, have shown that changes in B-BB permeability may be dependent on mechanical stresses caused by altered hemodynamics. It is possible that each of these factors is involved to a varying degree depending on the mechanism of seizure induction. Our results demonstrated that the [14C]AIBA autoradiographic method is a high-resolution, quantifiable technique for measuring B-BB changes in fully awake, nonparalyzed animals. They also indicated that B-BB changes should be considered when using radioactive tracer techniques to study seizurerelated phenomena. Theoretically, such alterations may influence newer strategies applicable to patients such as positron-emission tomography (PET) scanning. Furthermore, our findings have potential therapeutic implications as B-BB alterations in patients with prolonged seizures might aifect the delivery of therapeutic agents to the brain (25).

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ZUCKER, D. K. Neuropathology of sustained seizures induced by kainic acid, submitted for publication.