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Blood-brain barrier dysfunctions following systemic injection of kainic acid in the rat
Life Sciences, Vol. 51, pp. 467-477 Printed in the USA
Pergamon Press
BLOOD-BRAIN BARRIER DYSFUNCTIONS INJECTION OF KAINIC ACID A.
Saija,
FOLLOWING IN THE RAT
SYSTEMIC
*P. Princi, §A. Pisani, §G. S a n t o r o , Pasquale, AM. Massi and #G. Costa
R.
De
Dept. F a r m a c o - B i o l o g i c o (School of P h a r m a c y ) , *Centro Interdipartimentale di Informazioni Farmacotossicologiche, §Dept. Biomorfologia and #Inst. Pharmacology (School of M e d i c i n e ) , University of Messina; AInst. P h a r m a c o l o g y (School of P h a r m a c y ) , University of Camerino (Italy). (Received in final form June 4, 1992) SUMMARY C h a n g e s in b l o o d - b r a i n b a r r i e r (BBB) p e r m e a b i l i t y and cerebral metabolic activity following intravenous injection of kainic acid (KA; 6, 12 mg/Kg) in rats were a s s e s s e d by c a l c u l a t i n g r e s p e c t i v e l y a b l o o d - t o - b r a i n t r a n s f e r c o n s t a n t (Ki) for [ 1 4 C ] ~ - a m i n o i s o b u t y r i c acid and local cerebral glucose utilization (LCGU) values, at d i f f e r e n t times (i h, or acute seizure phase, and 48 h, or c h r o n i c p a t h o l o g y phase) after the induction of seizures. A significant increase in t h e local permeability of t h e B B B w a s o b s e r v e d 1 h after the injection of K A 6 m g / K g (eliciting no s i g n i f i c a n t c h a n g e s in c e r e b r a l m e t a b o l i c activity, except within t h e f r o n t a l c o r t e x and the h i p p o c a m p u s ) and 12 m g / K g (which i n d u c e d a m a r k e d and w i d e s p r e a d e n h a n c e m e n t of L C G U ) . On the c o n t r a r y , d u r i n g the p a t h o l o g y phase, persistent regional increases in Ki v a l u e s were e v i d e n c e d in rats t r e a t e d with the l o w e s t d o s e of the convulsant, but not in rats injected with KA 12 m g / K g (a dose able to cause extensive neuronal damage). Thus one can s p e c u l a t e that: i) K A - i n d u c e d r e g i o n a l c h a n g e s in the permeability of the BBB are not c o r r e l a t e d with changes in neuronal activity; 2) opening of the BBB is not r e l i a b l y associated with neuronal injury.
Kainic acid (KA) , a p o t e n t central nervous system (CNS) excitotoxin, p r o d u c e s an a c u t e or s u b a c u t e m o d e l of s e i z u r e s , l a s t i n g h o u r s to days. P r i m a r y i n t e r e s t in K A w a s d u e to its unusual propensity to c a u s e i r r e v e r s i b l e neuropathological c h a n g e s i n v o l v i n g not o n l y n e r v e c e l l s b u t a l s o glia, m y e l i n sheaths and blood vessels I-2. Besides, r e p o r t s p r o v i d e e v i d e n c e of the e x i s t e n c e of c h a n g e s in f u n c t i o n a l c h a r a c t e r i s t i c s of the blood-brain b a r r i e r (BBB) in KAi n j e c t e d rats, i n c l u d i n g e x t r a v a s a t i o n of f o r e i g n and e n d o g e n o u s serum p r o t e i n s and alterations of BBB p e r m e a b i l i t y to t r a c e r s ~-'. 0024-3205/92 $5.00 + .00 Cop~right © 1992 Pergamon Press Ltd All rights reserved.
468
Blood-Brain Barrier and Kainic Acid
However, the factors responsible permeability (and t h e i r s i g n i f i c a n c e ) are s t i l l far f r o m b e i n g clarified.
Vol. 51, No. 7, 1992
for changes in during KA-induced
the BBB seizures
In t h e p r e s e n t study, the permeability of t h e B B B t o [ 1 4 C ] ~ aminoisobutyric acid ([14C]AIB) and the local cerebral glucose utilization (LCGU) w e r e m e a s u r e d in s m a l l b r a i n a r e a s of r a t s systemically injected with different d o s e s of KA, at d i f f e r e n t times after the induction of s e i z u r e s . This should further characterize the possible relationship existing between BBB dysfunctions, alterations in n e u r o n a l functional activity and neuropathological changes accompanying KA-induced limbic seizures. MATERIALS
AND METHODS
AND CALCULATIONS: Modifications of B B B p e r m e a b i l i t y were determined using a BBB tracer technique, that employs a small molecular weight radiolabeled aminoacid, [I~C]AIB, to identify and quantify small local changes of B B B p e r m e a b i l i t y 8 - 1 3 The m o d e l u s e d for t h e e s t i m a t i o n of b r a i n v a s c u l a r p e r m e a b i l i t y was developed b y O h n o et al. 14. A b l o o d - t o - b r a i n unidirectional transfer constant (Ki) f o r [ 1 4 C ] A I B m a y be c a l c u l a t e d from the following relationship: THEORY
Ci(T) Ki
- V Cb(T)
=
T o
Cpdt
w h e r e Ci(T) is t h e b r a i n p a r e n c h y m a l tracer concentration at t h e time T (duration of the experiment) , Cp is the tracer concentration in t h e t o t a l a r t e r i a l plasma, V is t h e r e s i d u a l blood volume (~i/g) and Cb(T) is t h e w h o l e - b l o o d tracer concentration (nCi/ml) . Sucrose, a compound of very low permeability at t h e B B B , w a s u s e d t o c a l c u l a t e V as a ratio between the tracer concentration in t h e b r a i n a n d in t h e w h o l e b l o o d a c c o r d i n g to the f o l l o w i n g e q u a t i o n 9 : Ci(sucr) v (sucr)
-
Cb(sucr) w h e r e V(sucr) is t h e c e r e b r a l b l o o d v o l u m e (~i/g), c i ( s u c r ) is the tracer concentration (nCi/g) in t h e b r a i n a n d C b ( s u c r ) is its concentration (nCi/ml) in t h e w h o l e - b l o o d . B e c a u s e Ki is r e l a t e d to t h e p e r m e a b i l i t y - s u r f a c e area product (PS) a n d b l o o d - f l o w (F) by PS = F in (i - Ki/F), and PS << F is a c o n d i t i o n w e l l s a t i s f i e d for [14C]AIB, Ki - PS, is i n d e p e n d e n t of F a n d can be e x p r e s s e d in t e r m s of p l a s m a c l e a r a n c e (ml/g/min). LCGU w a s c o m p u t e d for e a c h b r a i n s a m p l e u s i n g t h e 2 - d e o x y g l u c o s e (2-DG) t e c h n i q u e and the m a t h e m a t i c a l m o d e l d e v e l o p e d by S o k o l o f f et al. 15, m o d i f i e d for the influence of v a r y i n g p l a s m a g l u c o s e c o n c e n t r a t i o n s 16. T h i s t e c h n i q u e p r o v i d e s a r e l i a b l e a n d e ~ i c i e n t s c r e e n i n a m e t h o d for n e u r o a c t i v e and n e u r o t o x i c c o m p o u n d s , such as K A 18-21 . The experiments were carried o u t on m a l e W i s t a r rats (320-350 g b.w.) . The animals were maintained under normal controlled lighting and temperature conditions and allowed free ANIMALS:
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Blood-Brain Barrier and Kainic Acid
469
to food and water until used.
EXPERIMENTAL D E S I G N : E x p e r i m e n t s w e r e c a r r i e d out one h o u r a f t e r c o n v u l s a n t i n j e c t i o n (acute s e i z u r e phase), w h e n r a t s h a v e p e a k symptoms of seizure activity, a n d 48 h o u r s following KA administration (chronic pathology phase), w h e n an e x t e n s i v e n e u r o p a t h o l o g y with cellular necrosis may appear in several brain r e g i o n s zz. As the d e v e l o p m e n t of b e h a v i o u r a l c h a n g e s (typical of status epilepticus) and neuropathology follow~g KA administration a r e r e l a t e d to t h e d o s e of n e u r o t o x i n ~=, t h e c o n v u l s a n t was injected at 2 different dose levels (6 m g / K g and 12 m g / K g ) . C o n t r o l a n i m a l s w e r e o n l y g i v e n the v e h i c l e (NaCI 0.9% solution, i ml/Kg).
Two p o l y e t h y l e n e catheters were chronically inserted in the right f e m o r a l v e i n and a r t e r y under light ether a n a e s t h e s i a ; KA or its vehicle were injected intravenously (iv) t h r o u g h t h e c a t h e t e r inserted in the femoral vein. The rats were d i v i d e d into 6 groups of 16 a n i m a l s each: c o n t r o l s , a c u t e s e i z u r e p h a s e ; K A 6 m g / K g , a c u t e s e i z u r e phase; KA 12 mg/Kg, acute s e i z u r e phase; c o n t r o l s , c h r o n i c p a t h o l o g y phase; KA 6 mg/Kg, c h r o n i c p a t h o l o g y phase; KA 12 mg/Kg, chronic pathology phase. SURGICAL PREPARATION FOR TRACER INJECTION: O n t h e d a y of t h e experiment, the animals were lightly anaesthetized with ether; short polyethylene c a t h e t e r s w e r e i n s e r t e d in t h e left f e m o r a l vein for tracer administration and in the left femoral artery for blood sampling. Cannulas were passed subdermally behind the leg to e x i t j u s t r o s t r a l to the b a s e of the tail and t h e w o u n d s w e r e sutured. The a n i m a l s were p a r t i a l l y f a s t e n e d from the w a i s t d o w n by m e a n s of l o o s e - f i t t i n g tape (placed along the flanks), permitting f r e e m o v e m e n t of h e a d a n d f o r e l i m b s ; t h e r e w a s no a p p a r e n t i n d i c a t i o n of d i s t r e s s or s e i z u r e t r i g g e r i n g in t h i s p a r t i a l l y r e s t r a i n e d c o n d i t i o n . M o r e o v e r , e v e r y p r e c a u t i o n was taken to m i n i m i z e or avoid pain and discomfort to the animals. The rats w e r e t h e n a l l o w e d to r e c o v e r from the a n e s t h e s i a for 3-4 h before tracer injection.
L C G U and Ki v a l u e s for [14C]AIB were c a l c u l a t e d s e p a r a t e l y r a t s of e a c h e x p e r i m e n t a l group; regional blood volumes e s t i m a t e d in 4 animals of each experimental group.
in 6 were
TRACER INJECTION: [14C]AIB or 2 - d e o x y - D - ( l - 1 4 C ) g l u c o s e ([14C]DG) were injected 1 h or 48 h a f t e r a d m i n i s t r a t i o n of K A or K A vehicle. [14C]Sucrose w a s i n j e c t e d iv 1 h or 48 h f o l l o w i n g i n j e c t i o n of KA 6 mg/Kg or the vehicle; in rats given KA 12 mg/Kg, the t r a c e r w a s i n j e c t e d i m m e d i a t e l y at the o n s e t of t h e f i r s t episode of t o n i c - c l o n i c convulsions (50-70 min following n e u r o t o x i n injection) or 48 h after injection of the convulsant. M~SUREMENT O F Ki F O R [ 14 C ] A I B O R L C G U : The e v a l u a t i o n of Ki for [ C]AIB ^aD¢ LCGU values was carried out as previously d e s c r i b e d I Z I I % . Briefly, the animals were injected iv w i t h a bolus of [ 14 C ] A I B (25 ~Ci/rat) or of [ 14 C]DG (60 ~ C i / r a t ) . Small b l o o d samples ( 8 0 - 1 0 0 ~i) w e r e c o l l e c t e d in p o l y e t h y l e n e t u b e s at predetermined intervals of time during the e x p e r i m e n t a l period. A large v o l u m e was w i t h d r a w n at the end to m e a s u r e the w h o l e - b l o o d i s o t o p e c o n c e n t r a t i o n only in rats given [14C]AIB. At the end of the experimental p e r i o d (20 m i n and 45 m i n r e s p e c t i v e l y after [14C]AIB and [14C]DG injection) all the rats w e r e s a c r i f i c e d by
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Blood-Brain Barrier and Kainic Acid
Vol. 51, No. 7, 1992
rapid iv injection of 1 ml saturated KCl solution. The d u r a t i o n of the e x p e r i m e n t a l time was c h o s e n to m i n i m i z e b o t h the e f f e c t of i n t r a v a s c u l a r tracer on brain 14C activity m e a s u r e d at the end of the e x p e r i m e n t and the e v e n t u a l b r a i n - t o - b l o o d r e f l u x of tracer. The b r a i n w a s t h e n r a p i d l y r e m o v e d and s m a l l t i s s u e s R e c i m e n s were d i s s e c t e d on dry ice, according to Paxinos and W a t s o n 23, from the f o l l o w i n g areas: frontal cortex, t e m p o r o p a r i e t a l (temp.par.) cortex, occipital c o r t e x ( i n c l u d i n g the e n t o r h i n a l cortex), striatum, hippocampus, thalamus, hypothalamus, cerebellum and brain-stem. Sample s o l u b i l i s a t i o n was a c c o m p l i s h e d by a d d i n g 1 ml of S o l u e n e i00 (Packard) and incubating the vials at 60°C overnight. E a c h v i a l w a s t h e n f i l l e d w i t h 4 ml of H i o n i c - F l u o r (Packard). B l o o d s a m p l e s were c e n t r i f u g e d ; then, 30 ~i of p l a s m a w e r e d i l u t e d in 0.5 ml d i s t i l l e d w a t e r and c o u n t e d in 6 ml of Hionic-Fluor. A 20 ~i w h o l e - b l o o d s a m p l e was d i g e s t e d in 0.5 ml Soluene/ethanol (1:2) s o l u t i o n , b l a n c h e d w i t h 0.3 ml h y d r o g e n p e r o x i d e (30% wt/vol) and c o u n t e d in 6 ml H i o n i c F l u o r a c i d i f i e d with 0.5 ml 0.5 N HCI. B e t a - c o u n t i n g was p e r f o r m e d by a P a c k a r d PL s c i n t i l l a t i o n counter. Single s a m p l e q u e n c h i n g was m o n i t o r e d by the e x t e r n a l s t a n d a r d m e t h o d . S a m p l e c o u n t s w e r e c o r r e c t e d for b a c k g r o u n d and quenching. Plasma glucose concentrations d e t e r m i n e d by the glucose oxidase
(for L C G U assay.
measurement)
were
BLOOD VOLUMES: Regional blood volumes were c a l c u l a t e d to i n t r o d u c e a c o r r e c t i o n for the r e s i d u a l i n t r a v a s c u l a r t r a c e r in the e s t i m a t i o n of Ki for [ 14 C]AIB (see T H E O R Y A N D C A L C U L A T I O N S ) . Blood v o l u m e (V) was defined as the [±~C]sucrose space at 1 minute a f t e r t h e iv i n j e c t i o n of t h e t r a c e r 8,9. A 20 ~ C i b o l u s of [14C]sucrose was injected iv. One minute later a blood sample was c o l l e c t e d and the animal sacrificed by decapitation. Brain samples and w h o l e - b l o o d w e r e d i g e s t e d a n d c o u n t e d in t h e s a m e w a y as [14C]AIB.
REGIONAL
AND PHYSIOLOGICAL PARAMETERS: Following KA or KA vehicle injection, the animals were observed for b e h a v i o u r a l changes, and various physiological parameters were monitored. Mean arterial blood p r e s s u r e was m e a s u r e d by connecting the arterial c a t h e t e r to a strain gauge transducer and both systolic and diastolic pressures were displayed on c h a n n e l s of a p o l i g r a p h (Basile, Italy). A r t e r i a l blood samples were w i t h d r a w n for d e t e r m i n a t i o n of pH, pO 2 a n d p C O 2 ( p H - b l o o d g a s a n a l y z e r , A V L AG, B i o m e d i c a l Instruments, Switzerland). BEHAVIOUR
ANALYSIS: Results are expressed as m e a n ± S.E.. Differences in K i v a l u e s for [14C]AIB, rates of glucose u t i l i z a t i o n and regional blood volume were t e s t e d for s t a t i s t i c a l significance by t w o - w a y A N O V A . Statistical significance was a c c e p t e d w h e r e P < 0.05. STATISTICAL
USED: [14C]AIB (59 m C i / m m o l ) , [ 1 4 C ] D G (59 m C i / m m o l ) and [14C]sucrose (540 mCi/mmol) were purchased from Amersham (England); k a i n i c acid was purchased from Sigma C h e m i c a l Co..
DRUGS
RESULTS BEHAVIOUR
injection,
AND
PHYSIOLOGICAL
specific
types
Following KA or KA v e h i c l e behaviour were noted, including:
PARAMETERS:
of
Vol. 51, No. 7, 1992
Blood-Brain Barrier and Kainic Acid
TABLE
ACUTE
SEIZURE
PHASE
CHRONIC
PATHOLOGY
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
I
(i h)
CONTROLS Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
471
2.334±0 2.512±0 3.053±0 1.702±0 1.079±0 0.843±0 4.867±0 2.417±0 2.627±0
PHASE
095 123 060 094 031 045 099 048 237
KA 6 m q / K q 3.554±0.216, 3.798±0.349* 4.685±0.441, 2.162±0.282 1.533±0.018, 1.039±0.139 5.990±0.375 3.253±0.265* 3.762±0.358
KA
12 m q / K q
7.941±0.850" 6.844±0.477* 8.234±0.680* 2.172±0.080" 2.579±0.017" 1.114±0.035" 5.715±0.300 3.973±0.264* 3.612±0.131"
(48 h_~
CONTROLS
KA 6 m g / K g
2.295±0.105 2.484±0.114 3.010±0.038 1.823±0.121 1.017±0.033 0.896±0.066 5.008±0.105 2.355±0.054 2.727±0.264
2 811±0.163 3 195±0.137, 3 927±0.235* 1 720±0.132 1 424±0.107, 0 597±0.043 4.824±0.342 3.125±0.176, 2.803±0.158
KA
12 m q / K q
1.770±0.312 2.026±0.180 2.386±0.287 1.531±0.103 1.230±0.263 0.686±0.159 4.597±0.563 2.411±0.243 1.998±0.154
Modifications of Ki for [ 1 4 C ] A I B ( m l / g / m i n x 10 -3 ) in r a t s injected iv w i t h k a i n i c a c i d (KA) 6 or 12 m g / K g , or g i v e n o n l y the v e h i c l e (controls). (* P < 0.05 v e r s u s c o n t r o l s )
normal for vehicle-treated animals; staring spells and "wet dog shakes" (WDS) for a n i m a l s i n j e c t e d w i t h KA 6 m g / K g ; s a l i v a t i o n , rearing, tonic-clonic convulsions and status epilepticus for a n i m a l s i n j e c t e d w i t h KA 12 mg/Kg. T h r o u g h o u t the two day period after KA injection, the a n i m a l s m a n i f e s t e d a b n o r m a l b e h a v i o u r and were hyperexcitable; besides, rats i n j e c t e d w i t h the h i g h e s t d o s e of K A w e r e a p h a g i c and lost weight. During the experimental p e r i o d , if m a i n t a i n e d u n d e r n o n s t r e s s f u l conditions, the KA-injected rats used in our s t u d y s h o w e d no a b n o r m a l r e s p o n s e to the e x p e r i m e n t a l p r o t o c o l ; v a l u e s of a r t e r i a l b l o o d pH, p O ~ and p C O 2 w e r e w i t h i n normal limits (data n o t shown). P r i o r to i s o t o p e i n j e c t i o n a n d t h r o u g h o u t t h e t w o d a y s f o l l o w i n g the injection of t h e n e u r o t o x i n , the physiological parameters (plasma glucose values, blood pressure, hematocrit, rectal temperature, a r t e r i a l b l o o d g a s e s a n d pH) d i d n o t s i g n i f i c a n t l y d i f f e r b e t w e e n e x p e r i m e n t a l g r o u p s (data not shown). ACUTE
SEIZURE
PHASE
BBB permeability (table I): the i n j e c t i o n significant enhancement of K i v a l u e s
of K A 6 m g / K g e l i c i t e d a calculated within the
472
Blood-Brain Barrier and Kainic Acid
TABLE
ACUTE
SEIZURE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
CHRONIC
PHASE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
II
(i h)
CONTROLS
KA 6 m g / K g
68.1±2.924 73.4±2.377 72.2±5.205 71.2±0.308 50.4±1.779 69.3±4.289 46.0±2.305 46.1±5.323 46.2±2.864
78.8±1.057, 83 3±2.322 78 8 ± 3 . 0 8 9 76 0 ± 2 . 7 3 1 78 0 ± 4 . 4 1 2 , 77 6±3.097 53 4 ± 0 . 8 5 1 51 5 ± 1 . 1 2 9 50 9 ± 1 . 0 5 1
PATHOLOGY
Vol. 51, No. 7, 1992
PHASE
KA
12 m q / K q
94.4±2.522* 100.6±2.952" 94.6±1.899" 93.0±1.864, 153.6±0.886, 86.9±1.421, 70.1±1.435, 54.0±1.071, 55.3±0.957*
(48 h)
CONTROLS
KA 6 m q / K g
KA
12 m g / K g
69.0±5.000 74.6±4.135 73.2±5.241 68.3±0.059 51.8±0.624 77.3±2.286 47.5±1.506 47.6±1.914 43.9±0.996
72.9±3.873 75.8±3.705 73.2±2.291 71.5±2.717 53.3±2.451 80.8±2.151 51.6±0.774 49.1±2.549 49.9±1.087
41.9±0.661, 47.4±0.926* 47.0±1.212, 50.3±1.420, 37.3±0.494* 54.1±1.188, 39.0±1.347, 35.5±0.755* 38.0±1.037,
Modifications of L C G U (~mol/100 g/min) in r a t s i n j e c t e d iv w i t h k a i n i c a c i d (KA) 6 or 12 m g / K g , or g i v e n o n l y t h e v e h i c l e (controls). (* P < 0.05 v e r s u s c o n t r o l s )
frontal, temporoparietal and occipital cortex, hippocampus and c e r e b e l l u m . M o r e m a r k e d and w i d e s p r e a d i n c r e a s e s of Ki v a l u e s w e r e i n d u c e d by a d m i n i s t r a t i o n of the h i g h e s t d o s e of K A (12 mg/Kg) in all b r a i n r e g i o n s examined, e x c e p t the h y p o t h a l a m u s . Cerebral glucose u s e ( t a b l e II) : W i t h a d o s e of 6 m g / K g of KA, several brain areas showed a slight increase of L C G U values, but this effect was statistically significant only within the f r o n t a l c o r t e x and the h i p p o c a m p u s . A s t r i k i n g w i d e s p r e a d i n c r e a s e in L C G U w a s o b s e r v e d f o l l o w i n g t h e i n j e c t i o n of K A 12 m g / K g . T h e g r e a t e s t i n c r e a s e in L C G U was w i t h i n the h i p p o c a m p u s , the proposed epileptogenic focus 21,22, f o l l o w i n g b o t h d o s e s t e s t e d of KA. Regional blood volumes ( t a b l e III): T h e r e w a s no d i f f e r e n c e in b l o o d r e g i o n a l v o l u m e b e t w e e n c o n t r o l s and r a t s g i v e n K A 6 mg/Kg. Conversely, blood regional volumes increased significantly in a n i m a l s i n j e c t e d w i t h t h e h i g h e s t d o s e of n e u r o t o x i n (12 m g / K g ) w i t h i n all b r a i n r e g i o n s examined. CHRONIC
PATHOLOGY
PHASE
BBB permeability (table there was a statistically
I) : In r a t s i n j e c t e d with KA 6 mg/Kg, s i g n i f i c a n t i n c r e a s e in Ki v a l u e s w i t h i n
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the temporoparietal and occipital cortex, hippocampus and cerebellum; however, in a n i m a l s g i v e n K A 12 m g / K g , no obvious m o d i f i c a t i o n of [14C]AIB t r a n s p o r t a c r o s s the BBB w a s o b s e r v e d . C e r e b r a l g l u c o s e u s e (table II): Controls (vehicle) a n d a n i m a l s g i v e n K A 6 m g / K g s h o w e d no s i g n i f i c a n t d i f f e r e n c e in L C G U values. On the contrary, L C G U v a l u e s in r a t s i n j e c t e d w i t h K A 12 m g / K g w e r e m a r k e d l y d e p r e s s e d in a w i d e s p r e a d way. Regional blood volume ( t a b l e III) : T h e r e w a s n o s i g n i f i c a n t difference in r e g i o n a l blood volumes between controls and rats g i v e n 6 or 12 m g / K g of KA. TABLE
ACUTE
SEIZURE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
CHRONIC
PHASE
(50-70 min)
CONTROLS
KA 6 m q / K g
KA
37.46±2.55 29 07±2.26 32 40±1.68 19 61±1.10 26 83±1.59 26 77±2.29 30 31±2.70 39 16±1.14 26.36±1.05
32.50±2.41 34.18±2.64 34.77±2.33 24.99±1.95 25.92±1.88 27.34±1.97 31.53±2.56 41.92±2.30 28.56±1.72
53.04±3.21, 57.11±3.35" 56.60±4.16, 39.59±1.92" 52.74±2.27* 61.37±3.49" 51.91±2.76" 90.55±4.24* 37.93±2.55*
KA 6 m q / K g
KA
33.41±2.56 32.95±2.34 33.27±1.99 21.20±1.13 28.24±1.71 34.97±2.94 31.24±2.34 40.11±2.25 29.03±2.15
31.34±2.24 35.04±2.18 35.85±2.65 23.79±1.72 30.25±2.41 40.76±3.11 32.55±2.33 43.68±2.51 33.37±2.72
PATHOLOGY
PHASE
37.55±2 30.11±2 31.95±1 20.13±1 27.54±1 27.10±2 30.88±2 38.55±1 27.27±1
12 m q / K q
(48 h)
CONTROLS Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
III
43 35 77 22 56 46 48 91 24
12 m q / K g
Regional b l o o d v o l u m e s ° (~i/g) in r a t s i n j e c t e d iv w i t h k a i n i c a c i d (KA) 6 or 12 mg/Kg, or g i v e n o n l y t h e v e h i c l e (controls). ( * P < 0.05 v e r s u s gontrols) ° B l o o d v o l u m e is d e f i n e d as t h e [ l ~ C ] s u c r o s e space 1 min a f t e r t h e i n t r a v e n o u s i n j e c t i o n of [ 14 C ] s u c r o s e . DISCUSSION A s i g n i f i c a n t i n c r e a s e in the local p e r m e a b i l i t y of t h e B B B in the r a t w a s o b s e r v e d 1 h a f t e r the i n t r a v e n o u s i n j e c t i o n of K A 6 m g / K g (eliciting mild behavioural alterations without significant c h a n g e s in L C G U changes, e x c e p t w i t h i n the f r o n t a l c o r t e x a n d the hippocampus) a n d 12 m g / K g (a d o s e i n d u c i n g a m a r k e d a n d w i d e s p r e a d enhancement of c e r e b r a l m e t a b o l i c a c t i v i t y ) . H o w e v e r , d u r i n g t h e
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pathology phase, persistent regional alterations in BBB p e r m e a b i l i t y to [14C]AIB were evidenced in rats t r e a t e d w i t h the l o w e s t d o s e of the c o n v u l s a n t (inducing only m o d e r a t e c h a n g e s in brain m o r p h o l o g i c a l structures), but not in rats injected w i t h KA 12 m g / K g (which c a u s e s e x t e n s i v e n e u r o n a l damage). T h e s e r e s u l t s are at v a r i a n c e with those of Zucker et al 7, who also a s s e s s e d BBB permeability with [14C]AIB following KA. H o w e v e r , d u e to a different methodology (in p a r t i c u l a r the i n t r o d u c t i o n , in our experiments, of a c o r r e c t i o n for i n t r a v a s c u l a r tracer), it is d o u b t f u l if such results may be compared with our p r e s e n t data. In fact, in o r d e r to a v o i d i n c o r r e c t e s t i m a t i o n s of the a c t u a l BBB p e r m e a b i l i t y to the tracer, the introduction of a c o r r e c t i o n for intravascular tracer s h o u l d n o t be n e g l e c t e d , as K A - i n d u c e d seizures are a c c o m p a n i e d by transient increases in c e r e b r a l blood f l o w 24 a n d i n d u c e irreversible damages a l s o in b r a i n blood vessels 2 . The findings p r e s e n t e d herein emphasize two concepts: i) regional c h a n g e s in BBB p e r m e a b i l i t y do not parallel and/or are not related to s i g n i f i c a n t changes in n e u r o n a l metabolic activity; for example, in r a t s g i v e n KA 6 mg/Kg, d u r i n g the s e i z u r e p h a s e , a s i g n i f i c a n t increase in Ki values was observed w i t h i n the frontal and o c c i p i t a l c o r t e x and the cerebellum, in s p i t e of a b s e n c e of modifications in L C G U values, and, in a d d i t i o n , the percent i n c r e a s e (42.07%) in Ki v a l u e s w i t h i n the h i p p o c a m p u s (the only area w h e r e a s i g n i f i c a n t i n c r e a s e in L C G U (54.76%) w a s observed) was c o n s i s t e n t with that calculated within the cortex and the c e r e b e l l u m (34.59-53.45%); 2) K A - i n d u c e d n e u r o p a t h o l o g i c a l changes are not always accompanied by o b v i o u s modifications in B B B p e r m e a b i l i t y to [14C]AIB (see results obtained in rats given KA 12 m g / K g d u r i n g the p a t h o l o g y phase). W i t h r e g a r d to t h e f i r s t issue, t h e r e is c l e a r e v i d e n c e that neuronal activation p e r se is i n a d e q u a t e to a c c o u n t for the i n c r e a s e d p e r m e a b i l i t y of the BBB in K A - t r e a t e d rats 24. R e g i o n a l c h a n g e s in c e r e b r a l levels of n e u r o t r a n s m i t t e r a m i n o a c i d s =J, in norepinephrine and s e r o t o n i n t u r n o v e r 26 and in t h e a c t i v i t y of Na + ,K + - A T P a s e 27 are a s s o c i a t e d w i t h KA s e i z u r e s ; on t h e o t h e r hand, d i f f e r e n t p r o c e s s e s i n v o l v e d in the BBB can be s e l e c t i v e l y and temporarily altered in c o n s e q u e n c e of c h a n g e s in t h e functional activity of neuronal circuits within the CNS I0,12'28'29 Thus it is tempting to h y p o t h e s i z e that changes in BBB p e r m e a b i l i t y following KA systemic injection m i g h t be related, in p a r t , to a c h a l l e n g e of t h e n e u r o g e n i c and/or vasogenic c o m p o n e n t s c o n t r o l l i n g BBB c h a r a c t e r i s t i c s (perhaps at the level of n e u r o n a l innervation present in t h e c e r e b r a l capillary endothelium or d u e to a s t i m u l a t e d release of v a s o a c t i v e substances). As to the s e c o n d issue, the BBB o p e n i n g is not a l w a y s a s s o c i a t e d with neuronal damage 30,31 and the increased cerebrovascular p e r m e a b i l i t y to protein during seizures might not be a d e l e t e r i o u s m e c h a n i s m 3,32. Thus, the increase of the BBB p e r m e a b i l i t y o b s e r v e d d u r i n g the p a t h o l o g i c a l p h a s e in rats g i v e n KA 6 m g / K g d o e s not o c c u r c o n s e q u e n t l y to an actual BBB breakdown, but m i g h t o p e r a t e as a h o m e o s t a t i c mechanism. The absence of BBB o p e n i n g d u r i n g the p a t h o l o g y p h a s e f o l l o w i n g the i n j e c t i o n of KA 12 m g / K g m i g h t be due to derangements in t h e c o n t r o l of c e r e b r a l capillary permeability.
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A s . t h e i n d u c i n g a g e n t is still p r e s e n t d u r i n g the [ 1 4 C ] A I B and [ ± ~ C ] D G e x p e r i m e n t s c a r r i e d out d u r i n g the s e i z u r e p h a s e , the e f f e c t s of the s e i z u r e s per se on BBB p e r m e a b i l i t y and c e r e b r a l m e t a b o l i s m may be confounded by the direct effects of the inducing agent itself. M o d e r a t e to high levels of [3H]kainic acid specific b i n d i n g are found in several brain regions including the cerebral cortex, the hippocampus and the cerebellum 33, where a s i g n i f i c a n t K A - i n d u c e d i n c r e a s e of BBB p e r m e a b i l i t y was observed. Anyway, as KA is m e t a b o l i c a l l y rather inert and therefore capable of diffusing widely throughout t h e b r a i n , it is u n l i k e l y that r e g i o n a l s e l e c t i v e c h a n g e s in BBB c h a r a c t e r i s t i c s during the seizure phase (see results obtained in rats given KA 6 mg/Kg) may be c o n s e q u e n t to a d i r e c t a c t i o n of t h e n e u r o t o x i n on l o c a l m i c r o v a s c u l a r structures. An additional hypothesis might be that K A - i n d u c e d BBB opening is dependent on o t h e r p o s s i b l e e f f e c t s of t h e c o n v u l s a n t , for e x a m p l e m o d i f i c a t i o n s in systemic blood p r e s s u r e or in c e r e b r a l b l o o d flow. Due to the e x c e s s i v e f u n c t i o n a l n e u r o n a l a c t i v i t y 34, regional cerebral blood flow increases tremendously during seizures, in d i r e c t p r o p o r t i o n to the rate of g l u c o s e m e t a b o l i s m and c e r e b r a l o x y g e n c o n s u m p t i o n ; o n l y s l i g h t c h a n g e s in r a t arterial blood pressure occur during seizures following the i n j e c t i o n of h i g h d o s e s of KA 35. But b l o o d p r e s s u r e i n c r e a s e d u r i n g c o n v u l s i o n s does not always r e s u l t in BBB l e a k a g e 36 and, m o r e o v e r , e v i d e n c e has b e e n f o u n d of a d i s s o c i a t i o n between changes in s v s t e m i c blood pressure and alterations in B B B permeability 3T . Finally, in the m a t h e m a t i c a l m o d e l we used, Ki values for [14C]AIB are independent of cerebral blood flow values. Anyway, further experiments could clarify a possible r e l a t i o n s h i p b e t w e e n K A - i n d u c e d B B B o p e n i n g and c h a n g e s in s y s t e m i c b l o o d p r e s s u r e and cerebral blood flow. Finally, a brief methodological comment is n e e d e d . In a p r e l i m i n a r y series of e x p e r i m e n t s we found no s i g n i f i c a n t c h a n g e in r e g i o n a l b l o o d v o l u m e b e t w e e n c o n t r o l s and r a t s g i v e n KA 12 mg/Kg, e x c e p t d u r i n g the o c c u r r e n c e of an episode of tonicclonic seizures. Similarly, Makino et al. 34 showed that, in the cat, regional cerebral blood flow increases almost s i m u l t a n e o u s l y w i t h the d e v e l o p m e n t of K A - i n d u c e d s e i z u r e in the p r i m a r y focus and in t h e a r e a s w h e r e s e i z u r e p r o p a g a t i o n is o b s e r v e d , and r e t u r n s to b a s e l i n e value once the seizure has d i s a p p e a r e d . For this r e a s o n , in our c u r r e n t e x p e r i m e n t s , in r a t s g i v e n KA 12 m@~Kg the c o r r e c t i o n for i n t r a v a s c u l a r t r a c e r in Ki v a l u e s for [~C]AIB (regional blood volumes) was estimated during the o c c u r r e n c e of convulsive episodes. However, as this c o r r e c t i o n is probably greater than the actual one, it m a y cause an underestimation of Ki values for [14C]AIB in rats g i v e n KA 12 mg/Kg during the acute seizure phase. As to the LCGU determination, we attempted to take brain samples from h o m o g e n o u s gray matter areas. In structures such as the hippocampus, however, some substructural heterogeneity within the sample was unavoidable. As t h e k i n e t i c c o n s t a n t s of t h e L C G U m o d e l h a v e d i f f e r e n t values for gray and white matter, some of our values may not exactly reflect the true mean metabolic rate. The a d v a n t a g e of the p r e s e n t m e t h o d is the p o s s i b i l i t y to e s t i m a t e Ki v a l u e s for [ 1 4 C ] A I B and local r a t e s of g l u c o s e u t i l i z a t i o n u s i n g the s a m e m e t h o d of d i s s e c t i o n I0,II However, LCGU values c a l c u l a t e d in our c o n t r o l r a t s are c o n s i s t e n t w i t h t h o s e a l r e a d y r e p o r t e d a n d
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o b t a i n e d by a u t o r a d i o g r a p h s 38. F u r t h e r m o r e , in t h e c a l c u l a t i o n of L C G U w i t h t h e 2 - D G method, s e v e r a l s t r i n g e n t r e q u i r e m e n t s m u s t be met. In a n i m a l s w i t h i n t e n s e K A - i n d u c e d s e i z u r e s , s o m e of t h e s e c o n d i t i o n s m a y or m a y not be f u l f i l l e d a n d L C G U v a l u e s m a y t e n d to show exaggerated increases. However, the 2-DG method remains useful under a variety of extreme conditions (9^-q~ t r u e a l t e r a t i o n s in b l o o d and t i s s u e g l u c o s e c o n c e n t r a t i o n s ) ±~-z±. In c o n c l u s i o n , o n e c a n s p e c u l a t e t h a t K A - i n d u c e d B B B o p e n i n g is only partially due to seizure hyperactivity a n d is n o t r e l i a b l y a s s o c i a t e d w i t h n e u r o n a l injury. Of course, f u r t h e r i n v e s t i g a t i o n s n e e d to be u n d e r t a k e n to e l u c i d a t e m e c h a n i s m s i n v o l v e d in s e i z u r e r e l a t e d a l t e r a t i o n s in m i c r o v a s c u l a r c o n t r o l . REFERENCES I. G. SPERK, H. L A S S M A N N , H. BARAN, S.J. KISH, F. S E I T E L B E R G E R a n d O. H O R N Y K I E W I C Z , N e u r o s c i . iO 0 1 3 0 1 - 1 3 1 5 (1983). 2. H. LASSMANN, H. BARAN, U. PETSCHE, K. KITZ, G. SPERK, O. HORNYKIEWICZ and F. SEITELBERGER, Adv. Exp. Med. Biol. 203223-230 (1986). 3. R.E. RUTH and G.S. F E I N E R M A N , A c t a N e u r o p a t h o l . 7_66380-387
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Blood-Brain Barrier and Kainic Acid
(1983).
D.T. M O N A G H A N a n d C.W. COTMAN, B r a i n Res. 252 9 1 - 1 0 0 (1982). K. M A K I N O , T. T A N A K A and Y. Y O N E M A S U , E p i l e p s y Res. 2 2 6 0 - 2 6 8 (1988) . 35. R.C. C O L L I N S , E. L O T H M A N and J.W. OLNEY, Status epilepticus: mechanisms of brain damage and treatment, A.V. D e l g a d o E s c u e t a , C.G. W a s t e r l a i n , D.M. T r e i m a n a n d R.J. P o r t e r eds., pp. 2 7 7 - 2 8 0 , R a v e n press, N e w Y o r k (1983). 36. B. O Z T A S a n d M. KAYA, P h a r m a c o l . Res. 2_/3 4 1 - 4 6 (1991). 37. M.D. E L L I S O N , J.T. P O V L I S H O C K and R.L. HAYES, J. Cereb. B l o o d F l o w M e t a b . 6 4 7 1 - 4 8 0 (1986). 38. M.C. E V A N S a n d B.S. MELDRUM, B r a i n Res. 297 2 3 5 - 2 4 5 (1988).
Pergamon Press
BLOOD-BRAIN BARRIER DYSFUNCTIONS INJECTION OF KAINIC ACID A.
Saija,
FOLLOWING IN THE RAT
SYSTEMIC
*P. Princi, §A. Pisani, §G. S a n t o r o , Pasquale, AM. Massi and #G. Costa
R.
De
Dept. F a r m a c o - B i o l o g i c o (School of P h a r m a c y ) , *Centro Interdipartimentale di Informazioni Farmacotossicologiche, §Dept. Biomorfologia and #Inst. Pharmacology (School of M e d i c i n e ) , University of Messina; AInst. P h a r m a c o l o g y (School of P h a r m a c y ) , University of Camerino (Italy). (Received in final form June 4, 1992) SUMMARY C h a n g e s in b l o o d - b r a i n b a r r i e r (BBB) p e r m e a b i l i t y and cerebral metabolic activity following intravenous injection of kainic acid (KA; 6, 12 mg/Kg) in rats were a s s e s s e d by c a l c u l a t i n g r e s p e c t i v e l y a b l o o d - t o - b r a i n t r a n s f e r c o n s t a n t (Ki) for [ 1 4 C ] ~ - a m i n o i s o b u t y r i c acid and local cerebral glucose utilization (LCGU) values, at d i f f e r e n t times (i h, or acute seizure phase, and 48 h, or c h r o n i c p a t h o l o g y phase) after the induction of seizures. A significant increase in t h e local permeability of t h e B B B w a s o b s e r v e d 1 h after the injection of K A 6 m g / K g (eliciting no s i g n i f i c a n t c h a n g e s in c e r e b r a l m e t a b o l i c activity, except within t h e f r o n t a l c o r t e x and the h i p p o c a m p u s ) and 12 m g / K g (which i n d u c e d a m a r k e d and w i d e s p r e a d e n h a n c e m e n t of L C G U ) . On the c o n t r a r y , d u r i n g the p a t h o l o g y phase, persistent regional increases in Ki v a l u e s were e v i d e n c e d in rats t r e a t e d with the l o w e s t d o s e of the convulsant, but not in rats injected with KA 12 m g / K g (a dose able to cause extensive neuronal damage). Thus one can s p e c u l a t e that: i) K A - i n d u c e d r e g i o n a l c h a n g e s in the permeability of the BBB are not c o r r e l a t e d with changes in neuronal activity; 2) opening of the BBB is not r e l i a b l y associated with neuronal injury.
Kainic acid (KA) , a p o t e n t central nervous system (CNS) excitotoxin, p r o d u c e s an a c u t e or s u b a c u t e m o d e l of s e i z u r e s , l a s t i n g h o u r s to days. P r i m a r y i n t e r e s t in K A w a s d u e to its unusual propensity to c a u s e i r r e v e r s i b l e neuropathological c h a n g e s i n v o l v i n g not o n l y n e r v e c e l l s b u t a l s o glia, m y e l i n sheaths and blood vessels I-2. Besides, r e p o r t s p r o v i d e e v i d e n c e of the e x i s t e n c e of c h a n g e s in f u n c t i o n a l c h a r a c t e r i s t i c s of the blood-brain b a r r i e r (BBB) in KAi n j e c t e d rats, i n c l u d i n g e x t r a v a s a t i o n of f o r e i g n and e n d o g e n o u s serum p r o t e i n s and alterations of BBB p e r m e a b i l i t y to t r a c e r s ~-'. 0024-3205/92 $5.00 + .00 Cop~right © 1992 Pergamon Press Ltd All rights reserved.
468
Blood-Brain Barrier and Kainic Acid
However, the factors responsible permeability (and t h e i r s i g n i f i c a n c e ) are s t i l l far f r o m b e i n g clarified.
Vol. 51, No. 7, 1992
for changes in during KA-induced
the BBB seizures
In t h e p r e s e n t study, the permeability of t h e B B B t o [ 1 4 C ] ~ aminoisobutyric acid ([14C]AIB) and the local cerebral glucose utilization (LCGU) w e r e m e a s u r e d in s m a l l b r a i n a r e a s of r a t s systemically injected with different d o s e s of KA, at d i f f e r e n t times after the induction of s e i z u r e s . This should further characterize the possible relationship existing between BBB dysfunctions, alterations in n e u r o n a l functional activity and neuropathological changes accompanying KA-induced limbic seizures. MATERIALS
AND METHODS
AND CALCULATIONS: Modifications of B B B p e r m e a b i l i t y were determined using a BBB tracer technique, that employs a small molecular weight radiolabeled aminoacid, [I~C]AIB, to identify and quantify small local changes of B B B p e r m e a b i l i t y 8 - 1 3 The m o d e l u s e d for t h e e s t i m a t i o n of b r a i n v a s c u l a r p e r m e a b i l i t y was developed b y O h n o et al. 14. A b l o o d - t o - b r a i n unidirectional transfer constant (Ki) f o r [ 1 4 C ] A I B m a y be c a l c u l a t e d from the following relationship: THEORY
Ci(T) Ki
- V Cb(T)
=
T o
Cpdt
w h e r e Ci(T) is t h e b r a i n p a r e n c h y m a l tracer concentration at t h e time T (duration of the experiment) , Cp is the tracer concentration in t h e t o t a l a r t e r i a l plasma, V is t h e r e s i d u a l blood volume (~i/g) and Cb(T) is t h e w h o l e - b l o o d tracer concentration (nCi/ml) . Sucrose, a compound of very low permeability at t h e B B B , w a s u s e d t o c a l c u l a t e V as a ratio between the tracer concentration in t h e b r a i n a n d in t h e w h o l e b l o o d a c c o r d i n g to the f o l l o w i n g e q u a t i o n 9 : Ci(sucr) v (sucr)
-
Cb(sucr) w h e r e V(sucr) is t h e c e r e b r a l b l o o d v o l u m e (~i/g), c i ( s u c r ) is the tracer concentration (nCi/g) in t h e b r a i n a n d C b ( s u c r ) is its concentration (nCi/ml) in t h e w h o l e - b l o o d . B e c a u s e Ki is r e l a t e d to t h e p e r m e a b i l i t y - s u r f a c e area product (PS) a n d b l o o d - f l o w (F) by PS = F in (i - Ki/F), and PS << F is a c o n d i t i o n w e l l s a t i s f i e d for [14C]AIB, Ki - PS, is i n d e p e n d e n t of F a n d can be e x p r e s s e d in t e r m s of p l a s m a c l e a r a n c e (ml/g/min). LCGU w a s c o m p u t e d for e a c h b r a i n s a m p l e u s i n g t h e 2 - d e o x y g l u c o s e (2-DG) t e c h n i q u e and the m a t h e m a t i c a l m o d e l d e v e l o p e d by S o k o l o f f et al. 15, m o d i f i e d for the influence of v a r y i n g p l a s m a g l u c o s e c o n c e n t r a t i o n s 16. T h i s t e c h n i q u e p r o v i d e s a r e l i a b l e a n d e ~ i c i e n t s c r e e n i n a m e t h o d for n e u r o a c t i v e and n e u r o t o x i c c o m p o u n d s , such as K A 18-21 . The experiments were carried o u t on m a l e W i s t a r rats (320-350 g b.w.) . The animals were maintained under normal controlled lighting and temperature conditions and allowed free ANIMALS:
Vol. 51, No. 7, 1992
access
Blood-Brain Barrier and Kainic Acid
469
to food and water until used.
EXPERIMENTAL D E S I G N : E x p e r i m e n t s w e r e c a r r i e d out one h o u r a f t e r c o n v u l s a n t i n j e c t i o n (acute s e i z u r e phase), w h e n r a t s h a v e p e a k symptoms of seizure activity, a n d 48 h o u r s following KA administration (chronic pathology phase), w h e n an e x t e n s i v e n e u r o p a t h o l o g y with cellular necrosis may appear in several brain r e g i o n s zz. As the d e v e l o p m e n t of b e h a v i o u r a l c h a n g e s (typical of status epilepticus) and neuropathology follow~g KA administration a r e r e l a t e d to t h e d o s e of n e u r o t o x i n ~=, t h e c o n v u l s a n t was injected at 2 different dose levels (6 m g / K g and 12 m g / K g ) . C o n t r o l a n i m a l s w e r e o n l y g i v e n the v e h i c l e (NaCI 0.9% solution, i ml/Kg).
Two p o l y e t h y l e n e catheters were chronically inserted in the right f e m o r a l v e i n and a r t e r y under light ether a n a e s t h e s i a ; KA or its vehicle were injected intravenously (iv) t h r o u g h t h e c a t h e t e r inserted in the femoral vein. The rats were d i v i d e d into 6 groups of 16 a n i m a l s each: c o n t r o l s , a c u t e s e i z u r e p h a s e ; K A 6 m g / K g , a c u t e s e i z u r e phase; KA 12 mg/Kg, acute s e i z u r e phase; c o n t r o l s , c h r o n i c p a t h o l o g y phase; KA 6 mg/Kg, c h r o n i c p a t h o l o g y phase; KA 12 mg/Kg, chronic pathology phase. SURGICAL PREPARATION FOR TRACER INJECTION: O n t h e d a y of t h e experiment, the animals were lightly anaesthetized with ether; short polyethylene c a t h e t e r s w e r e i n s e r t e d in t h e left f e m o r a l vein for tracer administration and in the left femoral artery for blood sampling. Cannulas were passed subdermally behind the leg to e x i t j u s t r o s t r a l to the b a s e of the tail and t h e w o u n d s w e r e sutured. The a n i m a l s were p a r t i a l l y f a s t e n e d from the w a i s t d o w n by m e a n s of l o o s e - f i t t i n g tape (placed along the flanks), permitting f r e e m o v e m e n t of h e a d a n d f o r e l i m b s ; t h e r e w a s no a p p a r e n t i n d i c a t i o n of d i s t r e s s or s e i z u r e t r i g g e r i n g in t h i s p a r t i a l l y r e s t r a i n e d c o n d i t i o n . M o r e o v e r , e v e r y p r e c a u t i o n was taken to m i n i m i z e or avoid pain and discomfort to the animals. The rats w e r e t h e n a l l o w e d to r e c o v e r from the a n e s t h e s i a for 3-4 h before tracer injection.
L C G U and Ki v a l u e s for [14C]AIB were c a l c u l a t e d s e p a r a t e l y r a t s of e a c h e x p e r i m e n t a l group; regional blood volumes e s t i m a t e d in 4 animals of each experimental group.
in 6 were
TRACER INJECTION: [14C]AIB or 2 - d e o x y - D - ( l - 1 4 C ) g l u c o s e ([14C]DG) were injected 1 h or 48 h a f t e r a d m i n i s t r a t i o n of K A or K A vehicle. [14C]Sucrose w a s i n j e c t e d iv 1 h or 48 h f o l l o w i n g i n j e c t i o n of KA 6 mg/Kg or the vehicle; in rats given KA 12 mg/Kg, the t r a c e r w a s i n j e c t e d i m m e d i a t e l y at the o n s e t of t h e f i r s t episode of t o n i c - c l o n i c convulsions (50-70 min following n e u r o t o x i n injection) or 48 h after injection of the convulsant. M~SUREMENT O F Ki F O R [ 14 C ] A I B O R L C G U : The e v a l u a t i o n of Ki for [ C]AIB ^aD¢ LCGU values was carried out as previously d e s c r i b e d I Z I I % . Briefly, the animals were injected iv w i t h a bolus of [ 14 C ] A I B (25 ~Ci/rat) or of [ 14 C]DG (60 ~ C i / r a t ) . Small b l o o d samples ( 8 0 - 1 0 0 ~i) w e r e c o l l e c t e d in p o l y e t h y l e n e t u b e s at predetermined intervals of time during the e x p e r i m e n t a l period. A large v o l u m e was w i t h d r a w n at the end to m e a s u r e the w h o l e - b l o o d i s o t o p e c o n c e n t r a t i o n only in rats given [14C]AIB. At the end of the experimental p e r i o d (20 m i n and 45 m i n r e s p e c t i v e l y after [14C]AIB and [14C]DG injection) all the rats w e r e s a c r i f i c e d by
470
Blood-Brain Barrier and Kainic Acid
Vol. 51, No. 7, 1992
rapid iv injection of 1 ml saturated KCl solution. The d u r a t i o n of the e x p e r i m e n t a l time was c h o s e n to m i n i m i z e b o t h the e f f e c t of i n t r a v a s c u l a r tracer on brain 14C activity m e a s u r e d at the end of the e x p e r i m e n t and the e v e n t u a l b r a i n - t o - b l o o d r e f l u x of tracer. The b r a i n w a s t h e n r a p i d l y r e m o v e d and s m a l l t i s s u e s R e c i m e n s were d i s s e c t e d on dry ice, according to Paxinos and W a t s o n 23, from the f o l l o w i n g areas: frontal cortex, t e m p o r o p a r i e t a l (temp.par.) cortex, occipital c o r t e x ( i n c l u d i n g the e n t o r h i n a l cortex), striatum, hippocampus, thalamus, hypothalamus, cerebellum and brain-stem. Sample s o l u b i l i s a t i o n was a c c o m p l i s h e d by a d d i n g 1 ml of S o l u e n e i00 (Packard) and incubating the vials at 60°C overnight. E a c h v i a l w a s t h e n f i l l e d w i t h 4 ml of H i o n i c - F l u o r (Packard). B l o o d s a m p l e s were c e n t r i f u g e d ; then, 30 ~i of p l a s m a w e r e d i l u t e d in 0.5 ml d i s t i l l e d w a t e r and c o u n t e d in 6 ml of Hionic-Fluor. A 20 ~i w h o l e - b l o o d s a m p l e was d i g e s t e d in 0.5 ml Soluene/ethanol (1:2) s o l u t i o n , b l a n c h e d w i t h 0.3 ml h y d r o g e n p e r o x i d e (30% wt/vol) and c o u n t e d in 6 ml H i o n i c F l u o r a c i d i f i e d with 0.5 ml 0.5 N HCI. B e t a - c o u n t i n g was p e r f o r m e d by a P a c k a r d PL s c i n t i l l a t i o n counter. Single s a m p l e q u e n c h i n g was m o n i t o r e d by the e x t e r n a l s t a n d a r d m e t h o d . S a m p l e c o u n t s w e r e c o r r e c t e d for b a c k g r o u n d and quenching. Plasma glucose concentrations d e t e r m i n e d by the glucose oxidase
(for L C G U assay.
measurement)
were
BLOOD VOLUMES: Regional blood volumes were c a l c u l a t e d to i n t r o d u c e a c o r r e c t i o n for the r e s i d u a l i n t r a v a s c u l a r t r a c e r in the e s t i m a t i o n of Ki for [ 14 C]AIB (see T H E O R Y A N D C A L C U L A T I O N S ) . Blood v o l u m e (V) was defined as the [±~C]sucrose space at 1 minute a f t e r t h e iv i n j e c t i o n of t h e t r a c e r 8,9. A 20 ~ C i b o l u s of [14C]sucrose was injected iv. One minute later a blood sample was c o l l e c t e d and the animal sacrificed by decapitation. Brain samples and w h o l e - b l o o d w e r e d i g e s t e d a n d c o u n t e d in t h e s a m e w a y as [14C]AIB.
REGIONAL
AND PHYSIOLOGICAL PARAMETERS: Following KA or KA vehicle injection, the animals were observed for b e h a v i o u r a l changes, and various physiological parameters were monitored. Mean arterial blood p r e s s u r e was m e a s u r e d by connecting the arterial c a t h e t e r to a strain gauge transducer and both systolic and diastolic pressures were displayed on c h a n n e l s of a p o l i g r a p h (Basile, Italy). A r t e r i a l blood samples were w i t h d r a w n for d e t e r m i n a t i o n of pH, pO 2 a n d p C O 2 ( p H - b l o o d g a s a n a l y z e r , A V L AG, B i o m e d i c a l Instruments, Switzerland). BEHAVIOUR
ANALYSIS: Results are expressed as m e a n ± S.E.. Differences in K i v a l u e s for [14C]AIB, rates of glucose u t i l i z a t i o n and regional blood volume were t e s t e d for s t a t i s t i c a l significance by t w o - w a y A N O V A . Statistical significance was a c c e p t e d w h e r e P < 0.05. STATISTICAL
USED: [14C]AIB (59 m C i / m m o l ) , [ 1 4 C ] D G (59 m C i / m m o l ) and [14C]sucrose (540 mCi/mmol) were purchased from Amersham (England); k a i n i c acid was purchased from Sigma C h e m i c a l Co..
DRUGS
RESULTS BEHAVIOUR
injection,
AND
PHYSIOLOGICAL
specific
types
Following KA or KA v e h i c l e behaviour were noted, including:
PARAMETERS:
of
Vol. 51, No. 7, 1992
Blood-Brain Barrier and Kainic Acid
TABLE
ACUTE
SEIZURE
PHASE
CHRONIC
PATHOLOGY
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
I
(i h)
CONTROLS Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
471
2.334±0 2.512±0 3.053±0 1.702±0 1.079±0 0.843±0 4.867±0 2.417±0 2.627±0
PHASE
095 123 060 094 031 045 099 048 237
KA 6 m q / K q 3.554±0.216, 3.798±0.349* 4.685±0.441, 2.162±0.282 1.533±0.018, 1.039±0.139 5.990±0.375 3.253±0.265* 3.762±0.358
KA
12 m q / K q
7.941±0.850" 6.844±0.477* 8.234±0.680* 2.172±0.080" 2.579±0.017" 1.114±0.035" 5.715±0.300 3.973±0.264* 3.612±0.131"
(48 h_~
CONTROLS
KA 6 m g / K g
2.295±0.105 2.484±0.114 3.010±0.038 1.823±0.121 1.017±0.033 0.896±0.066 5.008±0.105 2.355±0.054 2.727±0.264
2 811±0.163 3 195±0.137, 3 927±0.235* 1 720±0.132 1 424±0.107, 0 597±0.043 4.824±0.342 3.125±0.176, 2.803±0.158
KA
12 m q / K q
1.770±0.312 2.026±0.180 2.386±0.287 1.531±0.103 1.230±0.263 0.686±0.159 4.597±0.563 2.411±0.243 1.998±0.154
Modifications of Ki for [ 1 4 C ] A I B ( m l / g / m i n x 10 -3 ) in r a t s injected iv w i t h k a i n i c a c i d (KA) 6 or 12 m g / K g , or g i v e n o n l y the v e h i c l e (controls). (* P < 0.05 v e r s u s c o n t r o l s )
normal for vehicle-treated animals; staring spells and "wet dog shakes" (WDS) for a n i m a l s i n j e c t e d w i t h KA 6 m g / K g ; s a l i v a t i o n , rearing, tonic-clonic convulsions and status epilepticus for a n i m a l s i n j e c t e d w i t h KA 12 mg/Kg. T h r o u g h o u t the two day period after KA injection, the a n i m a l s m a n i f e s t e d a b n o r m a l b e h a v i o u r and were hyperexcitable; besides, rats i n j e c t e d w i t h the h i g h e s t d o s e of K A w e r e a p h a g i c and lost weight. During the experimental p e r i o d , if m a i n t a i n e d u n d e r n o n s t r e s s f u l conditions, the KA-injected rats used in our s t u d y s h o w e d no a b n o r m a l r e s p o n s e to the e x p e r i m e n t a l p r o t o c o l ; v a l u e s of a r t e r i a l b l o o d pH, p O ~ and p C O 2 w e r e w i t h i n normal limits (data n o t shown). P r i o r to i s o t o p e i n j e c t i o n a n d t h r o u g h o u t t h e t w o d a y s f o l l o w i n g the injection of t h e n e u r o t o x i n , the physiological parameters (plasma glucose values, blood pressure, hematocrit, rectal temperature, a r t e r i a l b l o o d g a s e s a n d pH) d i d n o t s i g n i f i c a n t l y d i f f e r b e t w e e n e x p e r i m e n t a l g r o u p s (data not shown). ACUTE
SEIZURE
PHASE
BBB permeability (table I): the i n j e c t i o n significant enhancement of K i v a l u e s
of K A 6 m g / K g e l i c i t e d a calculated within the
472
Blood-Brain Barrier and Kainic Acid
TABLE
ACUTE
SEIZURE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
CHRONIC
PHASE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
II
(i h)
CONTROLS
KA 6 m g / K g
68.1±2.924 73.4±2.377 72.2±5.205 71.2±0.308 50.4±1.779 69.3±4.289 46.0±2.305 46.1±5.323 46.2±2.864
78.8±1.057, 83 3±2.322 78 8 ± 3 . 0 8 9 76 0 ± 2 . 7 3 1 78 0 ± 4 . 4 1 2 , 77 6±3.097 53 4 ± 0 . 8 5 1 51 5 ± 1 . 1 2 9 50 9 ± 1 . 0 5 1
PATHOLOGY
Vol. 51, No. 7, 1992
PHASE
KA
12 m q / K q
94.4±2.522* 100.6±2.952" 94.6±1.899" 93.0±1.864, 153.6±0.886, 86.9±1.421, 70.1±1.435, 54.0±1.071, 55.3±0.957*
(48 h)
CONTROLS
KA 6 m q / K g
KA
12 m g / K g
69.0±5.000 74.6±4.135 73.2±5.241 68.3±0.059 51.8±0.624 77.3±2.286 47.5±1.506 47.6±1.914 43.9±0.996
72.9±3.873 75.8±3.705 73.2±2.291 71.5±2.717 53.3±2.451 80.8±2.151 51.6±0.774 49.1±2.549 49.9±1.087
41.9±0.661, 47.4±0.926* 47.0±1.212, 50.3±1.420, 37.3±0.494* 54.1±1.188, 39.0±1.347, 35.5±0.755* 38.0±1.037,
Modifications of L C G U (~mol/100 g/min) in r a t s i n j e c t e d iv w i t h k a i n i c a c i d (KA) 6 or 12 m g / K g , or g i v e n o n l y t h e v e h i c l e (controls). (* P < 0.05 v e r s u s c o n t r o l s )
frontal, temporoparietal and occipital cortex, hippocampus and c e r e b e l l u m . M o r e m a r k e d and w i d e s p r e a d i n c r e a s e s of Ki v a l u e s w e r e i n d u c e d by a d m i n i s t r a t i o n of the h i g h e s t d o s e of K A (12 mg/Kg) in all b r a i n r e g i o n s examined, e x c e p t the h y p o t h a l a m u s . Cerebral glucose u s e ( t a b l e II) : W i t h a d o s e of 6 m g / K g of KA, several brain areas showed a slight increase of L C G U values, but this effect was statistically significant only within the f r o n t a l c o r t e x and the h i p p o c a m p u s . A s t r i k i n g w i d e s p r e a d i n c r e a s e in L C G U w a s o b s e r v e d f o l l o w i n g t h e i n j e c t i o n of K A 12 m g / K g . T h e g r e a t e s t i n c r e a s e in L C G U was w i t h i n the h i p p o c a m p u s , the proposed epileptogenic focus 21,22, f o l l o w i n g b o t h d o s e s t e s t e d of KA. Regional blood volumes ( t a b l e III): T h e r e w a s no d i f f e r e n c e in b l o o d r e g i o n a l v o l u m e b e t w e e n c o n t r o l s and r a t s g i v e n K A 6 mg/Kg. Conversely, blood regional volumes increased significantly in a n i m a l s i n j e c t e d w i t h t h e h i g h e s t d o s e of n e u r o t o x i n (12 m g / K g ) w i t h i n all b r a i n r e g i o n s examined. CHRONIC
PATHOLOGY
PHASE
BBB permeability (table there was a statistically
I) : In r a t s i n j e c t e d with KA 6 mg/Kg, s i g n i f i c a n t i n c r e a s e in Ki v a l u e s w i t h i n
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the temporoparietal and occipital cortex, hippocampus and cerebellum; however, in a n i m a l s g i v e n K A 12 m g / K g , no obvious m o d i f i c a t i o n of [14C]AIB t r a n s p o r t a c r o s s the BBB w a s o b s e r v e d . C e r e b r a l g l u c o s e u s e (table II): Controls (vehicle) a n d a n i m a l s g i v e n K A 6 m g / K g s h o w e d no s i g n i f i c a n t d i f f e r e n c e in L C G U values. On the contrary, L C G U v a l u e s in r a t s i n j e c t e d w i t h K A 12 m g / K g w e r e m a r k e d l y d e p r e s s e d in a w i d e s p r e a d way. Regional blood volume ( t a b l e III) : T h e r e w a s n o s i g n i f i c a n t difference in r e g i o n a l blood volumes between controls and rats g i v e n 6 or 12 m g / K g of KA. TABLE
ACUTE
SEIZURE
Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
CHRONIC
PHASE
(50-70 min)
CONTROLS
KA 6 m q / K g
KA
37.46±2.55 29 07±2.26 32 40±1.68 19 61±1.10 26 83±1.59 26 77±2.29 30 31±2.70 39 16±1.14 26.36±1.05
32.50±2.41 34.18±2.64 34.77±2.33 24.99±1.95 25.92±1.88 27.34±1.97 31.53±2.56 41.92±2.30 28.56±1.72
53.04±3.21, 57.11±3.35" 56.60±4.16, 39.59±1.92" 52.74±2.27* 61.37±3.49" 51.91±2.76" 90.55±4.24* 37.93±2.55*
KA 6 m q / K g
KA
33.41±2.56 32.95±2.34 33.27±1.99 21.20±1.13 28.24±1.71 34.97±2.94 31.24±2.34 40.11±2.25 29.03±2.15
31.34±2.24 35.04±2.18 35.85±2.65 23.79±1.72 30.25±2.41 40.76±3.11 32.55±2.33 43.68±2.51 33.37±2.72
PATHOLOGY
PHASE
37.55±2 30.11±2 31.95±1 20.13±1 27.54±1 27.10±2 30.88±2 38.55±1 27.27±1
12 m q / K q
(48 h)
CONTROLS Cortex frontal temp.par. occipital Striatum Hippocampus Thalamus Hypothalamus Cerebellum Brain-stem
III
43 35 77 22 56 46 48 91 24
12 m q / K g
Regional b l o o d v o l u m e s ° (~i/g) in r a t s i n j e c t e d iv w i t h k a i n i c a c i d (KA) 6 or 12 mg/Kg, or g i v e n o n l y t h e v e h i c l e (controls). ( * P < 0.05 v e r s u s gontrols) ° B l o o d v o l u m e is d e f i n e d as t h e [ l ~ C ] s u c r o s e space 1 min a f t e r t h e i n t r a v e n o u s i n j e c t i o n of [ 14 C ] s u c r o s e . DISCUSSION A s i g n i f i c a n t i n c r e a s e in the local p e r m e a b i l i t y of t h e B B B in the r a t w a s o b s e r v e d 1 h a f t e r the i n t r a v e n o u s i n j e c t i o n of K A 6 m g / K g (eliciting mild behavioural alterations without significant c h a n g e s in L C G U changes, e x c e p t w i t h i n the f r o n t a l c o r t e x a n d the hippocampus) a n d 12 m g / K g (a d o s e i n d u c i n g a m a r k e d a n d w i d e s p r e a d enhancement of c e r e b r a l m e t a b o l i c a c t i v i t y ) . H o w e v e r , d u r i n g t h e
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pathology phase, persistent regional alterations in BBB p e r m e a b i l i t y to [14C]AIB were evidenced in rats t r e a t e d w i t h the l o w e s t d o s e of the c o n v u l s a n t (inducing only m o d e r a t e c h a n g e s in brain m o r p h o l o g i c a l structures), but not in rats injected w i t h KA 12 m g / K g (which c a u s e s e x t e n s i v e n e u r o n a l damage). T h e s e r e s u l t s are at v a r i a n c e with those of Zucker et al 7, who also a s s e s s e d BBB permeability with [14C]AIB following KA. H o w e v e r , d u e to a different methodology (in p a r t i c u l a r the i n t r o d u c t i o n , in our experiments, of a c o r r e c t i o n for i n t r a v a s c u l a r tracer), it is d o u b t f u l if such results may be compared with our p r e s e n t data. In fact, in o r d e r to a v o i d i n c o r r e c t e s t i m a t i o n s of the a c t u a l BBB p e r m e a b i l i t y to the tracer, the introduction of a c o r r e c t i o n for intravascular tracer s h o u l d n o t be n e g l e c t e d , as K A - i n d u c e d seizures are a c c o m p a n i e d by transient increases in c e r e b r a l blood f l o w 24 a n d i n d u c e irreversible damages a l s o in b r a i n blood vessels 2 . The findings p r e s e n t e d herein emphasize two concepts: i) regional c h a n g e s in BBB p e r m e a b i l i t y do not parallel and/or are not related to s i g n i f i c a n t changes in n e u r o n a l metabolic activity; for example, in r a t s g i v e n KA 6 mg/Kg, d u r i n g the s e i z u r e p h a s e , a s i g n i f i c a n t increase in Ki values was observed w i t h i n the frontal and o c c i p i t a l c o r t e x and the cerebellum, in s p i t e of a b s e n c e of modifications in L C G U values, and, in a d d i t i o n , the percent i n c r e a s e (42.07%) in Ki v a l u e s w i t h i n the h i p p o c a m p u s (the only area w h e r e a s i g n i f i c a n t i n c r e a s e in L C G U (54.76%) w a s observed) was c o n s i s t e n t with that calculated within the cortex and the c e r e b e l l u m (34.59-53.45%); 2) K A - i n d u c e d n e u r o p a t h o l o g i c a l changes are not always accompanied by o b v i o u s modifications in B B B p e r m e a b i l i t y to [14C]AIB (see results obtained in rats given KA 12 m g / K g d u r i n g the p a t h o l o g y phase). W i t h r e g a r d to t h e f i r s t issue, t h e r e is c l e a r e v i d e n c e that neuronal activation p e r se is i n a d e q u a t e to a c c o u n t for the i n c r e a s e d p e r m e a b i l i t y of the BBB in K A - t r e a t e d rats 24. R e g i o n a l c h a n g e s in c e r e b r a l levels of n e u r o t r a n s m i t t e r a m i n o a c i d s =J, in norepinephrine and s e r o t o n i n t u r n o v e r 26 and in t h e a c t i v i t y of Na + ,K + - A T P a s e 27 are a s s o c i a t e d w i t h KA s e i z u r e s ; on t h e o t h e r hand, d i f f e r e n t p r o c e s s e s i n v o l v e d in the BBB can be s e l e c t i v e l y and temporarily altered in c o n s e q u e n c e of c h a n g e s in t h e functional activity of neuronal circuits within the CNS I0,12'28'29 Thus it is tempting to h y p o t h e s i z e that changes in BBB p e r m e a b i l i t y following KA systemic injection m i g h t be related, in p a r t , to a c h a l l e n g e of t h e n e u r o g e n i c and/or vasogenic c o m p o n e n t s c o n t r o l l i n g BBB c h a r a c t e r i s t i c s (perhaps at the level of n e u r o n a l innervation present in t h e c e r e b r a l capillary endothelium or d u e to a s t i m u l a t e d release of v a s o a c t i v e substances). As to the s e c o n d issue, the BBB o p e n i n g is not a l w a y s a s s o c i a t e d with neuronal damage 30,31 and the increased cerebrovascular p e r m e a b i l i t y to protein during seizures might not be a d e l e t e r i o u s m e c h a n i s m 3,32. Thus, the increase of the BBB p e r m e a b i l i t y o b s e r v e d d u r i n g the p a t h o l o g i c a l p h a s e in rats g i v e n KA 6 m g / K g d o e s not o c c u r c o n s e q u e n t l y to an actual BBB breakdown, but m i g h t o p e r a t e as a h o m e o s t a t i c mechanism. The absence of BBB o p e n i n g d u r i n g the p a t h o l o g y p h a s e f o l l o w i n g the i n j e c t i o n of KA 12 m g / K g m i g h t be due to derangements in t h e c o n t r o l of c e r e b r a l capillary permeability.
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A s . t h e i n d u c i n g a g e n t is still p r e s e n t d u r i n g the [ 1 4 C ] A I B and [ ± ~ C ] D G e x p e r i m e n t s c a r r i e d out d u r i n g the s e i z u r e p h a s e , the e f f e c t s of the s e i z u r e s per se on BBB p e r m e a b i l i t y and c e r e b r a l m e t a b o l i s m may be confounded by the direct effects of the inducing agent itself. M o d e r a t e to high levels of [3H]kainic acid specific b i n d i n g are found in several brain regions including the cerebral cortex, the hippocampus and the cerebellum 33, where a s i g n i f i c a n t K A - i n d u c e d i n c r e a s e of BBB p e r m e a b i l i t y was observed. Anyway, as KA is m e t a b o l i c a l l y rather inert and therefore capable of diffusing widely throughout t h e b r a i n , it is u n l i k e l y that r e g i o n a l s e l e c t i v e c h a n g e s in BBB c h a r a c t e r i s t i c s during the seizure phase (see results obtained in rats given KA 6 mg/Kg) may be c o n s e q u e n t to a d i r e c t a c t i o n of t h e n e u r o t o x i n on l o c a l m i c r o v a s c u l a r structures. An additional hypothesis might be that K A - i n d u c e d BBB opening is dependent on o t h e r p o s s i b l e e f f e c t s of t h e c o n v u l s a n t , for e x a m p l e m o d i f i c a t i o n s in systemic blood p r e s s u r e or in c e r e b r a l b l o o d flow. Due to the e x c e s s i v e f u n c t i o n a l n e u r o n a l a c t i v i t y 34, regional cerebral blood flow increases tremendously during seizures, in d i r e c t p r o p o r t i o n to the rate of g l u c o s e m e t a b o l i s m and c e r e b r a l o x y g e n c o n s u m p t i o n ; o n l y s l i g h t c h a n g e s in r a t arterial blood pressure occur during seizures following the i n j e c t i o n of h i g h d o s e s of KA 35. But b l o o d p r e s s u r e i n c r e a s e d u r i n g c o n v u l s i o n s does not always r e s u l t in BBB l e a k a g e 36 and, m o r e o v e r , e v i d e n c e has b e e n f o u n d of a d i s s o c i a t i o n between changes in s v s t e m i c blood pressure and alterations in B B B permeability 3T . Finally, in the m a t h e m a t i c a l m o d e l we used, Ki values for [14C]AIB are independent of cerebral blood flow values. Anyway, further experiments could clarify a possible r e l a t i o n s h i p b e t w e e n K A - i n d u c e d B B B o p e n i n g and c h a n g e s in s y s t e m i c b l o o d p r e s s u r e and cerebral blood flow. Finally, a brief methodological comment is n e e d e d . In a p r e l i m i n a r y series of e x p e r i m e n t s we found no s i g n i f i c a n t c h a n g e in r e g i o n a l b l o o d v o l u m e b e t w e e n c o n t r o l s and r a t s g i v e n KA 12 mg/Kg, e x c e p t d u r i n g the o c c u r r e n c e of an episode of tonicclonic seizures. Similarly, Makino et al. 34 showed that, in the cat, regional cerebral blood flow increases almost s i m u l t a n e o u s l y w i t h the d e v e l o p m e n t of K A - i n d u c e d s e i z u r e in the p r i m a r y focus and in t h e a r e a s w h e r e s e i z u r e p r o p a g a t i o n is o b s e r v e d , and r e t u r n s to b a s e l i n e value once the seizure has d i s a p p e a r e d . For this r e a s o n , in our c u r r e n t e x p e r i m e n t s , in r a t s g i v e n KA 12 m@~Kg the c o r r e c t i o n for i n t r a v a s c u l a r t r a c e r in Ki v a l u e s for [~C]AIB (regional blood volumes) was estimated during the o c c u r r e n c e of convulsive episodes. However, as this c o r r e c t i o n is probably greater than the actual one, it m a y cause an underestimation of Ki values for [14C]AIB in rats g i v e n KA 12 mg/Kg during the acute seizure phase. As to the LCGU determination, we attempted to take brain samples from h o m o g e n o u s gray matter areas. In structures such as the hippocampus, however, some substructural heterogeneity within the sample was unavoidable. As t h e k i n e t i c c o n s t a n t s of t h e L C G U m o d e l h a v e d i f f e r e n t values for gray and white matter, some of our values may not exactly reflect the true mean metabolic rate. The a d v a n t a g e of the p r e s e n t m e t h o d is the p o s s i b i l i t y to e s t i m a t e Ki v a l u e s for [ 1 4 C ] A I B and local r a t e s of g l u c o s e u t i l i z a t i o n u s i n g the s a m e m e t h o d of d i s s e c t i o n I0,II However, LCGU values c a l c u l a t e d in our c o n t r o l r a t s are c o n s i s t e n t w i t h t h o s e a l r e a d y r e p o r t e d a n d
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o b t a i n e d by a u t o r a d i o g r a p h s 38. F u r t h e r m o r e , in t h e c a l c u l a t i o n of L C G U w i t h t h e 2 - D G method, s e v e r a l s t r i n g e n t r e q u i r e m e n t s m u s t be met. In a n i m a l s w i t h i n t e n s e K A - i n d u c e d s e i z u r e s , s o m e of t h e s e c o n d i t i o n s m a y or m a y not be f u l f i l l e d a n d L C G U v a l u e s m a y t e n d to show exaggerated increases. However, the 2-DG method remains useful under a variety of extreme conditions (9^-q~ t r u e a l t e r a t i o n s in b l o o d and t i s s u e g l u c o s e c o n c e n t r a t i o n s ) ±~-z±. In c o n c l u s i o n , o n e c a n s p e c u l a t e t h a t K A - i n d u c e d B B B o p e n i n g is only partially due to seizure hyperactivity a n d is n o t r e l i a b l y a s s o c i a t e d w i t h n e u r o n a l injury. Of course, f u r t h e r i n v e s t i g a t i o n s n e e d to be u n d e r t a k e n to e l u c i d a t e m e c h a n i s m s i n v o l v e d in s e i z u r e r e l a t e d a l t e r a t i o n s in m i c r o v a s c u l a r c o n t r o l . REFERENCES I. G. SPERK, H. L A S S M A N N , H. BARAN, S.J. KISH, F. S E I T E L B E R G E R a n d O. H O R N Y K I E W I C Z , N e u r o s c i . iO 0 1 3 0 1 - 1 3 1 5 (1983). 2. H. LASSMANN, H. BARAN, U. PETSCHE, K. KITZ, G. SPERK, O. HORNYKIEWICZ and F. SEITELBERGER, Adv. Exp. Med. Biol. 203223-230 (1986). 3. R.E. RUTH and G.S. F E I N E R M A N , A c t a N e u r o p a t h o l . 7_66380-387
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