Brain Research, 577 (1992) 121-126 Elsevier

121

BRES 17592

Early cellular swelling during cerebral ischemia in vivo is mediated by excitatory amino acids released from nerve terminals Yoichi K a t a y a m a a't'*, Toru T a m u r a a, D o n a l d P. B e c k e r a and Takashi Tsubokawa b "Division of Neurosurgery, UCLA School of Medicine, University of California at Los Angeles, California 90024 (USA) and bDepartrnent of Neurological Surgery, Nihon University School of Medicine, Tokyo 173 (Japan) (Accepted 19 November 1991) Key words: Cerebral ischemia; Cellular swelling; Excitatory amino acid; Potassium ion; Microdialysis; Spreading depression

This study demonstrates ischemic cellular swelling in vivo detected as changes in the concentration of 14C-sucrose pre-perfused into the extracellular space (ECS) as an ECS marker. Microdialysis was utilized as a means of perfusion and measurement of the extracellular concentration of 14C-sucrose ([14C-sucrose]e). Concomitant with an abrupt increase in [K+]e at 1-3 min following the ischemia induction, [14Csucrose]e was also rapidly elevated. Since sucrose is not taken up by either cells or capillaries, the absolute amount of *4C-sucrose in the ECS must be unchanged. The increase therefore appears to represent a relative decrease in water volume in the ECS resulting from a movement of water into the cells, i.e. cellular swelling. Ca2+-free perfusate containing Co2+, which has been shown to block excitatory amino acid release during cerebral ischemia, significantly delayed the increase in [14C-sucrose]c and [K÷]e. Kynurenic acid, a broad-spectrum antagonist of excitatory amino acids, administered in situ through the dialysis probe also significantly delayed the increase in [14C-sucrose]cand [K+]c. These findings indicate that the early cellular swelling occurring during cerebral ischemia is a result of massive ionic fluxes mediated by excitatory amino acids which are released by a Ca2+-dependent exocytotic process from the nerve terminals. INTRODUCTION The occurrence of cellular swelling and resultant shrinkage of the extracellular space (ECS) during cerebral ischemia or spreading depression has been repeatedly demonstrated in in vitro ~'t9 as well as in vivo 23'24 studies by various methods, including the measurement of ECS markers 9'2°. When rapid cellular swelling occurs, water moves from the ECS into the cells and causes a decrease in water volume in the ECS. In contrast, the ECS markers do not move into the cells and are left behind. The cellular swelling and ECS shrinkage can thus be detected as an increase in concentration of the markers. As ECS markers, tetraethylammonium, tetraethyltris- methylammonium and choline have been employed 9' 20 These markers are introduced into the ECS by a superfusion technique, and changes in their ECS concentration are monitored by employing electrodes sensitive to ammonium ions 9"2°. In the present study, we attempted to detect cellular swelling during cerebral ischemia using microdialysis based on similar principles, laC-sucrose was pre-perfused into the ECS through the dialysis probe. This substance has been widely used in in vitro studies as an ECS marker. Changes in the extracellular concentration of

14C-sucrose ([14C-surcrose]e) were determined from the dialysate. Such a technique can provide a unique opportunity to examine the mechanism of cellular swelling during cerebral ischemia in vivo, by measuring other neurochemical changes occurring in the same dialysate fractions and by manipulating neurochemical processes by administering various agents in situ through the dialysis probe. The rapid water movements causing cellular swelling during cerebral ischemia is mainly a consequence of massive ionic fluxes across the plasma membrane. Since the ionic fluxes cause the osmotic pressure of intracellular impermeable anions no longer to be counterbalanced, water moves into the cells 8'9A4. The cellular swelling therefore develops concomitantly with ionic shifts 8'9'14. We first determined whether an increase in [laC-sucrose]e during cerebral ischemia, if observed, is concomitant with the onset of sudden ionic fluxes. Preliminary data from pilot experiments have been reported in abstract form elsewhere 1~. In addition, the present study tested the hypothesis that the early cellular swelling during cerebral ischemia is mediated by excitatory amino acids (EAAs). E A A s have been found in vitro to produce pronounced cellular swelling as a result of massive ionic shifts 4'5'15'18.

* On leave from the Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan. Correspondence: Y. Katayama, Department of Neurological Surgery, Nihon University School of Medicine, ltabashi-ku, Tokyo 173, Japan.

122 C a 2 + - d e p e n d e n t E A A release has b e e n s h o w n to begin

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ing c e r e b r a l ischemia ~3. We h a v e f u r t h e r d e m o n s t r a t e d that the o n s e t of a large increase in [K+]~, which r e p r e -

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bral ischemia. We t h e r e f o r e e x a m i n e d w h e t h e r or not the cellular swelling during c e r e b r a l ischemia, if d e t e c t e d ,

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could be influenced by inhibition of C a 2 + - d e p e n d e n t E A A release e m p l o y i n g Ca2+-free dialysis with a perfusate c o n t a i n i n g C o 2+ or by b l o c k i n g E A A through

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35

Fig. 1. The time course of changes in [ 1 4 C - s u c r o s e ] d following termination of '4C-sucrose perfusion for 20 min through the dialysis probe. Filled circles, control dialysis (n = 18); open squares, dialysis with Ca-'+-free perfusate containing Co -'+ (10 mM, n = 9); open circles, dialysis with kynurenic acid (KYN, 10 mM, n = 9). Mean _+ S.E.M.

MATERIALS AND METHODS Young adult Sprague-Dawley rats (n = 18) weighing 180-240 g were used. The animals were maintained in an environmentally controlled room with a 12-h light/dark cycle and given free access to food and water. They were anesthetized with a mixture of nitrous oxide (66%), oxygen (33%) and enflurane (1%), and placed in a stereotaxic frame with a nosebar setting of 2.5 mm below the interaural level. The skin wounds and pressure points were infiltrated with I% xylocaine. The rectal temperature was maintained at 37-38°C with a heating pad. A pair of dialysis probes (CMA/10, Bioanalytical System Inc.; O.D., 500/~m; effective length, 3 mm; cut off, 20,000 Mw were lowered vertically through small skull holes into the brain, placing the tip in the hippocampus (3.8-4.8 mm caudal to the bregma, 2.0 mm lateral to the midline and 3.5 mm below the surface of the dura). Thus, approximately 50% of the effective length of the probes was located within the hippocampus. The probes were initially perfused with modified Ringer solution containing 14C-sucrose (10 mM, 4.8 mCi/mmol) and 3 mM Ca -'+ at a rate of 5.0/A/min. In one of the 2 probes chosen at random, the effects of Ca 2+free perfusate containing Co 2÷ (cobalt chloride) or in situ administration of KYN (sodium kynurenate) were tested (test probe). Based on previous dose-response studies, the smallest perfusate concentration of Co 2÷ or KYN which demonstrated near maximum effects on [K+]e were employed (10 mM Co 2÷, n = 9, or 10 mM KYN, n = 9). The test probe was perfused with Ca2+-free perfusate containing Co 2+ or a perfusate containing KYN throughout the experiment. The dialysis through the other probe served as the control (control probe). At 20 min after initiation of the 14C-sucrose perfusion, the perfusate was switched to K'-free Ringer solution without 14C-sucrose, and measurements of the K + and ~4C-sucrose concentrations of each dialysate ([K+]d and [J4C-sucrose]d) were initiated. Details concerning the characteristics of the estimation of [K+]e with K +free dialysis have been given elsewhere t2. The osmolarity of each perfusate was maintained constant by adjusting the concentration of sodium chloride. The K + concentration and the radioactivity of each dialysate fraction were measured with a K+-sensitive electrode and a scintillation counter, respectively. Cerebral ischcmia was induced by decapitation following initiation of the measurements of [K+]d. The temperature of the perfusate was set at 37°C, and this temperature was maintained by using

a chamber filled with water at 37°C in which the whole length of the sections of inlet tubing was placed. The dialysate fractions were collected at 1-min intervals. The length of the outlet tube was adjusted to a length which resulted in the dead space of the probe and tubing being 5.0 ill. Thus, there was a 1-min delay between the changes observed in the dialysate and the actual changes occurring within the brain. The brain was removed at 35 min after termination of the 14C-sucrose perfusion and processed for autoradiography. RESULTS F o l l o w i n g t e r m i n a t i o n of the L4C-sucrose perfusion, [~4C-sucrose]d d e c r e a s e d rapidly during an initial p e r i o d of a few m i n u t e s and d e c r e a s e d slowly t h e r e a f t e r (Fig. 1). T h e r e was no effect of C a 2 * - f r e e p e r f u s a t e containing C o 2÷ (10 m M ) o r in situ a d m i n i s t r a t i o n of K Y N (10 m M ) t h r o u g h the dialysis p r o b e on this d e c a y curve. T h e d e c a y c u r v e b e f o r e ischemia induction reflected the condition of the 14C-sucrose p e r f u s i o n which was later confirmed by a u t o r a d i o g r a p h y .

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s h o w i n g typical d e c a y curves. A u t o r a d i o g r a m s of these animals d e m o n s t r a t e d that an a r e a of the h i p p o c a m p u s and o v e r l y i n g c e r e b r a l c o r t e x , a p p r o x i m a t e l y 1.5 m m distant f r o m the p r o b e , was p e r f u s e d by t4C-sucrose (Fig. 2). As the t i m e of ischemia induction, 17 min following the t e r m i n a t i o n of the t4C-sucrose p e r f u s i o n was c h o s e n , since by this time the slope of the c u r v e b e c a m e less steep. A s u d d e n and m a r k e d increase in [K+]o was o b s e r v e d at I - 3 min after the induction of c e r e b r a l ischemia (Fig. 3 A ) . [t4C-sucrose]d invariably increased c o n c o m i t a n t l y

123

Fig. 2. Representative example of autoradiography of the rat brain perfused with 14C-sucrose for 20 min through the dialysis probe placed in the hippocampus (left). The same autoradiography was superimposed, using a computer, on the adjacent slice processed by thionine staining (right). CC, corpus callosum; LH, lateral hypothalamus; MH, medial habenular nucleus. The arrow indicates the tip of the dialysis probe.

with this increase in [K+]d, usually remaining elevated for 2-4 min and decreasing thereafter (Fig. 3B). The largest increase as compared to the preceding fraction in each animal was noted mostly at the fraction which demonstrated the largest increase in [K+]d . The difference was at best one fraction. [~4C-sucrose]d increased to a level which varied from 1.2- to 1.9-fold the concentration in the preceding fraction (1.4 + 0.1-fold, m + / 1.8

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Fig. 4. Representative examples of changes in {14C-sucrose]d during cerebral ischemia, showing the effect of Ca2+-free perfusate containing Co 2+ (10 mM). Filled circles, control probe: open circles, test probe perfused with Ca2+-free perfusate containing Co 2÷ (10 mM). Data from 3 representative experiments (3 pairs of control and test probes) have been superimposed.

icantly delayed by dialysis with Ca2+-free perfusate containing Co 2+ (10 mM). Thus, the latency of the fraction which displayed the largest increase (L,) as compared to the immediately preceding fraction was significantly prolonged (P < 0.05, n = 9, Fig. 4 and Table I). The latency was also prolonged by in situ administration of KYN (10 mM) (P < 0.05, n = 9, Fig. 5 and Table I).

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