Co-variation of free amino acids in brain interstitial fluid during pentylenetetrazole-induced convulsive status epilepticus

Brain Research 764 Ž1997. 230–236

Short communication

Co-variation of free amino acids in brain interstitial fluid during pentylenetetrazole-induced convulsive status epilepticus a, )

GianPietro Sechi

, Giulio Rosati a , Giovanni A. Deiana a , Valentino Petruzzi b , Franca Deriu a , Paola Correddu a , Pier Luigi De Riu c a

Neurological Clinic, UniÕersity of Sassari, Viale S. Pietro 10, 07100 Sassari, Italy Veterinary Surgery Department, UniÕersity of Sassari, Via Vienna 2, 07100 Sassari, Italy Chair of Neurological Rehabilitation, UniÕersity of Torino, Via Zuretti 29, 10126 Torino, Italy b

c

Accepted 8 April 1997

Abstract Effects of pentylenetetrazole ŽPTZ.-induced convulsive status epilepticus on free amino acids changes in venous blood, CSF and interstitial fluid ŽIF. of the brain were examined in dogs. A volume of brain IF sufficient for analysis was obtained by chronically implanted tissue cages. The onset of PTZ-induced convulsive seizures seemed mainly related to a marked increase of glutamate, aspartate, taurine, glycine and phosphoserine while, the maintenance and frequency of seizures seemed related to a marked increase of serine and glycine, in combination with a moderate rise of glutamate. L-a-Aminoadipate was recovered in moderate amount in epileptic brain IF, while, in controls, this compound was present in minimal amount. The observed complex temporal variation of the amino acidic pattern may play a role in PTZ-induced seizures and, possibly, in pharmacological kindling and brain structural alterations induced by PTZ. q 1997 Elsevier Science B.V. Keywords: Free amino acid; Brain interstitial fluid; Pentylenetetrazole

Pentylenetetrazole ŽPTZ. is a chemical convulsant, often used in high doses to generate clonic or tonic-clonic convulsive seizures in animals and man w62x. Its mechanism of action is only partially understood. A selective antagonism of GABA-mediated post-synaptic inhibition has been suggested w34,61x or an impairment of GABArbenzodiazepine-coupled chloride channel activity w6x, but pharmacologic antagonists to benzodiazepines do not appear able to block seizures from PTZ, w5x and also has been suggested a direct effect of this compound on excitability and stability of neuronal cell membranes w7,26x. Moreover, recent experimental evidences indicate that the selective activation of some cortical areas may play a role w51x. Previous studies, on the other hand, suggested for PTZ, preferentially, a subcortical locus of action w37,57x. In addition, studies in vitro, w8x and through selective N-methyl-D-aspartate ŽNMDA. receptor agonists and antagonists indicate that the excitatory amino acids probably contribute to the convulsive effects of PTZ

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Corresponding author. Fax: q49 Ž79. 22-8423.

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 8 7 - 3

w13,16,17,50,58x. Their contributions, however, seem to be indirect, and related to the reduction by PTZ of the inhibitory GABAergic input w8,62x. To further elucidate the mechanism of action of PTZ, as a convulsant, we evaluated, in adult mongrel dogs of both sexes, the contemporary variation in time Žco-variation., in relation to convulsive behavior, of free amino acids levels in plasma, cerebrospinal fluid ŽCSF., and interstitial fluid ŽIF. of the brain. The animals, anesthetized with sodium pentobarbital Ž40 mgrkg intravenous Ži.v.., with supplementary doses given as required later., were divided into two groups. Group A, rendered epileptic till development of convulsive generalized status by administering PTZ I.V. at 200–300 mgrkg. Group B, anesthetized with sodium pentobarbital only. Throughout the experiments the acidbase status and blood gases in arterial blood, blood pressure, rectal temperature, and blood glucose concentrations were intermittently checked. After PTZ administration, the animals were observed closely with respect to signs of generalized clonic, or tonic-clonic seizure activity. The frequency of generalized seizures were recorded and related to time after PTZ administration. Seizure activity was monitored visually and

G. Sechi et al.r Brain Research 764 (1997) 230–236

by means of a Grass 8-channel electroencephalograph, for at least 60 min after PTZ administration. Lateral and medial electrodes were placed in both the right and left cranium. Recordings from all areas were performed with reference to an electrode glued to the midline of the posterior occipital-cervical area. Simultaneous samples of venous blood, CSF and brain IF were taken, at different times Ž5, 10, 30 and 60 min., after the appearance of convulsive status, in each animal, between 9.30 h and 11.00 h, after 12 h of fasting and overnight sleep. In each group, different dogs Ž2–3. were used at each time. A volume of whole-brain IF sufficient for analysis Žabout 0.2 ml. was obtained through a sterilized, hollow, multiperforated, polypropilene sphere, approx. 10 mm in diameter, implanted for 4 weeks into the left parieto-temporal region. These capsules, also known as ‘tissue cages’, are similar to those used by Guyton et al. for measurement of negative IF pressures, w18x and to those used by Waterman and Kastan for measurement of antibiotic levels in IF w60x. Recently, we applied this technique to study the biodisposition of phenytoin in brain IF, w47x and to study the physicochemical properties of the wholebrain IF w48x. CSF samples were obtained through a 21-gauge needle positioned in the subarachnoid space of the foramen magnum. Brain IF samples were withdrawn from the implanted capsule by a small syringe with a fine needle. Specimens of plasma, CSF Ž1 ml. and brain IF Žf 0,2 ml. were deproteinized within 1 h of collection with sulphosalicylic acid, centrifuged and the supernatant analysed immediately or stored at y708C prior to analysis. Amino acid analysis was carried out using the Beckman System Gold TM HPLC, a system optimized for the analysis of free amino acids. The system utilizes ion-exchange chromatography incorporating post-column reaction with ninhydrin and subsequent detection in the visible region of the spectrum. The concentrations of the following amino acids were comparatively evaluated in plasma, CSF and brain IF in both groups: phosphoserine, taurine, aspartate, hydroxyproline, threonine, serine, asparagine, glutamate, glutamine, aaminoadipate, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine and lysine. Data were analyzed by Student’s t-test for paired samples between mean values " Standard Deviation ŽS.D.., at the various times, and the level of significance was set at P - 0.05. In group A, 3–5 min after PTZ injection in all the animals the epileptic behavioral manifestations gradually progressed from clonic seizures to tonic-clonic seizures. The frequency of seizures was variable: in the first 30 min the animals had 1–4 clonic seizures every 5 min, with intermixed tonic-clonic seizures Ž2–4.. The frequency of clonic seizures and tonic-clonic seizures decreased progressively from 30 to 60 min. The EEG changes, following I.V. PTZ injection, consisted of isolated bilateral spikes within the first 30 s, followed by bursts of spikes, more

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prolonged during the clonic seizures, separated by variable periods of EEG suppression. During the tonic-clonic seizures, paroxysmal generalized repetitive spike discharges, which lasted 50–60 s, were observed. There were no seizures in the control animals. Moreover, in group A, during the epileptic status neither hypoglycemia nor significant relative hypotension, in comparison with the baseline values, were noticed. A mild significant hypoxemia Ž pO 2 , from 104 " 4 to 92 " 5, P - 0.05., in combination with a significant plasma acidosis ŽpH, from 7.4 " 0.02 to 7.15 " 0.05, P - 0.005., was observed at 30 and 60 min. In addition, at 10 and 30 min, a significant rise in rectal temperature, in comparison with the baseline values, was noticed Ž8C, from 36.5 " 0.3 to 37 " 0.05, P - 0.05.. During the first 10 min of convulsive seizures, in the brain IF of group A, with respect to group B, was noticed a significant increase of the concentrations of glutamate Ž12–50-fold., aspartate Ž14–33-fold., phosphoserine Ž6.5– 11-fold., glycine Ž3–15-fold.. taurine Ž5.5–8.5-fold. and glutamine Ž3–5.5-fold.. a-Aminoadipate not detectable in the brain IF of group B, had values of 18 " 5 mMrl at 5 min and of 16 " 3 mMrl at 10 min, in the brain IF of group A. At 10 min taurine was significantly higher Ž P 0.05. in plasma of group A, with respect to group B. The levels of amino acids in the CSF of group A did not differ significantly from that of group B. However, there was a trend towards decreased levels, in the CSF of group A, with respect to group B, of phosphoserine, aspartate, threonine, OH-proline, serine, glutamine, glycine, alanine, methionine, isoleucine, leucine, tyrosine, phenylalanine and lisine. At 30 min, except for taurine, the concentrations of all other amino acids increased significantly in the brain IF of group A, with respect to group B. Phosphoserine, aspartate, OH-proline, threonine, asparagine, glutamine, alanine, valine, methionine, tyrosine, ornithine and lysine increased 2–4-fold, serine, glutamate, isoleucine, leucine, phenylalanine 5–9-fold, and glycine up to 20-fold. aAminoadipate not detectable in the brain IF of group B, had values of 8.9 " 2 mMrl in brain IF of group A. The concentration of OH-proline and glycine were significantly higher Ž P - 0.02 to P - 0.01. in plasma of group A, with respect to group B. In the CSF of group A, the concentrations of glycine and glutamate, were significantly higher Ž P - 0.01. with respect to the values in group B. The concentrations of other amino acids in the CSF, did not differ significantly in the two groups. In the brain IF of group A, at 60 min the concentrations of glutamate, isoleucine and ornithine increased significantly, 4–6-fold, and glutamine, glycine, valine, leucine and lysine 2–3.5fold. The concentrations of glutamine and ornithine increased significantly Ž P - 0.001. also in plasma of group A dogs, with respect, to group B. Moreover, in the CSF of group A, glutamate and ornithine concentrations increased significantly Ž P - 0.001., with respect, to group B. The temporal course of glutamate and aspartate concentrations in the CSF of group A dogs, with respect to group B, is

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Fig. 1. Time course of CSF concentrations of L-a-glutamate and L-a-aspartate in dogs with PTZ-induced convulsive SE ŽI—I. and in controls Ž`—`.. Two to three animals were used at each time. Bars are SD. ) P - 0.005.

shown in Fig. 1. Complex amino acid changes in the CSF of rats following PTZ-induced convulsions have been reported by Halonen et al. w19x. This study is the first report in vivo on the complex extracellular changes of free amino acid levels during experimental generalized convulsive status epilepticus induced by PTZ. In addition, for the first time, in this study, the sampling of brain IF in experimental epilepsy has been made through chronically implanted tissue cages. This method does not permit frequent sampling, but limits the occurrence of acute damage of various cellular structures, owing to implantation of a probe, such as in intracranial dialysis, w9,54x and it also permits to collect whole-brain IF for physicochemical studies w48x. A serious criticism to this method is that it has the disadvantage of measuring cage fluid and not true interstitial fluid. In this paper, we used the terms brain IF rather than brain cage fluid, since our previous findings on this experimental model indicated that this cage fluid is strongly representative of the physiological brain IF w48x. In this series of measurements, the temporal profile of the changes of amino acid levels, in relation to convulsive behavior, shows that there is a dramatic increase of the amino acid neurotransmitters glutamate and aspartate in brain IF, during the first 10 min of convulsive seizures induced by PTZ ŽFig. 2.. From moderate to marked elevations of extracellular phosphoserine, glycine, taurine and glutamine were also found. Through microdialysis, a similar increase of glutamate in brain IF has also been found in several animal models of focal epilepsy Že.g., intracerebral application of cobalt, folate, kainate, and in electrically kindled amygdala., w11,31,41,59x but not during seizures induced by systemic kainate, bicuculline w30x and picrotoxin w36x. In addition, either by intracranial dialysis, or by push-pull cannulation, during intraoperative recording, an increase in brain IF aspartate and glutamate has been found in human brain during focal spontaneous seizures, w2,12x or following electrical stimulation w10x.

Glutamate and aspartate are synthesized from glucose and other precursors by several biochemical routes within brain w35x. Moreover, metabolic studies have revealed the existence of several different pools or compartments for both glutamate and aspartate w14x. In our study, the dramatic increase of these neurotransmitters amino acids in brain IF during the first 10 min of seizures could be due either to excessive synaptic release, or to activation of different biochemical pathways, in both neuronal cells or glia w23x. For glutamate, in particular, the simultaneous moderate rise of glutamine in brain IF, indicates that

Fig. 2. Time course of brain IF concentrations of glutamate, aspartate, glycine and taurine in dogs with PTZ-induced convulsive SE ŽI—I. and in controls Ž`—`.. Two to three animals were used at each time. Bars are SD. ) P - 0.005; ) ) P - 0.02; ) ) ) P - 0.05.

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elevation of this amino acid content may partly result from increased detoxification of ammonia in the pathway from a-ketoglutarate through glutamate to glutamine w40x. The finding that epileptic seizures are accompanied by an increase in brain ammonia concentration fits this possibility w3x. Moreover, a putative presynaptic mechanism of action of PTZ as a convulsant is an increase in the amount of excitatory transmitter, released due to removal of recurrent inhibition on excitatory pathways brought about by GABA w62x. Therefore, in our study, a contributing factor for the observed increase of glutamate in brain IF during PTZ convulsive seizures may have been the blockade of GABA receptors presynaptically located on these excitatory neurons. Recently, increased content of glycine, w21x taurine and glycine, w44x alanine, glycine and phosphoethanolamine, w20x or glutamate, glycine and aspartate w40,49x in epileptogenic regions Žin interictal state. of human brain has been reported, as also a marked increase of glutamate, aspartate, serine and glycine in association with human focal seizures w3x. The increased glycine concentration in epileptogenic human brain confirms the earlier findings of Van Gelder et al. w55x. Taurine is thought to be an inhibitory neurotransmitter or neuromodulator w42x. An increase in extracellular cerebral taurine has been reported during acute status epilepticus after kainic acid injected into rabbit hippocampus, w29x and in rats during seizures induced by systemic bicuculline w4x. The rise of taurine content in epileptic brain IF in the first 10 min, in our experimental model ŽFig. 2., could represent a defence mechanism because this compound has been shown to have antiepileptic properties w56x. A similar mechanism has been postulated by Benveniste et al. w1x in cerebral ischemia. Our data indicate that there is a strictly complex relationship between phosphoserine, serine and glycine content in epileptic brain IF. ŽFig. 2, Fig. 3. Indeed, during the first 10 min of convulsive seizures, there is a marked increase of phosphoserine, while at 30 min the phosphoserine levels fall and a marked rise in serine and glycine

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content is observed. Since studies using radioactive precursors suggest that much of the serine in brain is derived from de novo synthesis from glucose through phosphoserine, w35x and glycine from serine, with which it is interconvertible, w35x it is likely that the activation of these metabolic pathways may play an important role in seizure activity induced by PTZ. The finding of a 3–6-fold increase in the rate of glycolysis during epileptic seizures fits this possibility w3x. Glycine is now known to greatly potentiate the response to excitatory amino acid agonists, via action on the glycine modulatory site of the NMDA receptor w25,53x. Thus, elevated brain IF glycine levels at 30 min in our epileptic model, in combination with a moderate rise in glutamate concentrations, may explain the persistence of convulsive seizures from this time onwards. Together with the major changes so far discussed, at 30 and 60 min, in the epileptic brain IF we found less pronounced changes for most of the other amino acids considered Žabout 1–5 times the control levels.. In our opinion, this finding may be unrelated to specific metabolic changes and, possibly, due either to a substantial decrease Ž20–30%. of the extracellular space during seizure activity, w22x or to a breakdown of the blood–brain barrier induced by PTZ seizures w32x. In agreement with Ronne-Engstrom et al. w43x until the net effect of these phenomena are better understood, we regard any increase in brain IF concentrations of amino acids below a factor of two as potentially insignificant in terms of specific metabolic changes due to PTZ convulsive seizures. A new and intriguing finding in our study is the discovery that L-a-aminoadipic acid is recovered in moderate amounts in epileptic brain IF. In brain IF of controls, instead, this compound is not detectable or present in minimal amounts. ŽFig. 4. L-a-aminoadipic acid, a sixcarbon homologue of glutamate, is derived from lysine and it is a natural metabolite of brain w52x. This amino acid is now known to have gliotoxic action at micromolar concentrations and, partly, neurotoxic effects on different regions of the central nervous system

Fig. 3. Time course of brain IF concentrations of phosphoserine and serine in dogs with PTZ-induced convulsive SE ŽI—I. and in controls Ž`—`.. Two to three animals were used at each time. Bars are SD. ) P - 0.005; ) ) P - 0.02.

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Fig. 4. Time course of brain IF concentrations of L-a-aminoadipate in dogs with PTZ-induced convulsive SE ŽI—I. and in controls Ž`—`.. Two to three animals were used at each time. Bars are SD. ) P - 0.005.

w15,38x. In our study, the rise of a-aminoadipate content in epileptic brain IF parallels that of glutamate, Ži.e., it is greater in the first 10 min of convulsive status. thus, it is likely that it may be involved together with glutamate, in neuronal cells damage and astrocytic swelling observed in the brain during sustained PTZ seizures w24x. Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter w46x. In convulsive status epilepticus induced by PTZ, failure of astrocytic glutamate uptake, due to a-aminoadipate toxicity, might enhance dramatically the neurotoxicity of glutamate. Moreover, it is noteworthy that after exposure to toxic levels of a-aminoadipate the functional impairment of glial cells can last even two months w27x. Consequently, after PTZ treatment, the extracellular glutamate concentration and its synaptic action might remain enhanced for the same period. A similar mechanism might play a role in pharmacological kindling induced by repeated doses of PTZ w33x and, perhaps, in other models of epileptogenesis. The finding that in rats pretreatment with MK-801, an antagonist of the NMDA receptor, completely prevents the convulsions, the kindling process and changes in biochemical parameters induced by PTZ fits this possibility w16,17x. Moreover, the brain IF content of amino acids and in particular of glutamate and aspartate, may be influenced by several factors including ischemia w1x and hypoglycemia w45x. In our experiments, the dogs convulsed freely, however neither hypoglycemia, nor significant relative hypotension were noticed. In all animals, instead, mainly after 30 min of convulsions, a mild arterial hypoxemia occurred, likely due to vascular or pulmonary problems. Hypoxia without ischemia is well tolerated by brain w39x. Moreover during recurrent seizures, it is known that the oxygen insufficiency seems to occur only after several seizures, when a transition from an increase in cortical

pO 2 to a decrease in cortical pO 2 took place w28x. Since in our experimental model the dramatic increase of glutamate and aspartate occurs in the first 10 min of the epileptic status, a significant influence of ischemia or hypoxemia on our results seems unlikely. In conclusion, this investigation shows that an enhanced excitatory neurotrasmission may play a role in the action of PTZ as a convulsant. During PTZ-induced convulsive status epilepticus, in brain IF, a complex temporal variation of the amino acidic pattern occurs. In particular, the onset of convulsive seizures seems mainly related to a marked increase of glutamate, aspartate, taurine and fosphoserine; while the maintenance and frequency of seizures seemed related mainly to a marked increase of glycine in combination with a moderate rise of glutamate. Furthermore, the recovery of moderate amount of a-amino-adipate in epileptic brain IF suggests a role for this compound, together with glutamate, both in PTZ seizure related brain damage and in the pharmacological kindling induced by repeated doses of PTZ.

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