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The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection
Neuroscience Letters, 90 (1988) 208 212 Elsevier Scientific Publishers Ireland Ltd
208
NSL 05447
The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection Peter Andin61, Anders L e h m a n n 1, Katarina Ellr6n 1, Elisabet Wennberg 2, Ingemar Kjellmer3, Thorkild Nielsen4 and Henrik Hagberg !'4 Ilnstitute of Neurobiotogy, 2Department of Anesthesiology and 3Department of Pediatrics, University of G6teborg, G6teborg (Sweden) and 4Department of Gynecology and Obstetrics, Borhs Hospital, Bor~s (Sweden) (Received I February 1988; Revised version received 5 April 1988; Accepted 5 April 1988)
Key words: Excitatory amino acid; Kynurenic acid; Hypoxia; Ischemia; Neuroprotection; Neonatal The neuroprotective effect of kynurenic acid, an unspecific antagonist of excitatory amino acid receptors, was evaluated in a model of hypoxic-ischemia in neonatal rats. One-week-old rats were subjected to ligation of the left carotid artery and exposure to 7.7 % 02/92.3 % N2 for 2 h. Kynurenic acid (300 mg/kg) was administered i.p. immediately after the period of hypoxic-ischemia in one group (n = 32) and compared with saline-treated (n =27). After 2 weeks the rats were sacrificed and the brain damage evaluated by comparing the weight of the lesioned and unlesioned hemispheres. In rats receiving kynurenic acid the reduction in weight of the lesioned hemisphere was 25.4_+3.3% as compared to 37.8_+3.6% in salinetreated controls (P<0.001). The results suggest that excitatory amino acids are involved in the development of postischemic damage in the immature brain.
Experimental data suggest that excitatory amino acids (EAA) are involved in the development of ischemic neuronal degeneration in adult animals [15, 18]. There is, e.g., a pronounced increase of extracellular EAA in the brain during ischemia [1, 12]. Interruption at any point of the trisynaptic pathway of the hippocampus [14] or administration of an N-methyl-D-aspartate (NMDA) receptor antagonist [8] attenuates ischemic neuronal damage in the CA I subfield. Recent observations suggest a role for EAA in the genesis of ischemic damage also in the immature brain: there is a high density of glutamate receptors in the basal ganglia of newborn infants and immature rats [10] and quisqualate and N M D A are potent excitotoxins in the 7-day-old rat brain [5, 21]. Glutamate and aspartate increase multi-fold during hypoxic-ischemia in the brain of immature lambs [11]. Ischemic damage in the newborn rat is preceded by a loss of glutamate receptors [22]. Recently it was also shown that MK-801, a non-competitive N M D A receptor antagonist, Correspondence: H. Hagberg, Institute of Neurobiology, P.O. Box 33031, 40033 G6teborg, Sweden. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
209
administered to 7-day-old rats before and during hypoxic-ischemia attenuates the brain damage [17]. In the clinical setting it is difficult to predict and pretreat cases of fetal asphyxia and it is therefore of interest to evaluate the effect of treatment with EAA-antagonists after the ischemic insult. The effect of post-ischemic treatment with kynurenic acid (Kyn), an unspecific antagonist of EAA receptors, was studied. The modified Levine model [16, 19] was used for induction of unilateral hypoxic-ischemia which allows long-term survival after the insult. In most animals unilateral damage is seen in the striatum, cortex and hippocampus [19]. Brain damage was estimated by weighing the hemispheres. This simple way of evaluating the damage [17] was used since loss of brain weight in this model shows a close relationship to morphometric and electrophysiologic criteria of brain damage (Andin6 et al., in preparation). Seven-day-old rats of either sex (n = 63) were anesthetized with halothane (2% for induction and 0.8% for maintenance) in O2/N20, 30%/70%. The left carotid artery was exposed through a midline incision, ligated with a 9~0-prolene suture and electrocauterized. After closure of the wounds the pups were returned to their dam and left to recover for at least 2 h. Each litter (9 12 rats) was then exposed to 7.70 ± 0.01% 02 in N? for 2h at 36°C. Immediately after the 2-h-period of hypoxic-ischemia, Kyn was administered i.p. (300 mg/kg, 1.6 mmol/kg; dissolved in saline with NaOH and pH adjusted with HC1; injection volume: 0.10-0.15 ml) to half of the pups of each litter ('hypoxic-ischemia + kynurenic acid'; n=33). The other pups recieved 0.10 0.15 ml of saline i.p. ('hypoxic-ischemia controls'; n=30). The pups were weightmatched in pairs within each litter and the statistical evaluation was made between the 27 surviving pairs using Student's t-test. At 3 weeks of age the rats were sacrificed and the brain was displaced from the skull. The cerebellum and the brainstem including the mesencephalon were removed, a midline incision was made separating the two TABLE I THE MEAN LOSS OF W E I G H T OF L E S I O N E D HEMISPHERES, THE N U M B E R OF A N I M A L S (n) A N D THE N U M B E R OF DEATHS A F T E R I t Y P O X I C - I S C H E M I A (MORTALITY) IN EACH LITTER OF THE H Y P O X I C - I S C H E M I C C O N T R O L S A N D H Y P O X I C - I S C H E M I C RATS T R E A T E D WITH K Y N U R E N I C ACID The hemispheric loss of weight is given as % reduction + S.E.M. Litter no.
Hypoxic-ischemia Mean
S.E.M.
1 2 3 4 5 6
32.9 23.7 49.9 32.3 57.9 32.0
6.0 7.2 5.0 12.9 5.5 7.2
Total
37.8
3.6
Hypoxic-ischemia + kynurenic acid (300 mg/kg) n
Mortality
Mean
S.E.M.
n
Mortality
4 5 5 4 4 5
0 0 l 1 0 1
7.2 l 1.4 35. l 34.6 48.4 16.1
1.9 7.7 4.9 6.5 4.2 4.2
5 5 5 6 5 6
0 0 1 0 0 0
27
3
25.4
3.3
32
1
210
J
i
Fig. 1. Seven-day-oldrats were exposed to hypoxic-ischemia(unilateral carotid artery occlusion and 2 h of hypoxia). After 2 weeks the animals were sacrifiedand the brains (A,B) were examined. A: typical unilateral brain damage in a rat recievingsaline after the period of hypoxic-ischemia. B: the brain of an animal that recieved kynurenic acid (300 mg/kg, i.p.) immediatelyafter the hypoxic-ischemicperiod demonstrating a significant reduction of the brain damage.
forebrain hemispheres and they were weighed on a high precision balance (Mettler, sensitivity: ___0.1 mg). The brain damage for each animal was expressed as % reduction of weight in lesioned compared to the contralateral unlesioned hemisphere. Control animals subjected to left carotid ligation without exposure to hypoxia exhibited no brain damage (0.0 + 0.2 %; n = 7). The weights of the animals in the two groups were similar on the day of hypoxic exposure and sacrifice (Kyn group: 12.6+0.3 and 33.7+ 1.0; saline-treated group: 12.5 + 0.35 and 33.9 + 1. i). The unlesioned hemispheric weight was 471 + 9 mg in the Kyn-treated group compared to 484___ 8 mg in the saline-treated group (not statistically significant). The respiratory frequency and the general behaviour after treatment did not differ between the groups. There was a somewhat higher mortality in the saline-treated group compared to the Kyn-treated group (not significant; Table I). The mean hemispheric weight loss was 25.4+ 3.3% in Kyn-treated rats which was lower (P < 0.001" paired t-test) compared to saline treated controls 37.8 + 3.6% (Fig. I). The data for each litter are presented in Table I. Kyn given immediately after the 2-h period of hypoxic-ischemia thus offers a significant but not complete neuroprotection which supports the excitotoxic hypothesis of cell death in the immature brain. Kyn is a tryptophane metabolite endogenous to the brain [25], and considering its rather lipophilic structure, Kyn would be expected to cross the blood--brain barrier [25]. The exact concentration of Kyn reached at the receptor sites in the brain after an i.p. injection is unknown. However, concentrations high enough to antagonize EAA receptors are most probably attained, cf. concentrations as low as 200/tmol/1 of Kyn reduce EAA excitation by 50 ?6 [3]. In addition, the same dose of Kyn as used in the present study, administered i.p., reduces postischemic edema in neonatal rats [23] and decreases stroke size in adult rats [7]. Kyn is an EAA receptor antagonist with broad spectrum, i.e. it blocks the electrophysiologic action of kainate, N M D A and quisqualate [13, 25]. The neurotoxic effects of kainate and N M D A are also inhibited by Kyn [6]. Because of
211
these properties Kyn was the EAA-receptor blocker of choice for our purposes considering the high density of EAA-receptors in the 7-day-old rat brain which probably are of the quisqualate and NMDA type [5, 10, 21]. Pretreatment with MK-801 has been shown to attenuate ischemic damage in neonatal rats [17]. Concerning treatment after iscbemia, postischemic deafferentation [14] or postischemic administration of D-2-amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist [2], limit pyramidal damage in CAI of the adult hippocampus. The present study shows that treatment given in the posthypoxic-ischemic phase only, attenuates brain damage also in neonatal rats. The mechanisms behind this neuroprotection remain obscure. It is known that the content of EAA increases extracellularly during ischemia in immature lambs [11] but the levels normalize within 15 min of reflow similar to results obtained from the adult rat and rabbit brain [1, 12]. Short exposures of nervous tissue to EAAs could induce cell death with delayed appearance [4]. Such a delayed brain damage may be enhanced by electrical 'hyperactivity' in the postischemic neonatal brain with resultant neurotoxic effects [24]. These processes during the postischemic period may be inhibited by Kyn, e.g. EAA antagonists administered to neuronal cultures aJ?er the exposure with EAA reduce the excitotoxic cell injury [20]. Kyn may have other effects not related to excitotoxicity. Information is lacking if Kyn for instance affect hemodynamics, pH regulation and respiratory control. This study was supported by the Swedish Medical Research Council (2591, 12x00164, 17x-05416), Barnhusfonden, F6reningen Margaretahemmet, Ragnar och Torsten S6derbergs stiftelser, Laerdals foundations, Tore Nilsons fond, Wibergs stiftelser and Bergvalls stiftelser. Thanks to Prof. Anders Hamberger for support and valuable criticism.
I Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippoeampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369 1374. 2 Boast, C.A., Gerhardt, S.C. and Janak, P., Systemic AP7 reduces ischemic brain damage in gerbils. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1986, pp. 249 252. 3 Cotman, C.W., Flatman, J.A., Ganong, A.H. and Perkins, M.N., Effects of excitatory amino acid antagonists on evoked and spontaneous excitatory potentials in guinea-pig hippocampus, J. Physiol. (Lond.), 378 (1986) 403~.15. 4 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A.R., Glutamate neurotoxicity in cortical cell cul ture, J. Neurosci., 7 (1987) 357 368. 5 Foster, A.C., Collins, J.F. and Schwarcz, R., On the excitotoxic properties of quinolinic acid, 2,3piperidine dicarboxylic acids and structurally related compounds, Neuropharmacology, 22 (1983) 1331 1342. 6 Foster, A.C., Vezzani, A., French, E.D. and Schwarcz, R., Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid, Neurosci. Lett., 48 (1984) 273 278. 7 Germano, I.M., Pins, L.H., Meldrum, B.S., Bartkowski, H.M. and Simon, R.P., Kynurenate inhibition of cell excitation decreases stroke size and deficits, Ann. Neurol., 22 (1987) 730 734.
212 8 Gill, R., Foster, A.C. and Woodruff', G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343 3349. 9 Greenamyre, J.T., The role of glutamate in neurotransmission and in neurologic disease, Arch. Neurol., 43 (1986) 1058 1063. 10 Greenamyre, T., Penney, J.B., Young, A.B., Hudson, C., Silverstein, F.S. and Johnston, M.V., Evidence for transient perinatal glutamatergic innervation of globus pallidus, J. Neurosci., 7 (1987) 1022 1030. 11 Hagberg, H., Andersson, P., Kjellmer, I., Thiringer, K. and Thordstein, M., Extracellular overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia, Neurosci. Lett, 78 (1987) 311 3t7. 12 Hagberg, H., Lehmann, A., Sandberg, M., Nystr6m, B., Jacobson, 1. and Hamberger, A., lschemiainduced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments, J. Cereb. Blood Flow Metab., 5 (19851413 419. 13 Herrling, P.L., Pharmacology of the corticocaudate excitatory postsynaptic potential in the cat: evidence for its mediation by quisqualate- or kainate-receptors, Neuroscience, 14 (1985) 417~126. 14 Johansen, F.F., J6rgensen, M.B. and Diemer, N.H., Ischemia induced delayed neuronal death in the CAI hippocampus is dependent on intact glutamatergic innervation. In T.P. Hicks, D Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1986, pp. 245248. 15 J6rgensen, M.B. and Diemer, N.H., Selective neuron loss after cerebral ischemia in the rat: possible role of transmitter glutamate, Acta Neurol. Scan&, 66 (1982) 536~546. 16 Levine, S., Anoxic-ischemic encephalopathy in rats, Am. J. Pathol., 36 (1960) 1 17. 17 McDonald, J.W., Silverstein, F.S. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359361. 18 Meldrum, B.S., Evans, M.C., Swan, J.tt. and Simon, R.P., Protection against hypoxic/ischaemic brain damage with excitatory amino acid antagonists, Med. Biol., 65 (1987) 153 157. 19 Rice, J.E., Vannucci, R.C. and Brierly, J.B., The influence of immaturity on hypoxic-ischemic brain damage in the rat, Ann. Neurol., 9 ( 1981) 131 141. 20 Rothman, S.M., Thurston, J.H. and Hauhart, R.E., Delayed neurotoxicity of excitatory amino acids in vitro, Neuroscience, 22 (1987) 471 480. 21 Silverstein, F.S., Chen, R. and Johnston, M.V., The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain, Neurosci. Lett., 71 (1986) 13-I 8. 22 Silverstein, F.S.. Torke, L., Barks, J. and Johnston, M.V., Hypoxia-ischemia produces local disruption of glutamate receptors in developing brain, Dev. Brain Res., 34 (1987) 33 39. 23 Simon, R.P., Young, R.S.K., Stout, S. and Cheng, J., Inhibition of excitatory neurotransmission with kynurenate reduces brain edema in neonatal anoxia, Neurosci. Lett., 71 (1986) 361 364. 24 Sloviter, R.S., "Epileptic" brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies, Brain Res. Bull., I0 (1983) 675 697. 25 Stone, T.W. and Connick, J.H, Quinolinic acid and other kynurenines in the central nervous system, Neuroscience, 15 (1985) 597 617.
208
NSL 05447
The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection Peter Andin61, Anders L e h m a n n 1, Katarina Ellr6n 1, Elisabet Wennberg 2, Ingemar Kjellmer3, Thorkild Nielsen4 and Henrik Hagberg !'4 Ilnstitute of Neurobiotogy, 2Department of Anesthesiology and 3Department of Pediatrics, University of G6teborg, G6teborg (Sweden) and 4Department of Gynecology and Obstetrics, Borhs Hospital, Bor~s (Sweden) (Received I February 1988; Revised version received 5 April 1988; Accepted 5 April 1988)
Key words: Excitatory amino acid; Kynurenic acid; Hypoxia; Ischemia; Neuroprotection; Neonatal The neuroprotective effect of kynurenic acid, an unspecific antagonist of excitatory amino acid receptors, was evaluated in a model of hypoxic-ischemia in neonatal rats. One-week-old rats were subjected to ligation of the left carotid artery and exposure to 7.7 % 02/92.3 % N2 for 2 h. Kynurenic acid (300 mg/kg) was administered i.p. immediately after the period of hypoxic-ischemia in one group (n = 32) and compared with saline-treated (n =27). After 2 weeks the rats were sacrificed and the brain damage evaluated by comparing the weight of the lesioned and unlesioned hemispheres. In rats receiving kynurenic acid the reduction in weight of the lesioned hemisphere was 25.4_+3.3% as compared to 37.8_+3.6% in salinetreated controls (P<0.001). The results suggest that excitatory amino acids are involved in the development of postischemic damage in the immature brain.
Experimental data suggest that excitatory amino acids (EAA) are involved in the development of ischemic neuronal degeneration in adult animals [15, 18]. There is, e.g., a pronounced increase of extracellular EAA in the brain during ischemia [1, 12]. Interruption at any point of the trisynaptic pathway of the hippocampus [14] or administration of an N-methyl-D-aspartate (NMDA) receptor antagonist [8] attenuates ischemic neuronal damage in the CA I subfield. Recent observations suggest a role for EAA in the genesis of ischemic damage also in the immature brain: there is a high density of glutamate receptors in the basal ganglia of newborn infants and immature rats [10] and quisqualate and N M D A are potent excitotoxins in the 7-day-old rat brain [5, 21]. Glutamate and aspartate increase multi-fold during hypoxic-ischemia in the brain of immature lambs [11]. Ischemic damage in the newborn rat is preceded by a loss of glutamate receptors [22]. Recently it was also shown that MK-801, a non-competitive N M D A receptor antagonist, Correspondence: H. Hagberg, Institute of Neurobiology, P.O. Box 33031, 40033 G6teborg, Sweden. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
209
administered to 7-day-old rats before and during hypoxic-ischemia attenuates the brain damage [17]. In the clinical setting it is difficult to predict and pretreat cases of fetal asphyxia and it is therefore of interest to evaluate the effect of treatment with EAA-antagonists after the ischemic insult. The effect of post-ischemic treatment with kynurenic acid (Kyn), an unspecific antagonist of EAA receptors, was studied. The modified Levine model [16, 19] was used for induction of unilateral hypoxic-ischemia which allows long-term survival after the insult. In most animals unilateral damage is seen in the striatum, cortex and hippocampus [19]. Brain damage was estimated by weighing the hemispheres. This simple way of evaluating the damage [17] was used since loss of brain weight in this model shows a close relationship to morphometric and electrophysiologic criteria of brain damage (Andin6 et al., in preparation). Seven-day-old rats of either sex (n = 63) were anesthetized with halothane (2% for induction and 0.8% for maintenance) in O2/N20, 30%/70%. The left carotid artery was exposed through a midline incision, ligated with a 9~0-prolene suture and electrocauterized. After closure of the wounds the pups were returned to their dam and left to recover for at least 2 h. Each litter (9 12 rats) was then exposed to 7.70 ± 0.01% 02 in N? for 2h at 36°C. Immediately after the 2-h-period of hypoxic-ischemia, Kyn was administered i.p. (300 mg/kg, 1.6 mmol/kg; dissolved in saline with NaOH and pH adjusted with HC1; injection volume: 0.10-0.15 ml) to half of the pups of each litter ('hypoxic-ischemia + kynurenic acid'; n=33). The other pups recieved 0.10 0.15 ml of saline i.p. ('hypoxic-ischemia controls'; n=30). The pups were weightmatched in pairs within each litter and the statistical evaluation was made between the 27 surviving pairs using Student's t-test. At 3 weeks of age the rats were sacrificed and the brain was displaced from the skull. The cerebellum and the brainstem including the mesencephalon were removed, a midline incision was made separating the two TABLE I THE MEAN LOSS OF W E I G H T OF L E S I O N E D HEMISPHERES, THE N U M B E R OF A N I M A L S (n) A N D THE N U M B E R OF DEATHS A F T E R I t Y P O X I C - I S C H E M I A (MORTALITY) IN EACH LITTER OF THE H Y P O X I C - I S C H E M I C C O N T R O L S A N D H Y P O X I C - I S C H E M I C RATS T R E A T E D WITH K Y N U R E N I C ACID The hemispheric loss of weight is given as % reduction + S.E.M. Litter no.
Hypoxic-ischemia Mean
S.E.M.
1 2 3 4 5 6
32.9 23.7 49.9 32.3 57.9 32.0
6.0 7.2 5.0 12.9 5.5 7.2
Total
37.8
3.6
Hypoxic-ischemia + kynurenic acid (300 mg/kg) n
Mortality
Mean
S.E.M.
n
Mortality
4 5 5 4 4 5
0 0 l 1 0 1
7.2 l 1.4 35. l 34.6 48.4 16.1
1.9 7.7 4.9 6.5 4.2 4.2
5 5 5 6 5 6
0 0 1 0 0 0
27
3
25.4
3.3
32
1
210
J
i
Fig. 1. Seven-day-oldrats were exposed to hypoxic-ischemia(unilateral carotid artery occlusion and 2 h of hypoxia). After 2 weeks the animals were sacrifiedand the brains (A,B) were examined. A: typical unilateral brain damage in a rat recievingsaline after the period of hypoxic-ischemia. B: the brain of an animal that recieved kynurenic acid (300 mg/kg, i.p.) immediatelyafter the hypoxic-ischemicperiod demonstrating a significant reduction of the brain damage.
forebrain hemispheres and they were weighed on a high precision balance (Mettler, sensitivity: ___0.1 mg). The brain damage for each animal was expressed as % reduction of weight in lesioned compared to the contralateral unlesioned hemisphere. Control animals subjected to left carotid ligation without exposure to hypoxia exhibited no brain damage (0.0 + 0.2 %; n = 7). The weights of the animals in the two groups were similar on the day of hypoxic exposure and sacrifice (Kyn group: 12.6+0.3 and 33.7+ 1.0; saline-treated group: 12.5 + 0.35 and 33.9 + 1. i). The unlesioned hemispheric weight was 471 + 9 mg in the Kyn-treated group compared to 484___ 8 mg in the saline-treated group (not statistically significant). The respiratory frequency and the general behaviour after treatment did not differ between the groups. There was a somewhat higher mortality in the saline-treated group compared to the Kyn-treated group (not significant; Table I). The mean hemispheric weight loss was 25.4+ 3.3% in Kyn-treated rats which was lower (P < 0.001" paired t-test) compared to saline treated controls 37.8 + 3.6% (Fig. I). The data for each litter are presented in Table I. Kyn given immediately after the 2-h period of hypoxic-ischemia thus offers a significant but not complete neuroprotection which supports the excitotoxic hypothesis of cell death in the immature brain. Kyn is a tryptophane metabolite endogenous to the brain [25], and considering its rather lipophilic structure, Kyn would be expected to cross the blood--brain barrier [25]. The exact concentration of Kyn reached at the receptor sites in the brain after an i.p. injection is unknown. However, concentrations high enough to antagonize EAA receptors are most probably attained, cf. concentrations as low as 200/tmol/1 of Kyn reduce EAA excitation by 50 ?6 [3]. In addition, the same dose of Kyn as used in the present study, administered i.p., reduces postischemic edema in neonatal rats [23] and decreases stroke size in adult rats [7]. Kyn is an EAA receptor antagonist with broad spectrum, i.e. it blocks the electrophysiologic action of kainate, N M D A and quisqualate [13, 25]. The neurotoxic effects of kainate and N M D A are also inhibited by Kyn [6]. Because of
211
these properties Kyn was the EAA-receptor blocker of choice for our purposes considering the high density of EAA-receptors in the 7-day-old rat brain which probably are of the quisqualate and NMDA type [5, 10, 21]. Pretreatment with MK-801 has been shown to attenuate ischemic damage in neonatal rats [17]. Concerning treatment after iscbemia, postischemic deafferentation [14] or postischemic administration of D-2-amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist [2], limit pyramidal damage in CAI of the adult hippocampus. The present study shows that treatment given in the posthypoxic-ischemic phase only, attenuates brain damage also in neonatal rats. The mechanisms behind this neuroprotection remain obscure. It is known that the content of EAA increases extracellularly during ischemia in immature lambs [11] but the levels normalize within 15 min of reflow similar to results obtained from the adult rat and rabbit brain [1, 12]. Short exposures of nervous tissue to EAAs could induce cell death with delayed appearance [4]. Such a delayed brain damage may be enhanced by electrical 'hyperactivity' in the postischemic neonatal brain with resultant neurotoxic effects [24]. These processes during the postischemic period may be inhibited by Kyn, e.g. EAA antagonists administered to neuronal cultures aJ?er the exposure with EAA reduce the excitotoxic cell injury [20]. Kyn may have other effects not related to excitotoxicity. Information is lacking if Kyn for instance affect hemodynamics, pH regulation and respiratory control. This study was supported by the Swedish Medical Research Council (2591, 12x00164, 17x-05416), Barnhusfonden, F6reningen Margaretahemmet, Ragnar och Torsten S6derbergs stiftelser, Laerdals foundations, Tore Nilsons fond, Wibergs stiftelser and Bergvalls stiftelser. Thanks to Prof. Anders Hamberger for support and valuable criticism.
I Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippoeampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369 1374. 2 Boast, C.A., Gerhardt, S.C. and Janak, P., Systemic AP7 reduces ischemic brain damage in gerbils. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1986, pp. 249 252. 3 Cotman, C.W., Flatman, J.A., Ganong, A.H. and Perkins, M.N., Effects of excitatory amino acid antagonists on evoked and spontaneous excitatory potentials in guinea-pig hippocampus, J. Physiol. (Lond.), 378 (1986) 403~.15. 4 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A.R., Glutamate neurotoxicity in cortical cell cul ture, J. Neurosci., 7 (1987) 357 368. 5 Foster, A.C., Collins, J.F. and Schwarcz, R., On the excitotoxic properties of quinolinic acid, 2,3piperidine dicarboxylic acids and structurally related compounds, Neuropharmacology, 22 (1983) 1331 1342. 6 Foster, A.C., Vezzani, A., French, E.D. and Schwarcz, R., Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid, Neurosci. Lett., 48 (1984) 273 278. 7 Germano, I.M., Pins, L.H., Meldrum, B.S., Bartkowski, H.M. and Simon, R.P., Kynurenate inhibition of cell excitation decreases stroke size and deficits, Ann. Neurol., 22 (1987) 730 734.
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