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A serotonin S2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil
Brain Research, 494 (1989) 387-390 Elsevier
387
BRES 23646
A serotonin S2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil Hideaki Fujikura, Hiroyuki Kato, Shinichi Nakano and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 18 April 1989) Key words: Cerebral ischemia; Repeated ischemia; Naftidrofuryl; Serotonin S2 antagonist; Gerbil
The effect of a serotonin S2 antagonist, naftidrofuryl, on ischemic neuronal damage was examined in the gerbil. Naftidrofuryl was injected i.p. 5 min prior to a single 5-min forebrain ischemia or immediately after each of three 2-min forebrain ischemic insults at 60-min intervals. In both groups the number of intact hippocampal CA1 neurons were significantly higher than in the saline-treated group. These results indicate that serotonin S2 antagonists have a protective effect against ischemic neuronal damage.
Since the report by Kirino et al. 9, ischemiainduced neuronal damage in the hippocampal CA1 sector has been known as 'delayed neuronal death', At present an excitatory neurotransmitter, glutamate, is considered to initiate the delayed neuronal death 15 and on the contrary, inhibitory neurotransmitters, G A B A 16 and adenosine 5, are known to ameliorate the neuronal damage. However, there has been no previous report on the serotonergic participation in the neuronal damage of the hippocampal CA1 sector. We investigated the effect of a serotonin S 2 antagonist, anftidrofuryi, on ischemic neuronal damage in the gerbil, Five-min forebrain ischemia in the gerbil has been well-known to destroy hippocampal CA1 neurons selectively. Recently Kato et al. 8 demonstrated that 2-min forebrain ischemia in the gerbil could not produce any neuronal damage but that 3 repeated insults of 2-min ischemia at 60-min intervals caused remarkable neuronal damage not only in the hippocampal CA1 sector but also in the dorsolateral crescent of the striatum, the thalamus and the substantia nigra. Repetitive transient ischemic attacks are c o m m o n clinical events and drug administration usually starts after the first ischemic insult,
Therefore, it is very important to study the effects of various compounds on the ischemia-induced neuronal damage using a repeated ischemia model. This is the first report on the application of a repeated ischemia model for the evaluation of protective effects of a compound against the neuronal damage. Male Mongolian gerbils, weighing 60-80 g, were used. They were anesthetized with a mixture of 2% halothane, 30% oxygen and 70% nitrous oxide and both c o m m o n carotid arteries were exposed. After anesthesia was discontinued and the animals regained consciousness, forebrain ischemia was induced by occlusion of the carotid arteries with Sugita No. 51 temporary aneurysm clips. The animals were divided into two groups. In the first group, a single 5-min ischemia was induced. Naftidrofuryl (50 mg/kg) or the same volume of saline was injected i.p. 5 min prior to the ischemia. In the second group, 3 episodes of 2-min ischemia were repeated at 60-min intervals. Naftidrofuryl (25 or 50 mg/kg) or saline was injected i.p. immediately after each ischemic insult. Seven days after the ischemic event, the animals were anesthetized with pentobarbital (40 mg/kg)i.p. and the brains were briefly washed by transcardiac
Correspondence: K. Kogure, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1, Seiryo-machi, Sendai 980, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
388 perfusion with heparinized saline followed by perfusion-fixation with 10% formalin for 20 min. The brains were removed and immersed in the same fixative until they were embedded in paraffin. Five-j~m-thick paraffin sections were obtained and stained with Cresyl violet or Hematoxylin/Eosin. The number of the pyramidal cells in the hippocampal CA1 sector per 1 mm linear length was counted under a light microscope. The average
Fig. 1. Representative photomicrographs of the hippocampal
number of right and left CAI neurons was taken as the neuronal density. Statistical analysis was carried out using the Mann-Whitney U-test. Representative photomicrographs of the hippocampal CA1 sector following 5-rain ischemia are shown in Fig. 1. The CA1 neurons in the salinetreated animals have nearly completely disappeared (Fig. lb). Naftidrofuryl-treated animals, however, showed almost complete preservation of the CA1 neurons (Fig. lc) when compared to the shamoperated animals (Fig. la). Fig. 2 shows representative photomicrographs of the CA1 sector following three 2-min ischemic episodes. The CA1 neurons in the saline-treated animals have almost completely disappeared (Fig. 2b). Naftidrofuryl-treated animals, however, exhibited a marked preservation of the CA1 neurons (Fig. 2c,d). The neuronal densities in the CA1 sector are summarized in Table I. Ischemic insults in both groups caused a reduction of about 85% in the number of CA1 neurons. The CA1 neurons in the naftidrofuryl (50 mg/kg)-treated animals with 5-min ischemia were significantly preserved (P < 0.01). Naftidrofuryl exhibited a dose-dependent protective effect in the animals with three 2-min ischemic episodes. Naftidrofuryl has been clinically used to improve cerebral circulation in European countries ~. This compound is rapidly metabolized and the biological half-life in the plasma is about 30 min in the rat 6 and about 1.5 h in humans 13. This compound easily penetrates the blood-brain barrier 6 and its concentration in the brain reaches its maximum 5 min after intraperitoneal administration (personal communication from Nippon Roussel Co.). Recently its serotonin S2 antagonistic action was confirmed by a binding study in the brain ~. The present study indicates that serotonin S,_ antagonists have a marked protective effect on ischemic hippocampal neuronal damage in both ischemia models in the gerbil. The increase in cerebral blood flow 2 or the inhibition of the platelet aggregation 7 of naftidrofuryl due to the S2 antagonistic action may play a role in the amelioration of the hippocampal neuronal damage. However, direct action to the brain also
CAa sector 7 days after 5-rain forebrain ischemia, a. shamoperated; b, saline-treated; c, naftidrofuryl (50 mg/kg)treated. Magnification: a-c × 70.
should be taken into consideration. The serotonin receptors in the central nervous
::
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k~ .... ~ . . . . . . . .
.... ~
~
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a
.......~;
.b
....
i
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I ................. ,,¢ :
~ ,
389
a
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Fig. 2. Representative photomicrographs of the hippocampal CA~ sector 7 days after three 2-min ischemic episodes, a, sham-operated; b, saline-treated; c, naftidrofuryl (25 mg/kg)-treated; d, naftidrofuryl (50 mg/kg)-treated. Magnification: a,c, × 7; b,d x 70.
system are divided into two major subclasses (S 1 and $2) based upon radioligand binding data. S: receptors are related to the serotonin-sensitive adenylate cyclase-cyclic A M P signal cascade, whereas S 2 receptors are linked to the inositol response. It has
been generally accepted that inhibitory and excitatory synaptic actions are mediated by S 1 and S2 receptors, respectively. Various serotonin-induced behaviors, such as tryptamine-induced clonic seizures m and 5-hydroxytryptophan-induced head
TABLE I
twitches TM have been known to be mediated by S 2 receptors. Autoradiographic studies with the S2selective ligand [3H]ketanserin have shown that the cerebral cortex contains a large number of S 2
Neur°naldensityinthehipp°campalCAIsect°r Values are means + S.E.M. Sample number is in the parentheses, Group
Neuronaldensity (~ram)
receptors but that only a small number of $2 receptors are observed in the hippocampal forma-
Sham-operatedcontr01 Saline Naftidrofuryl (25 mg/kg)
268+9**(5) 41 + 19 (8) -
Three 2-rain ischemic insults 268+9** (5) 49 + 17 (8) 161 + 34* (8)
Naftidrofuryl (50 mg/kg)
234 + 7** (9)
250 + 24** (9)
tion. Most of the S a receptors in the hippocampal formation are found in the deeper parts of the dentate hilar region 3. Furthermore, even ketanserin, a potent S2 antagonist, only partially inhibits the inositol response mediated by serotonin in the hippocampus 4. From these observations, the serotonin S 2 antagonist, naftidrofuryl, may not act on the hippocampus directly but on the upper neurons such
*P < 0.05, **P < 0.01 compared with saline-treated group (Mann-Whitney U-test).
as those in the cerebral cortex and the dentate gyrus. On the other hand, the hippocampal formation
5-rnin ischemia
390 receives a dense glutamatergic innervation. The entorhinal cortex provides a m a j o r excitatory input to the dentate gyrus via the fibers of the so-called perforant path, and the dentate gyrus provides a m a j o r excitatory input to the pyramidal cells of the h i p p o c a m p a l C A 3 sector via the mossy fibers. Further, the p y r a m i d a l cells of the hippocapal CA3 sector give rise to a powerful associational excitatory projection (the Schaffer collaterals) to the hippocampal CA1 sector. Glutamatergic neurotransmission is thought at present to play a m a j o r role in ischemic h i p p o c a m p a l neuronal damage, There is evidence that serotonin may modulate the glutamatergic excitatory transmission. A n ex-
techniques has shown that the excitatory action of serotonin in the facial m o t o n e u r o n s represents a facilitation of the excitatory effect of glutamate, instead of a direct excitatory action on these neurons 12. If these data can be e x t e n d e d to the h i p p o c a m p a l formation, modulation of s e r o t o n e r g i c - g l u t a m a t e r gic interaction by S 2 antagonists in the cerebral cortex or dentate gyrus m a y ameliorate the hippocampal neuronal damage. Though the detailed mechanisms of the protective effect of naftidrofuryl on the h i p p o c a m p a l neuronal damage were not clarified, this c o m p o u n d may have therapeutic value in the t r e a t m e n t of ischemic
p e r i m e n t using extracellular single-cell recording
neuronal damage.
1 Admani, A.K., New approch to treatment of recent stroke, Br. Med. J., 2 (1978) 1678-1679. 2 Bielenberg, G.W., Beck, T., Sauer, D., Burniol, M. and Krieglstein, J., Effects of cerebroprotective agents on cerebral blood flow and on postischemic energy metabolism in the rat brain, J. Cereb. Blood Flow Metab., 7 (1987) 480-488. 3 Bj6rklund, A., H6kfelt, T. and Swanson, L.W., Integrated systems of the CNS, part I Hypothalamus, Hippocampus, Amygdala, Retina. Handbook of Chemical Neuroanatomy, Vol. 5, Elsevier, Amsterdam, 1987, pp. 208-211. 4 Conn, P.J. and Sanders-Bush, E., Serotonin-stimulated phosphoinositide turnover: mediation by the Se binding site in rat cerebral cortex but not in subcortical regions, J. Pharmacol. Exp. Ther., 234 (1985) 195-203. 5 Dragunow, M. and Faull, R.L.M., Neuroprotective effects of adenosine, Trends Pharmacol. Sci., 9 (1988) 193-194. 6 Fujikura, H., Sakurai, Y., Okui, K., Nakanishi, K., Masada, M. and Nadai, T., Studies on the metabolic fate of naftidrofuryl (II) Distribution, metabolism and excretion in rats after intravenous administration, lyakuhin Kenkyu, 18 (1987)1014-1025. 7 Iizuka, H., Shimada, A., Kaneko, T., Tanimoto, Y. and Yanagita, T., General pharmacological effects of naftidrofuryl oxalate (LS-121), Preclin. Rep. Cent. Inst. Exp. Anita., 11 (1986) 313-324. 8 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain
Research, 479 (1989)366-370. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. t0 Leysen, J.E., Niemegeers, C.J.E., Tollenaere, J.P. and Laduron, P.M., Serotonergic component of neuroleptic receptors, Nature (Lond.), 272 (1978) 168-171. 11 Maloteaux, J.M., Haiech. J., De Campeneere, D., Berta, P. and Vidal, N., Biochemical and physiological evidences for antiserotonergic properties of naftidrofuryl, Arzneim. Forsch., 36 (1986) 1194-1198. 12 McCall, R.B. and Aghajanian, G.K., Serotonergic facilitation of facial motoneuron excitation, Brain Research, 169 (1979) 11-27. 13 Nishigaki, R., Umemura, K., Okui, K., Hayashi, T., Yamamoto, T. and Sakurai, Y., The pharmacokinetical analysis of the fate of naftidrofuryl oxalate (LS-121) in human subjects-II, Yakugaku Zasshi, 106 (1986)916-922. 14 Peroutka, S.J., Lebovitz, R.M. and Snyder, S.H., Two distinct central serotonin receptors with different physiological functions, Science, 212 (1981)827-829. 15 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 16 Sternau, L., Lust, W.D., Ricci, A.J. and Ratcheson, R.A., Pharmacological protection against delayed neuronal death elicited by ischemia, J. Cereb. Blood Flow Metab., 7 (1987) S147.
387
BRES 23646
A serotonin S2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil Hideaki Fujikura, Hiroyuki Kato, Shinichi Nakano and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 18 April 1989) Key words: Cerebral ischemia; Repeated ischemia; Naftidrofuryl; Serotonin S2 antagonist; Gerbil
The effect of a serotonin S2 antagonist, naftidrofuryl, on ischemic neuronal damage was examined in the gerbil. Naftidrofuryl was injected i.p. 5 min prior to a single 5-min forebrain ischemia or immediately after each of three 2-min forebrain ischemic insults at 60-min intervals. In both groups the number of intact hippocampal CA1 neurons were significantly higher than in the saline-treated group. These results indicate that serotonin S2 antagonists have a protective effect against ischemic neuronal damage.
Since the report by Kirino et al. 9, ischemiainduced neuronal damage in the hippocampal CA1 sector has been known as 'delayed neuronal death', At present an excitatory neurotransmitter, glutamate, is considered to initiate the delayed neuronal death 15 and on the contrary, inhibitory neurotransmitters, G A B A 16 and adenosine 5, are known to ameliorate the neuronal damage. However, there has been no previous report on the serotonergic participation in the neuronal damage of the hippocampal CA1 sector. We investigated the effect of a serotonin S 2 antagonist, anftidrofuryi, on ischemic neuronal damage in the gerbil, Five-min forebrain ischemia in the gerbil has been well-known to destroy hippocampal CA1 neurons selectively. Recently Kato et al. 8 demonstrated that 2-min forebrain ischemia in the gerbil could not produce any neuronal damage but that 3 repeated insults of 2-min ischemia at 60-min intervals caused remarkable neuronal damage not only in the hippocampal CA1 sector but also in the dorsolateral crescent of the striatum, the thalamus and the substantia nigra. Repetitive transient ischemic attacks are c o m m o n clinical events and drug administration usually starts after the first ischemic insult,
Therefore, it is very important to study the effects of various compounds on the ischemia-induced neuronal damage using a repeated ischemia model. This is the first report on the application of a repeated ischemia model for the evaluation of protective effects of a compound against the neuronal damage. Male Mongolian gerbils, weighing 60-80 g, were used. They were anesthetized with a mixture of 2% halothane, 30% oxygen and 70% nitrous oxide and both c o m m o n carotid arteries were exposed. After anesthesia was discontinued and the animals regained consciousness, forebrain ischemia was induced by occlusion of the carotid arteries with Sugita No. 51 temporary aneurysm clips. The animals were divided into two groups. In the first group, a single 5-min ischemia was induced. Naftidrofuryl (50 mg/kg) or the same volume of saline was injected i.p. 5 min prior to the ischemia. In the second group, 3 episodes of 2-min ischemia were repeated at 60-min intervals. Naftidrofuryl (25 or 50 mg/kg) or saline was injected i.p. immediately after each ischemic insult. Seven days after the ischemic event, the animals were anesthetized with pentobarbital (40 mg/kg)i.p. and the brains were briefly washed by transcardiac
Correspondence: K. Kogure, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1, Seiryo-machi, Sendai 980, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
388 perfusion with heparinized saline followed by perfusion-fixation with 10% formalin for 20 min. The brains were removed and immersed in the same fixative until they were embedded in paraffin. Five-j~m-thick paraffin sections were obtained and stained with Cresyl violet or Hematoxylin/Eosin. The number of the pyramidal cells in the hippocampal CA1 sector per 1 mm linear length was counted under a light microscope. The average
Fig. 1. Representative photomicrographs of the hippocampal
number of right and left CAI neurons was taken as the neuronal density. Statistical analysis was carried out using the Mann-Whitney U-test. Representative photomicrographs of the hippocampal CA1 sector following 5-rain ischemia are shown in Fig. 1. The CA1 neurons in the salinetreated animals have nearly completely disappeared (Fig. lb). Naftidrofuryl-treated animals, however, showed almost complete preservation of the CA1 neurons (Fig. lc) when compared to the shamoperated animals (Fig. la). Fig. 2 shows representative photomicrographs of the CA1 sector following three 2-min ischemic episodes. The CA1 neurons in the saline-treated animals have almost completely disappeared (Fig. 2b). Naftidrofuryl-treated animals, however, exhibited a marked preservation of the CA1 neurons (Fig. 2c,d). The neuronal densities in the CA1 sector are summarized in Table I. Ischemic insults in both groups caused a reduction of about 85% in the number of CA1 neurons. The CA1 neurons in the naftidrofuryl (50 mg/kg)-treated animals with 5-min ischemia were significantly preserved (P < 0.01). Naftidrofuryl exhibited a dose-dependent protective effect in the animals with three 2-min ischemic episodes. Naftidrofuryl has been clinically used to improve cerebral circulation in European countries ~. This compound is rapidly metabolized and the biological half-life in the plasma is about 30 min in the rat 6 and about 1.5 h in humans 13. This compound easily penetrates the blood-brain barrier 6 and its concentration in the brain reaches its maximum 5 min after intraperitoneal administration (personal communication from Nippon Roussel Co.). Recently its serotonin S2 antagonistic action was confirmed by a binding study in the brain ~. The present study indicates that serotonin S,_ antagonists have a marked protective effect on ischemic hippocampal neuronal damage in both ischemia models in the gerbil. The increase in cerebral blood flow 2 or the inhibition of the platelet aggregation 7 of naftidrofuryl due to the S2 antagonistic action may play a role in the amelioration of the hippocampal neuronal damage. However, direct action to the brain also
CAa sector 7 days after 5-rain forebrain ischemia, a. shamoperated; b, saline-treated; c, naftidrofuryl (50 mg/kg)treated. Magnification: a-c × 70.
should be taken into consideration. The serotonin receptors in the central nervous
::
..........
~i
k~ .... ~ . . . . . . . .
.... ~
~
""
a
.......~;
.b
....
i
~
I ................. ,,¢ :
~ ,
389
a
:
Fig. 2. Representative photomicrographs of the hippocampal CA~ sector 7 days after three 2-min ischemic episodes, a, sham-operated; b, saline-treated; c, naftidrofuryl (25 mg/kg)-treated; d, naftidrofuryl (50 mg/kg)-treated. Magnification: a,c, × 7; b,d x 70.
system are divided into two major subclasses (S 1 and $2) based upon radioligand binding data. S: receptors are related to the serotonin-sensitive adenylate cyclase-cyclic A M P signal cascade, whereas S 2 receptors are linked to the inositol response. It has
been generally accepted that inhibitory and excitatory synaptic actions are mediated by S 1 and S2 receptors, respectively. Various serotonin-induced behaviors, such as tryptamine-induced clonic seizures m and 5-hydroxytryptophan-induced head
TABLE I
twitches TM have been known to be mediated by S 2 receptors. Autoradiographic studies with the S2selective ligand [3H]ketanserin have shown that the cerebral cortex contains a large number of S 2
Neur°naldensityinthehipp°campalCAIsect°r Values are means + S.E.M. Sample number is in the parentheses, Group
Neuronaldensity (~ram)
receptors but that only a small number of $2 receptors are observed in the hippocampal forma-
Sham-operatedcontr01 Saline Naftidrofuryl (25 mg/kg)
268+9**(5) 41 + 19 (8) -
Three 2-rain ischemic insults 268+9** (5) 49 + 17 (8) 161 + 34* (8)
Naftidrofuryl (50 mg/kg)
234 + 7** (9)
250 + 24** (9)
tion. Most of the S a receptors in the hippocampal formation are found in the deeper parts of the dentate hilar region 3. Furthermore, even ketanserin, a potent S2 antagonist, only partially inhibits the inositol response mediated by serotonin in the hippocampus 4. From these observations, the serotonin S 2 antagonist, naftidrofuryl, may not act on the hippocampus directly but on the upper neurons such
*P < 0.05, **P < 0.01 compared with saline-treated group (Mann-Whitney U-test).
as those in the cerebral cortex and the dentate gyrus. On the other hand, the hippocampal formation
5-rnin ischemia
390 receives a dense glutamatergic innervation. The entorhinal cortex provides a m a j o r excitatory input to the dentate gyrus via the fibers of the so-called perforant path, and the dentate gyrus provides a m a j o r excitatory input to the pyramidal cells of the h i p p o c a m p a l C A 3 sector via the mossy fibers. Further, the p y r a m i d a l cells of the hippocapal CA3 sector give rise to a powerful associational excitatory projection (the Schaffer collaterals) to the hippocampal CA1 sector. Glutamatergic neurotransmission is thought at present to play a m a j o r role in ischemic h i p p o c a m p a l neuronal damage, There is evidence that serotonin may modulate the glutamatergic excitatory transmission. A n ex-
techniques has shown that the excitatory action of serotonin in the facial m o t o n e u r o n s represents a facilitation of the excitatory effect of glutamate, instead of a direct excitatory action on these neurons 12. If these data can be e x t e n d e d to the h i p p o c a m p a l formation, modulation of s e r o t o n e r g i c - g l u t a m a t e r gic interaction by S 2 antagonists in the cerebral cortex or dentate gyrus m a y ameliorate the hippocampal neuronal damage. Though the detailed mechanisms of the protective effect of naftidrofuryl on the h i p p o c a m p a l neuronal damage were not clarified, this c o m p o u n d may have therapeutic value in the t r e a t m e n t of ischemic
p e r i m e n t using extracellular single-cell recording
neuronal damage.
1 Admani, A.K., New approch to treatment of recent stroke, Br. Med. J., 2 (1978) 1678-1679. 2 Bielenberg, G.W., Beck, T., Sauer, D., Burniol, M. and Krieglstein, J., Effects of cerebroprotective agents on cerebral blood flow and on postischemic energy metabolism in the rat brain, J. Cereb. Blood Flow Metab., 7 (1987) 480-488. 3 Bj6rklund, A., H6kfelt, T. and Swanson, L.W., Integrated systems of the CNS, part I Hypothalamus, Hippocampus, Amygdala, Retina. Handbook of Chemical Neuroanatomy, Vol. 5, Elsevier, Amsterdam, 1987, pp. 208-211. 4 Conn, P.J. and Sanders-Bush, E., Serotonin-stimulated phosphoinositide turnover: mediation by the Se binding site in rat cerebral cortex but not in subcortical regions, J. Pharmacol. Exp. Ther., 234 (1985) 195-203. 5 Dragunow, M. and Faull, R.L.M., Neuroprotective effects of adenosine, Trends Pharmacol. Sci., 9 (1988) 193-194. 6 Fujikura, H., Sakurai, Y., Okui, K., Nakanishi, K., Masada, M. and Nadai, T., Studies on the metabolic fate of naftidrofuryl (II) Distribution, metabolism and excretion in rats after intravenous administration, lyakuhin Kenkyu, 18 (1987)1014-1025. 7 Iizuka, H., Shimada, A., Kaneko, T., Tanimoto, Y. and Yanagita, T., General pharmacological effects of naftidrofuryl oxalate (LS-121), Preclin. Rep. Cent. Inst. Exp. Anita., 11 (1986) 313-324. 8 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain
Research, 479 (1989)366-370. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. t0 Leysen, J.E., Niemegeers, C.J.E., Tollenaere, J.P. and Laduron, P.M., Serotonergic component of neuroleptic receptors, Nature (Lond.), 272 (1978) 168-171. 11 Maloteaux, J.M., Haiech. J., De Campeneere, D., Berta, P. and Vidal, N., Biochemical and physiological evidences for antiserotonergic properties of naftidrofuryl, Arzneim. Forsch., 36 (1986) 1194-1198. 12 McCall, R.B. and Aghajanian, G.K., Serotonergic facilitation of facial motoneuron excitation, Brain Research, 169 (1979) 11-27. 13 Nishigaki, R., Umemura, K., Okui, K., Hayashi, T., Yamamoto, T. and Sakurai, Y., The pharmacokinetical analysis of the fate of naftidrofuryl oxalate (LS-121) in human subjects-II, Yakugaku Zasshi, 106 (1986)916-922. 14 Peroutka, S.J., Lebovitz, R.M. and Snyder, S.H., Two distinct central serotonin receptors with different physiological functions, Science, 212 (1981)827-829. 15 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 16 Sternau, L., Lust, W.D., Ricci, A.J. and Ratcheson, R.A., Pharmacological protection against delayed neuronal death elicited by ischemia, J. Cereb. Blood Flow Metab., 7 (1987) S147.