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Effects of indeloxazine on hippocampal CA1 pyramidal cell damage following transient cerebral ischemia in the gerbil
NeuroPharmacology Vol. 32, No. 3, pp. 307-310, 1993 Printed in Great Britain
0028-3908/93$6.00+ 0.00 PergamonPress Ltd
EFFECTS OF INDELOXAZINE ON HIPPOCAMPAL CA1 PYRAMIDAL CELL D A M A G E FOLLOWING TRANSIENT CEREBRAL ISCHEMIA IN THE GERBIL T. KANO,* Y. KATAYAMA,S. MIYAZAKI,K. KINOSHITA,T. KAWAMATAand T. TSUBOKAWA Department of Neurological Surgery, Nihon University School of Medicine, Tokyo 173, Japan (Accepted 5 June 1992)
Summary--The effects of indeloxazineon the ischemia-induceddeath of hippocampal CA 1 pyramidal cells following transient cerebral ischemia were examined in the mongolian gerbil. Increased survival of CAI pyramidal cells was demonstrated in animals pre- and post-treated with indeloxazine. Increased survival of CA1 pyramidal cells was, however, not demonstrated in animals post-treated but not pre-treated with indeloxazine. A previous study has demonstrated that indeloxazine increases the glucose and adenosine triphosphate (ATP) contents in the brain probably through an enhanced capability of oxidative phosphorylation. It has been reported that increases in the glucose and ATP contents in the brain before ischemia delay the onset of massive ionic fluxes during ischemia. The delay in onset of this ionic event may help to protect these cells from death. The present data suggest that energy state before ischemia may play an important role in the protective effect of indeloxazine. Key words--indeloxazine hydrochloride, hippocampus, ischemia, glucose, adenosine triphosphate.
Indeloxazine is an indene derivative [( + )-2-[(inden-7yloxy)methyi]morpholine hydrochloride (YM0805~-)] (Kojima, Niigata, Fujikura, Tachikawa, Nozaki, Kagami and Takahashi, 1985) which inhibits the uptake of noradrenaline and serotonin by synaptosomes (Harada and Maeno, 1979), and increases the content of noradrenaline and serotonin in the rat brain (Yamaguchi, Harada and Nagano, 1985). This compound therefore displays antidepressant properties. ]En addition, indeloxazine has been shown to inhibit the amnesia induced by scopolamine and to facilitate the acquisition of learned behavior in the mouse (Yamamoto and Shimizu, 1987b). Based on these pharmacological effects, indeloxazine is being used in the treatment of human patients with dementiat. It has been reported recently that indeloxazine can significantly prolong the survival time of mice subjected to anoxia (Yamamoto and Shimizu, 1987a). The decreases in brain glucose utilization rate and adenosine triphosphate (ATP) content in a 4-vessel occlusion rat model of cerebral ischemia were inhibited by indeloxazine (Sakamoto, Ohtomo and Kogure, 1986). This agent prevented the impairment of passive and active avoidance learning after anoxic insult in rats (Yamamoto and Shimizu, 1987a). These findings suggest that indeloxazine can protect the brain against anoxic or ischemic damage. Indeloxazine increases the glucose and ATP contents *To whom correspondence should be addressed. tlnformation is available from Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan.
in the mouse brain without an increase in lactate (Harada, Terai, Kuroiwa and Yamaguchi, 1987). An enhanced capability of oxidative phosphorylation has been suggested to underlie the reported effect of indeioxazine on anoxia or ischemia. The present study examined the effects of indeloxazine on the ischemic damage of the hippocampal CA 1 pyramidal cells following transient cerebral ischemia in the mongolian gerbil (Kirino, 1982). METHODS
Male gerbils (60-80 g, n = 58) were used for the experiments. The animals were anesthetized with 1% halothane and 2:1 N20 in O2. The femoral artery was exposed and cannulated for monitoring the systemic arterial blood pressure. The common carotid arteries were exposed bilaterally for later occlusion. The core temperature was maintained at 37°C with a heating pad. In one group of animals (n = 6), a sham-operation was performed. Vascular clamps were placed on both common carotid arteries. The electroencephalograms quickly became isoelectric. Inhalation of halothane was then discontinued, since the animals exhibited signs of coma during ischemia. After 5 min had elapsed from the time when the isoelectric recording was observed, the vascular clamps were removed and the neck incision was closed with a suture. The animals were placed in cages and allowed free access to water and food. Indeloxazine hydrochloride was dissolved in saline before use. The animals were divided into 2 groups. In one group (Group I, n = 28), indeloxazine was administered at 30 min and immediately before, and
307
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immediately after the ischemic insult. In the other group (Group II, n = 18), administration of indeloxazine was performed three times at 30-min intervals beginning immediately after the ischemic insult. Indeloxazine was administered twice a day thereafter in both groups. In each animal, either 2 or 10 mg/kg was injected intraperitoneally on each occasion. In an untreated control group (n = 6), vehicle alone was injected at 30 min and immediately before the ischemic insult, three times at 30-min intervals beginning immediately after the ischemic insult, and twice a day thereafter. Three days after the ischemic insult (30 rain after the final administration of indeloxazine), the animals were anesthetized with pentobarbital and then perfused transcardially with 3.5% formaldehyde solution. The brains were removed and each brain was cut into 7-mm thick coronal sections which contained the hippocampal area. Post-fixation was performed for 48 hr in the same solution. The brain sections were then dehydrated with graded ethanol and embedded in paraffin. Serial sections of 5 #m in thickness were prepared on a microtome from the dorsal hippocampus and processed for Nissl staining. The number of surviving pyramidal cells which had wellpreserved perikarya and nuclei was counted in each CA1 region under a light microscope. Five coronal sections were photographed at approximately 20-pm intervals from the region 1.5ram posterior to the bregma, and the length of the CA1 region was measured. The number of surviving CAI pyramidal cells was again confirmed from the photographs. The density of surviving CA1 pyramidal cells (number/1 mm horizontal length) was calculated from the cell number and the length of the CA1 region. The average of the data obtained from the right and left was regarded as the cell density in each animal. The cell densities of CA 1 pyramidal cells were then compared between the groups by employing the Scheffe F-test. RESULTS AND DISCUSSION The typical change in the hippocampus caused by the transient forebrain ischemia was selective death of CA1 pyramidal cells [Fig. I(B)]. In sham-operated control animals, ischemic changes were never observed [Fig. l(A)]. Death of CA1 pyramidal cells
occurred in all groups subjected to ischemia, Thus, the density of surviving CA1 pyramidal cells was significantly lower in all the ischemia groups, regardless of whether or not they were treated with indeloxazine, as compared to the sham-operated control group (P < 0.01; Table 1). In the Group I animals, however, extensive death of CA 1 pyramidal cells of the hippocampus was often not observed [Fig. I(C)]. Significantly more CA1 pyramidal cells survived in the Group I animals, treated with either 2 or 10mg/kg indeloxazine, as compared to the saline-treated animals (P <0.01; Table 1). A significant difference in this protective effect of indeloxazine was noted between 2 and 10 mg/kg indeloxazine (P < 0.05). Such a protective effect was not detected in the Group II animals. Thus, extensive death of CA1 pyramidal cells of the hippocampus was always observed [Fig. I(D)]. There was no significant difference in density of surviving CAI pyramidal cells between the Group II animals, treated with either 2 or 10 mg/kg indeloxazine, and the saline-treated animals (Table 1). The hippocampal pyramidal cells in the CA I region are selectively vulnerable to transient ischemia (Kirino, 1982; Pulsinelli, Brierley and Plum, 1982). Although the mechanisms of ischemic neuronal damage are still not completely understood, it is generally considered that uncontrolled release of excitatory amino acids, such as glutamate, may play a crucial role. However, recent evidence have suggested that the magnitude of neuronal damage may be dependent on the balance between excitatory and inhibitory neurotransmission during and after ischemic insult, and many metabolic processes may also be involved in leading these cells to death. An increase in surviving CAI pyrimidal cells was demonstrated in the Group I animals but not in the Group II animals. The increased survival of CA1 pyramidal cells observed in the Group I animals may thus be attributable to the pre-treatment with indeloxazine. Indeloxazine has been found to exert no cerebral vasodilator action or central depressant activities (Yamamoto and Shimizu, 1987b), suggesting that neither an increased delivery of energy substances nor a decreased energy demand can explain the effects of indeloxazine. Indeloxazine, unlike barbiturate, activates electroencephalograms in normal
Table 1. Effectsof indeloxazineon hippocampalCA1 pyramidalcell death followingtransientcerebral ischemia SurvivingCA1 pyramidalcells Comparisonwith Comparisonwith Group n (number/lmm) sham control untreatedgroup Sham control 6 191.8+_4.9 Ischemia Untreated group 6 56.8 _+4.8 P < 0.01 Group I 2 mg/kg 14 140.6+_14.4 P < 0.01 P < 0.01 10mg/kg 14 155.2+ 10.7 P <0.01 P < 0.01 Group II 2 mg/kg 10 69.4 + 7.1 P < 0.01 NS 10mg/kg 8 70.6+ 18.3 P < 0.01 NS Mean +_SD. F-test. Pre 2 mg/kg vs pre 10mg/kg, P < 0.05.
Effects of indeloxazine on ischemic hippocampal damage
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D Fig. I. Representative photomicrographs of the hippocampus at 3 days after transient forebrain ischemia. (A) Sham-operated control; (B) saline-treated; (C) pre- as well as post-treated with I0 mg/kg indeloxazine: (D) post-treated with 10 mg/kg indeloxazine.
310
T. KANO et al.
as well as post-ischemia rats (Yamamoto and Shimizu, 1987b), suggesting that the increases in glucose and A T P content may not be induced by a decrease in energy demand. An enhanced oxidative phosphorylation has been suggested to underlie the accelerated ATP production because of a lack of increase in lactate content (Harada et al., 1987). It has been reported that exposure of juvenile rats to hypoxia for 24 hr increases the glucose content in the brain from 1.3 to 2.9/~mol/g and ATP content from 2.4 to 2.6Hmol/g, and delays the onset of massive ionic fluxes for approximately 5 rain (Hansen and Nordstrom, 1979). The increased surviving CA1 pyramidal cells demonstrated in the G r o u p I animals may potentially be accounted for by the delay in onset of the massive ionic fluxes. Energy failure develops rapidly around the onset of the massive ionic shifts (Hansen and Nordstrom, 1979). The delay in onset of this ionic event reduces the period for which the C A I pyramidal cells are exposed to global energy failure during 5-min transient ischemia, and may thereby help to protect these cells from death. Although further studies are needed to clarify the precise mechanism involved, the present data suggest that energy state before ischemia may play an important role in the protective effect of indeloxazine. In summary, the present results show that indeloxazine can protect against the death of CA 1 pyramidal cells following 5-min transient ischemia, when the drug is administered before the ischemia. Such findings are consistent with the results of previous studies which have demonstrated that the survival time of mice subjected to anoxia is prolonged (Yamamoto and Shimizu, 1987a), changes in cerebral metabolism after ischemia are attenuated (Sakamoto et al., 1986) and anoxia-induced impairment of passive and active avoidance learning in rats is prevented by indeloxazine ( Y a m a m o t o and Shimizu, 1987al.
The authors wish to thank Dr M1 Yamamoto, Department of Pharmacology, Central Research Laboratories, Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan, for supplying the indeloxazine. Acknowledgements
REFERENCES
Hansen A. J. and Nordstrom C.-H. (1979) Brain extracellular potassium and energy metabolism during ischemia in juvenile rats after exposure to hypoxia for 24 h. J. New'ochem. 32:915 920. Harada M. and Maeno H. (19793 Biochemical characteristics of a potential antidepressant, 2-(7-indenyloxymethyl) morpholine hydrochloride (YM-08054-13. Biochem. Pharmac. 28:2645 2651. Harada M., Terai M., Kuroiwa I. and Yamaguchi 1". (19873 Effects of indeloxazine hydrochloride (YM-080543 on glucose and energy metabolism in mouse brain. Yakuri lo Chirvo 1 5 : 5 1 2 3 5130 {in Japanese with English abstractl. Kirino T. (1982) Delayed neuronal death m the gerbal hippocampus following ischemia. Brain Res. 239: 57 69, Kojima T., Niigata K., Fujikura T., Tachikawa 5;.. Nozaki Y., Kagami S. and Takahashi K. (19853 Syntheses of ( + )-2-[( inden-7-yloxy)methyllmorpholine hydrochlo ride (YM-08054, indeloxazine hydrochloride) and its derivatives with potential cerebral-activating and antidepressive properties. Chem. Pharm. Bull. 33:3766 3744. Pulsinelli W. A,. Brierley J. B. and Plum F. (I 982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11,491 498. Sakamoto N., Ohtomo H. and Kogure K. (1986) Effecls indeloxazine hydrochloride on brain energy and glucose metabolism in four-vessel occlusion rat model. Brain Nerre 38" 387 391 (in Japanese with English abstract). Yamaguchi T., Hirada M. and Nagano H. (19853 Effects of indeloxazine HC1 (YM-080543 on monoamines and their metabolite contents in rat brain. Biogenic Amines 3: 21 32. Yamamoto M. and Shimizu M. (1987a) Effects of indeloxazine hydrochloride (YM-080543 on anoxia. Arch. Int. Pharmacodyn. Ther. 286:272 281. Yamamoto M. and Shimizu M. (1987b) Cerebral activating properties of indeloxazine hydrochloride. Neuropharmacolo~,y 26:761 770,
0028-3908/93$6.00+ 0.00 PergamonPress Ltd
EFFECTS OF INDELOXAZINE ON HIPPOCAMPAL CA1 PYRAMIDAL CELL D A M A G E FOLLOWING TRANSIENT CEREBRAL ISCHEMIA IN THE GERBIL T. KANO,* Y. KATAYAMA,S. MIYAZAKI,K. KINOSHITA,T. KAWAMATAand T. TSUBOKAWA Department of Neurological Surgery, Nihon University School of Medicine, Tokyo 173, Japan (Accepted 5 June 1992)
Summary--The effects of indeloxazineon the ischemia-induceddeath of hippocampal CA 1 pyramidal cells following transient cerebral ischemia were examined in the mongolian gerbil. Increased survival of CAI pyramidal cells was demonstrated in animals pre- and post-treated with indeloxazine. Increased survival of CA1 pyramidal cells was, however, not demonstrated in animals post-treated but not pre-treated with indeloxazine. A previous study has demonstrated that indeloxazine increases the glucose and adenosine triphosphate (ATP) contents in the brain probably through an enhanced capability of oxidative phosphorylation. It has been reported that increases in the glucose and ATP contents in the brain before ischemia delay the onset of massive ionic fluxes during ischemia. The delay in onset of this ionic event may help to protect these cells from death. The present data suggest that energy state before ischemia may play an important role in the protective effect of indeloxazine. Key words--indeloxazine hydrochloride, hippocampus, ischemia, glucose, adenosine triphosphate.
Indeloxazine is an indene derivative [( + )-2-[(inden-7yloxy)methyi]morpholine hydrochloride (YM0805~-)] (Kojima, Niigata, Fujikura, Tachikawa, Nozaki, Kagami and Takahashi, 1985) which inhibits the uptake of noradrenaline and serotonin by synaptosomes (Harada and Maeno, 1979), and increases the content of noradrenaline and serotonin in the rat brain (Yamaguchi, Harada and Nagano, 1985). This compound therefore displays antidepressant properties. ]En addition, indeloxazine has been shown to inhibit the amnesia induced by scopolamine and to facilitate the acquisition of learned behavior in the mouse (Yamamoto and Shimizu, 1987b). Based on these pharmacological effects, indeloxazine is being used in the treatment of human patients with dementiat. It has been reported recently that indeloxazine can significantly prolong the survival time of mice subjected to anoxia (Yamamoto and Shimizu, 1987a). The decreases in brain glucose utilization rate and adenosine triphosphate (ATP) content in a 4-vessel occlusion rat model of cerebral ischemia were inhibited by indeloxazine (Sakamoto, Ohtomo and Kogure, 1986). This agent prevented the impairment of passive and active avoidance learning after anoxic insult in rats (Yamamoto and Shimizu, 1987a). These findings suggest that indeloxazine can protect the brain against anoxic or ischemic damage. Indeloxazine increases the glucose and ATP contents *To whom correspondence should be addressed. tlnformation is available from Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan.
in the mouse brain without an increase in lactate (Harada, Terai, Kuroiwa and Yamaguchi, 1987). An enhanced capability of oxidative phosphorylation has been suggested to underlie the reported effect of indeioxazine on anoxia or ischemia. The present study examined the effects of indeloxazine on the ischemic damage of the hippocampal CA 1 pyramidal cells following transient cerebral ischemia in the mongolian gerbil (Kirino, 1982). METHODS
Male gerbils (60-80 g, n = 58) were used for the experiments. The animals were anesthetized with 1% halothane and 2:1 N20 in O2. The femoral artery was exposed and cannulated for monitoring the systemic arterial blood pressure. The common carotid arteries were exposed bilaterally for later occlusion. The core temperature was maintained at 37°C with a heating pad. In one group of animals (n = 6), a sham-operation was performed. Vascular clamps were placed on both common carotid arteries. The electroencephalograms quickly became isoelectric. Inhalation of halothane was then discontinued, since the animals exhibited signs of coma during ischemia. After 5 min had elapsed from the time when the isoelectric recording was observed, the vascular clamps were removed and the neck incision was closed with a suture. The animals were placed in cages and allowed free access to water and food. Indeloxazine hydrochloride was dissolved in saline before use. The animals were divided into 2 groups. In one group (Group I, n = 28), indeloxazine was administered at 30 min and immediately before, and
307
308
T. KANOet al.
immediately after the ischemic insult. In the other group (Group II, n = 18), administration of indeloxazine was performed three times at 30-min intervals beginning immediately after the ischemic insult. Indeloxazine was administered twice a day thereafter in both groups. In each animal, either 2 or 10 mg/kg was injected intraperitoneally on each occasion. In an untreated control group (n = 6), vehicle alone was injected at 30 min and immediately before the ischemic insult, three times at 30-min intervals beginning immediately after the ischemic insult, and twice a day thereafter. Three days after the ischemic insult (30 rain after the final administration of indeloxazine), the animals were anesthetized with pentobarbital and then perfused transcardially with 3.5% formaldehyde solution. The brains were removed and each brain was cut into 7-mm thick coronal sections which contained the hippocampal area. Post-fixation was performed for 48 hr in the same solution. The brain sections were then dehydrated with graded ethanol and embedded in paraffin. Serial sections of 5 #m in thickness were prepared on a microtome from the dorsal hippocampus and processed for Nissl staining. The number of surviving pyramidal cells which had wellpreserved perikarya and nuclei was counted in each CA1 region under a light microscope. Five coronal sections were photographed at approximately 20-pm intervals from the region 1.5ram posterior to the bregma, and the length of the CA1 region was measured. The number of surviving CAI pyramidal cells was again confirmed from the photographs. The density of surviving CA1 pyramidal cells (number/1 mm horizontal length) was calculated from the cell number and the length of the CA1 region. The average of the data obtained from the right and left was regarded as the cell density in each animal. The cell densities of CA 1 pyramidal cells were then compared between the groups by employing the Scheffe F-test. RESULTS AND DISCUSSION The typical change in the hippocampus caused by the transient forebrain ischemia was selective death of CA1 pyramidal cells [Fig. I(B)]. In sham-operated control animals, ischemic changes were never observed [Fig. l(A)]. Death of CA1 pyramidal cells
occurred in all groups subjected to ischemia, Thus, the density of surviving CA1 pyramidal cells was significantly lower in all the ischemia groups, regardless of whether or not they were treated with indeloxazine, as compared to the sham-operated control group (P < 0.01; Table 1). In the Group I animals, however, extensive death of CA 1 pyramidal cells of the hippocampus was often not observed [Fig. I(C)]. Significantly more CA1 pyramidal cells survived in the Group I animals, treated with either 2 or 10mg/kg indeloxazine, as compared to the saline-treated animals (P <0.01; Table 1). A significant difference in this protective effect of indeloxazine was noted between 2 and 10 mg/kg indeloxazine (P < 0.05). Such a protective effect was not detected in the Group II animals. Thus, extensive death of CA1 pyramidal cells of the hippocampus was always observed [Fig. I(D)]. There was no significant difference in density of surviving CAI pyramidal cells between the Group II animals, treated with either 2 or 10 mg/kg indeloxazine, and the saline-treated animals (Table 1). The hippocampal pyramidal cells in the CA I region are selectively vulnerable to transient ischemia (Kirino, 1982; Pulsinelli, Brierley and Plum, 1982). Although the mechanisms of ischemic neuronal damage are still not completely understood, it is generally considered that uncontrolled release of excitatory amino acids, such as glutamate, may play a crucial role. However, recent evidence have suggested that the magnitude of neuronal damage may be dependent on the balance between excitatory and inhibitory neurotransmission during and after ischemic insult, and many metabolic processes may also be involved in leading these cells to death. An increase in surviving CAI pyrimidal cells was demonstrated in the Group I animals but not in the Group II animals. The increased survival of CA1 pyramidal cells observed in the Group I animals may thus be attributable to the pre-treatment with indeloxazine. Indeloxazine has been found to exert no cerebral vasodilator action or central depressant activities (Yamamoto and Shimizu, 1987b), suggesting that neither an increased delivery of energy substances nor a decreased energy demand can explain the effects of indeloxazine. Indeloxazine, unlike barbiturate, activates electroencephalograms in normal
Table 1. Effectsof indeloxazineon hippocampalCA1 pyramidalcell death followingtransientcerebral ischemia SurvivingCA1 pyramidalcells Comparisonwith Comparisonwith Group n (number/lmm) sham control untreatedgroup Sham control 6 191.8+_4.9 Ischemia Untreated group 6 56.8 _+4.8 P < 0.01 Group I 2 mg/kg 14 140.6+_14.4 P < 0.01 P < 0.01 10mg/kg 14 155.2+ 10.7 P <0.01 P < 0.01 Group II 2 mg/kg 10 69.4 + 7.1 P < 0.01 NS 10mg/kg 8 70.6+ 18.3 P < 0.01 NS Mean +_SD. F-test. Pre 2 mg/kg vs pre 10mg/kg, P < 0.05.
Effects of indeloxazine on ischemic hippocampal damage
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D Fig. I. Representative photomicrographs of the hippocampus at 3 days after transient forebrain ischemia. (A) Sham-operated control; (B) saline-treated; (C) pre- as well as post-treated with I0 mg/kg indeloxazine: (D) post-treated with 10 mg/kg indeloxazine.
310
T. KANO et al.
as well as post-ischemia rats (Yamamoto and Shimizu, 1987b), suggesting that the increases in glucose and A T P content may not be induced by a decrease in energy demand. An enhanced oxidative phosphorylation has been suggested to underlie the accelerated ATP production because of a lack of increase in lactate content (Harada et al., 1987). It has been reported that exposure of juvenile rats to hypoxia for 24 hr increases the glucose content in the brain from 1.3 to 2.9/~mol/g and ATP content from 2.4 to 2.6Hmol/g, and delays the onset of massive ionic fluxes for approximately 5 rain (Hansen and Nordstrom, 1979). The increased surviving CA1 pyramidal cells demonstrated in the G r o u p I animals may potentially be accounted for by the delay in onset of the massive ionic fluxes. Energy failure develops rapidly around the onset of the massive ionic shifts (Hansen and Nordstrom, 1979). The delay in onset of this ionic event reduces the period for which the C A I pyramidal cells are exposed to global energy failure during 5-min transient ischemia, and may thereby help to protect these cells from death. Although further studies are needed to clarify the precise mechanism involved, the present data suggest that energy state before ischemia may play an important role in the protective effect of indeloxazine. In summary, the present results show that indeloxazine can protect against the death of CA 1 pyramidal cells following 5-min transient ischemia, when the drug is administered before the ischemia. Such findings are consistent with the results of previous studies which have demonstrated that the survival time of mice subjected to anoxia is prolonged (Yamamoto and Shimizu, 1987a), changes in cerebral metabolism after ischemia are attenuated (Sakamoto et al., 1986) and anoxia-induced impairment of passive and active avoidance learning in rats is prevented by indeloxazine ( Y a m a m o t o and Shimizu, 1987al.
The authors wish to thank Dr M1 Yamamoto, Department of Pharmacology, Central Research Laboratories, Yamanouchi Pharmaceutical Co. Ltd, Tokyo, Japan, for supplying the indeloxazine. Acknowledgements
REFERENCES
Hansen A. J. and Nordstrom C.-H. (1979) Brain extracellular potassium and energy metabolism during ischemia in juvenile rats after exposure to hypoxia for 24 h. J. New'ochem. 32:915 920. Harada M. and Maeno H. (19793 Biochemical characteristics of a potential antidepressant, 2-(7-indenyloxymethyl) morpholine hydrochloride (YM-08054-13. Biochem. Pharmac. 28:2645 2651. Harada M., Terai M., Kuroiwa I. and Yamaguchi 1". (19873 Effects of indeloxazine hydrochloride (YM-080543 on glucose and energy metabolism in mouse brain. Yakuri lo Chirvo 1 5 : 5 1 2 3 5130 {in Japanese with English abstractl. Kirino T. (1982) Delayed neuronal death m the gerbal hippocampus following ischemia. Brain Res. 239: 57 69, Kojima T., Niigata K., Fujikura T., Tachikawa 5;.. Nozaki Y., Kagami S. and Takahashi K. (19853 Syntheses of ( + )-2-[( inden-7-yloxy)methyllmorpholine hydrochlo ride (YM-08054, indeloxazine hydrochloride) and its derivatives with potential cerebral-activating and antidepressive properties. Chem. Pharm. Bull. 33:3766 3744. Pulsinelli W. A,. Brierley J. B. and Plum F. (I 982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11,491 498. Sakamoto N., Ohtomo H. and Kogure K. (1986) Effecls indeloxazine hydrochloride on brain energy and glucose metabolism in four-vessel occlusion rat model. Brain Nerre 38" 387 391 (in Japanese with English abstract). Yamaguchi T., Hirada M. and Nagano H. (19853 Effects of indeloxazine HC1 (YM-080543 on monoamines and their metabolite contents in rat brain. Biogenic Amines 3: 21 32. Yamamoto M. and Shimizu M. (1987a) Effects of indeloxazine hydrochloride (YM-080543 on anoxia. Arch. Int. Pharmacodyn. Ther. 286:272 281. Yamamoto M. and Shimizu M. (1987b) Cerebral activating properties of indeloxazine hydrochloride. Neuropharmacolo~,y 26:761 770,