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Effects of MK-801 and Pentobarbital on Cholinergic Terminal Damage and Delayed Neuronal Death in the Ischemic Gerbil Hippocampus
Brain Research Bulletin, Vol. 43, No. 1, pp. 81–85, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00347-4
Effects of MK-801 and Pentobarbital on Cholinergic Terminal Damage and Delayed Neuronal Death in the Ischemic Gerbil Hippocampus HIROHISA ISHIMARU, 1 AKIRA TAKAHASHI, YASUSHI IKARASHI AND YUJI MARUYAMA Department of Neuropsychopharmacology (Tsumura), Gunma University School of Medicine, 3-39-22 showa-machi, Maebashi-shi, Gunma 371, Japan
ABSTRACT: The present study covers both the effects of MK801, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, and pentobarbital on cholinergic terminal damage and delayed neuronal death (DND) in ischemic gerbil. To study the above effects, in vivo microdialysis, immunohistochemical, and morphological techniques were used. MK801 (3 mg/kg) or pentobarbital (50 mg/kg) were injected intraperitoneally 1 h or 30 min before 5 min ischemia, respectively. Each estimation was then carried out 4, 7, or 14 days after ischemia. Ischemia induced a significant decrease in acetylcholine (ACh) release and a disappearance of choline acetyltransferase (ChAT)-immunoreactivity in the hippocampus in addition to inducing DND. On day 4, MK-801 protected ischemia-induced DND in the hippocampal CA1 subfield. However, MK-801 had no effect against the decrease in ACh release in spite of protection of the decrease in ChAT-immunoreactivity. On day 7 and 14, no protective effect of MK801 was observed in all estimations. It became clear that the mechanism of cholinergic terminal dysfunction is different from that involved in pyramidal cell death, i.e., excitative neurotoxicity induced by overabundant extracellular glutamate. Pentobarbital also provided protection against DND. However, protective effects of pentobarbital on the decrease in ACh release and the low ChAT-immunoreactivity were incomplete. Our present study indicated a limitation on the efficacy of NMDA receptor antagonist and barbiturate against cerebral ischemia. Q 1997 Elsevier Science Inc.
the presynaptic terminals has so far been carried out. We, therefore, focused on the changes of presynaptic cholinergic function after ischemia, because the central cholinergic system may play a crucial role in learning and memory. Recently, we reported that the ability to release acetylcholine ( ACh ) and immunoreactivity for choline acetyltransferase ( ChAT ) in the hippocampal cholinergic terminals were gradually decreased prior to DND [ 9 – 11] . These findings show the importance and necessity of presynaptic neurochemical estimation in addition to postsynaptic morphological observation in the investigation of the mechanisms of cerebrovascular disease accompanied by dementia. MK-801, a noncompetitive N-methyl- D -aspartate ( NMDA ) receptor antagonist, prevents neurodegeneration induced by cerebral ischemia, hypoxic-ischemia, and carbon monoxide exposure [ 3,5,8,13 ] . Barbiturates are also known to prevent postischemic cell death in selected vulnerable regions including CA1 pyramidal cells in experimental animals [ 4,13,19 ] . Moreover, inducing coma treatment with barbiturates has been an effective therapeutic method for cerebral ischemia [17 ] . In the present study, we attempted to investigate whether MK-801 and pentobarbital, which have neuroprotective effects, protect presynaptic cholinergic terminal damage after ischemia.
KEY WORDS: Ischemia, Acetylcholine, Choline acetyltransferase, Microdialysis, Immunohistochemistry.
MATERIALS AND METHODS Animals and Experimental Schedule Male and female Mongolian gerbils weighing 50–70 g were used. The animals were housed under 12 h light/12 h dark conditions with ad lib access to food and water (except during the dialysis session) in a regulated room (23 { 17C, 55 { 5% humidity). In the neurochemical estimation, the guide cannula was installed in the brain. Two days after the guide cannula had been installed, transient cerebral ischemia was carried out. ( / ) MK801 hydrogen maleate (3 mg/kg, i.p.) or pentobarbital sodium (50 mg/kg, i.p.) were injected 1 h or 30 min before ischemia, respectively. These time periods were adopted according to previous reports that MK-801 or pentobarbital protected the isch-
INTRODUCTION Cerebral ischemic attack leads to delayed neuronal death ( DND ) in the CA1 pyramidal cell layer of hippocampus [14,18 ] . DND has received much attention in attempts to elucidate the pathogenesis of dementia in cerebrovascular disease, and there are some reports relating to the mechanism of ischemic cell death using various ischemic animal models. It has been reported, for example, that a massive release of excitatory amino acids is involved in the development of ischemic CA1 cell death [1] . However, no functional analysis of 1
To whom requests for reprints should be addressed.
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emic neuronal death [3,13]. In the vehicle-treated group, saline was injected intraperitoneally. The animals were divided into three groups for the neurochemical, morphological, and immunohistochemical estimations (five to seven animals were used for each group at each tested point). These estimations were carried out 4, 7, or 14 days after ischemia-recirculation. The experimental protocols were approved by the committee for Animal Experimentation at Gunma University, School of Medicine, and meet the guidelines of the Japanese Association for Laboratory Animal Science. Guide Cannula Installation Under pentobarbital anesthesia (50 mg/kg, i.p.), surgery was performed stereotaxically, in the right or left hemisphere, with reference to the bregma and dura, and with the incisor bar set at the level of the interaural line. The guide cannula was implanted using the following coordinates: A Å 02.6 mm, L Å 2.0 mm, and V Å 0.5 mm. Cerebral Ischemia The bilateral common carotid arteries (CCA) were exposed under 2.5% halothane anesthesia (20% O2 and 80% N2 ). Immediately after exposure of the CCA the halothane vaporizer was turned off and blood flow was completely arrested for a period of 5 min. In the vehicle- and MK-801–treated group, rectal temperature was maintained at 38.0 { 0.57C using a thermoregulated heating plate for the period of ischemia. However, in the pentobarbital-treated group, rectal temperature averaged 35.4 { 0.57C immediately after ischemia, in spite of the use of the heating plate. After surgery, the animals were placed in a heated plastic cage (377C) for 2 h. Control animals were treated in the same manner except for the absence of CCA occlusion. Neurochemistry For neurochemical estimations, changes in extracellular levels of ACh in the hippocampal formation were analyzed by in vivo microdialysis, coupled with high-performance liquid chromatography with electrochemical detection ( HPLCECD ) [ 9,10 ] . A microdialysis probe ( Carnegie Medicine, 2 mm in membrane length and 0.5 mm in diameter ) was slowly inserted via a guide cannula into the dentate gyrus-CA1 area of the hippocampal formation. This procedure was conducted on 4, 7, or 14 days after ischemia-recirculation. The tip of the probe was extended 2 mm below the end of the guide cannula ( 2.5 mm from the surface of the skull ) . The probe was continuously perfused with Ringer solution ( 147.2 mM NaCl, 4 mM KCl and 2.3 mM CaCl2 ) at a rate of 2 ml /min. Two hours after the probe was inserted, eserine sulfate ( 100 mM ) was added to the perfusion medium. According to Kametani and Kawamura [12 ] , ACh levels obtained from rats perfused with 100 mM eserine are higher by about 150% than those perfused with 10 mM eserine. Beginning with a sample 45 min after the start of eserine perfusion, 30 ml samples were collected every 15 min. For samples 1 – 20, perfusion fluid contained eserine. KCl ( 100 mM ) was added to the perfusion medium in samples 5 – 6, and atropine sulfate ( 3 mM ) was added to samples 13 – 14. Immediately after sampling, 25 pmol ethylhomocholine was added to each sample as an internal standard. Basal ACh release reflects the total amount of samples 1 – 4. The increases of ACh release induced by KCl and atropine were maintained during samples 5 – 9 and 14 – 19, respectively. Therefore, the effects of ischemia on KCl- and atropine-induced ACh release
were calculated according to the total amount of ACh in samples 5 – 9 and 14 – 19 ( with the mean basal release subtracted ) . Morphology The animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and killed by decapitation 4, 7, or 14 days after ischemiarecirculation. Coronal sections of 10 or 20 mm thickness were cut from the frozen brain in a cryostat at 0187C. A section of 2.0 mm in length of the hippocampal CA1 pyramidal cell layer was selected from the serial sections, because this area corresponded to the measuring site of the extracellular ACh. This section was then stained with hematoxylin and processed by standard procedure for light microscopic observations. Immunohistochemistry The presence of ChAT in the hippocampal cholinergic terminals was measured immnohistochemically. The animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 0.9% saline containing heparin (500 U/100 ml), followed by 4% paraformaldehyde in 0.2 M phosphate buffer (PB), pH 7.4, at a flow rate of 10 ml/min for 30 min. The brains were removed and kept in a solution of 30% sucrose in 1/ 15 M PB containing 0.9% NaCl (PBS) for 24 h to prevent damage during the freezing procedure. They were then cut serially on a freezing microtome at 20 mm. The avidin–biotin–peroxidase complex (ABC) method [7], using Vectastain (Vector Laboratories), was adopted. The staining procedure consisted of the incubation steps described below. Free floating sections were incubated in 3% normal goat serum (NGS) in PBS that contained 0.3% Triton X-100 for 2 h (first step). Next, sections were incubated in anti-ChAT polyclonal antibody (Chemicon International Inc., diluted 1:4000) for 2 h at room temperature, and then for an additional 20–22 h at 47C (second step). Then, the sections were incubated in biotinylated secondary antibody (diluted 1:200) for 1 h (third step). After that, the sections were incubated in ABC reagent in PB (Elite ABC reagent, Vectastain) containing 0.3% Triton X-100 and 0.5 M NaCl for 1 h (fourth step). The sections were then reacted for 5 min in a solution of 0.1% diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2 in 0.1 M Tris-HCl buffer (pH 7.5, fifth step). Finally, the sections were mounted on slides in glycerin and processed by the standard procedure for light-microscopic observations. Statistical Analysis Values are presented as means { SEM. Significant differences between the two groups were evaluated using the Bonferroni test after one-way analysis of variance for multiple comparison at each day tested. RESULTS The neurochemical data are shown in Figs. 1 and 2. In the control group, depolarization through addition of KCl to the perfusion fluid produced an approximately threefold increase, and perfusion of atropine resulted in a fivefold increase from the basal release level of ACh (each value of KCl- and atropine-induced release is expressed as the total amount of ACh with the mean basal release subtracted). A concentration of basal ACh release was about 115 nM. KCl- and atropine-induced release of ACh decreased on 4, 7, and 14 days after ischemia-recirculation. MK801 had no effect against the postischemic decrease in ACh release on day 4, 7, and 14 (Fig. 1). In the group treated with pentobarbital, no significant effect on the postischemic decrease in ACh release was observed on day 7 and 14 (Fig. 2).
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MK-801, PENTOBARBITAL, AND CHOLINERGIC DAMAGE
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FIG. 1. Release of acetylcholine (ACh) in the hippocampus in ischemic gerbil after treatment with MK-801. MK-801 (3 mg/kg) and vehicle (saline) were injected intraperitoneally 1 h before 5 min ischemia. Data are means { SEM from five animals. The control shows a nonischemic control. *p õ 0.05 vs. control (Bonferroni test).
Morphological and immunohistochemical changes are presented in Figs. 3 and 4. The morphological and immunohistochemical patterns were virtually identical among individual animals in each experimental group. In the control animals, pyramidal cells were clear and ChAT-positive structure (ChAT immunoreactivity) was seen preferentially in the stratum radiatum. In the ischemia-induced, vehicle-injected gerbils, from the fourth day, almost all pyramidal cells atrophied and disappeared, that is, DND and ChAT immunoreactivity also disappeared. MK801 protected ischemia-induced DND and disappearance in ChAT-immunoreactivity in the hippocampal CA1 subfield on day 4 (Fig. 3). On days 7 and 14, no protective effect of MK801 was observed (data not shown). In the pentobarbital-administered group, CA1 pyramidal cells survived on day 14, and a faint ChAT immunoreactivity was observed but not in the outer part of the stratum radiatum (Fig. 4). The same results were observed at 7 days after ischemia (data not shown).
DISCUSSION The change of extracellular ACh concentration indicates a dysfunction of the presynaptic terminal in the cholinergic neuron. ChAT immunoreactivity reveals a survival of the presynaptic terminal in the cholinergic neuron because ChAT is present in the cholinergic neuroterminal. We have reported that the presynaptic dysfunction and destruction of the cholinergic nerve terminal, following cerebral ischemia, precede the death of CA1 pyramidal cell [9–11]. A massive release of excitatory amino acids and an activation of NMDA receptor are involved in the development of ischemic CA1 cell death because cerebral ischemia elevates the extracellular concentration of glutamate [1] and MK-801 protects delayed neuronal death in various ischemic animals [3,5,8,13]. In the present study, MK-801 protected ischemiainduced DND and structural destruction of the cholinergic terminal on day 4. However, MK-801 could not improve the dys-
FIG. 2. Release of acetylcholine (ACh) in the hippocampus in ischemic gerbil after treatment with pentobarbital. Pentobarbital (50 mg/kg) and vehicle (saline) were injected intraperitoneally 30 min before 5 min ischemia. Data are means { SEM from seven animals. The control shows a nonischemic control. *p õ 0.05 vs. control (Bonferroni test).
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ISHIMARU ET AL. enhancement of the GABAA receptor binding [16]. In this study, rectal temperatures immediately after ischemia in the pentobarbital-treated group, were lower than those in the vehicle-treated group. Depression of brain energy metabolism, by means such as hypothermia, protects against postischemic cell damage [2,15]. It is, therefore, possible that the protective effect against CA1 cell death resulted from hypothermia induced by pentobarbital. However, pretreatment with pentobarbital improves neither dysfunction nor destruction of the cholinergic terminals. The results of our study clearly showed the inefficacy of both MK-801 and pentobarbital against postischemic dysfunction in the hippocampal cholinergic neuron in spite of the efficacy of these drugs against postischemic morphological changes. Although it
FIG. 3. Representative photomicrographs of pyramidal cells (left side) and choline acetyltransferase (ChAT)-positive structures (right side) in the hippocampal CA1 subfield in ischemic gerbil after treatment with MK-801. MK-801 (3 mg/kg) and vehicle (saline) were injected intraperitoneally 1 h before 5 min ischemia. Decapitation was carried out on the 4th day after ischemia. In the vehicle-treated animal, CA1 pyramidal cell death had occurred, and ChAT-immunoreactivity in the stratum radiatum was not seen. MK-801 protected CA1 pyramidal cell death and destruction of ChAT-positive structure. The control shows a nonischemic control. p: Stratum pyramidale. r: Stratum radiatum. Bar Å 100 mm.
function of the cholinergic terminals. From these results, we suggested that the cholinergic dysfunction may have no relation with the excitative neurotoxicity induced by overabundant extracellular glutamate. In addition, MK-801 did not protected DND on days 7 and 14. Such incomplete neuroprotective effect of MK801 may indicate a possibility that something else, which is different from the excitative neurotoxicity, that is, the postischemic terminal damage, is one factor of the mechanisms of DND. Pentobarbital resulted in complete protection against CA1 cell death in the hippocampal CA1 subfield on day 14. This result is in accordance with previous reports [4,13,19]. The neuroprotective mechanism of pentobarbital is generally considered to operate through the central nervous system depression or through
FIG. 4. Representative photomicrographs of pyramidal cells (left side) and choline acetyltransferase (ChAT)-positive structures (right side) in the hippocampal CA1 subfield in ischemic gerbil after treatment with pentobarbital. Pentobarbital (50 mg/kg) and vehicle (saline) were injected intraperitoneally 30 min before 5 min ischemia. Decapitation was carried out on the 14th day after ischemia. In the vehicle-treated animal, CA1 pyramidal cell death had occurred, and ChAT-immunoreactivity in the stratum radiatum was not seen. Pentobarbital protected CA1 pyramidal cell death, but did not quite protect destruction of ChAT-positive structure. The control shows a nonischemic control. p: Stratum pyramidale. r: Stratum radiatum. Bar Å 100 mm.
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MK-801, PENTOBARBITAL, AND CHOLINERGIC DAMAGE seems that the mechanism of cholinergic dysfunction may be different from that involved in postsynaptic cell death, it remains to explicate essentially the mechanism of postischemic cholinergic dysfunction. Recently, it has become clear that the survival of cholinergic neurons projecting to the hippocampus is maintained by nerve growth factor (NGF) [6]. The therapeutic effects of some neurotrophic factors on presynaptic dysfunction in the hippocampal cholinergic neurons following ischemia are to be expected because the damage of presynaptic terminals may be caused by a deficit of neurotrophic factors. In conclusion, our study indicates a limitation on the efficacy of NMDA receptor antagonist and barbiturate therapy, in that the hippocampal cholinergic system plays an important role in learning and memory. The development of neuroprotectants that have beneficial effects against cholinergic dysfunction is necessary for therapeutics of ischemic disease. REFERENCES 1. Benveniste, H.; Drejer, J.; Schousboe, A.; Diemer, N. H. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43:1369–1374; 1984. 2. Busto, R.; Dietrich, W. D.; Globus, M. Y.-T.; Valdes, I.; Scheinberg, P.; Ginsberg, M. D. Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J. Cereb. Blood Flow Metab. 7:729–738; 1987. 3. Gill, R.; Foster, A. C.; Woodruff, G. N. Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil. J. Neurosci. 7:3343–3349; 1987. 4. Hallmayer, J.; Hossmann, K. A.; Mies, G. Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes. Acta Neuropathol. 68:27–31; 1985. 5. Hattori, H.; Morin, A. M.; Schwartz, P. H.; Fujikawa, D. G.; Wasterlain, C. G. Posthypoxic treatment with MK-801 reduces hypoxicischemic damage in the neonatal rat. Neurology 39:713–718; 1989. 6. Hefti, F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transection. J. Neurosci. 6:2155–2162; 1986. 7. Hsu, S. M.; Raine, L.; Fanger, H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: A comparison
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between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577–580; 1981. Ishimaru, H.; Katoh, A.; Suzuki, H.; Fukuta, T.; Kameyama, T.; Nabeshima, T. Effects of N-methyl-D-aspartate receptor antagonists on carbon monoxide-induced brain damage in mice. J. Pharmacol. Exp. Ther. 261:349–352; 1992. Ishimaru, H.; Takahashi, A.; Ikarashi, Y.; Maruyama, Y. Temporal changes in extracellular acetylcholine and CA1 pyramidal cells in gerbil hippocampus following transient cerebral ischemia. Brain Res. 639:66–72; 1994. Ishimaru, H.; Takahashi, A.; Ikarashi, Y.; Maruyama, Y. Effect of transient cerebral ischemia on acetylcholine release in the gerbil hippocampus. Neuroreport 5:601–604; 1994. Ishimaru, H.; Takahashi, A.; Ikarashi, Y.; Maruyama, Y. Immunohistochemical and neurochemical studies of hippocampal cholinergic neurons after ischaemia. Neuroreport 6:557–560; 1995. Kametani, H.; Kawamura, H. Alterations in acetylcholine release in the rat hippocampus during sleep-wakefulness detected by intracerebral dialysis. Life Sci. 47:421–426; 1990. Kato, H.; Araki, T.; Kogure, K. Role of the excitotoxic mechanism in the deveropment of neuronal damage following repeated brief cerebral ischemia in the gerbil: Protective effects of MK-801 and pentobarbital. Brain Res. 516:175–179; 1990. Kirino, T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239:57–69; 1982. Okada, Y.; Tanimoto, M.; Yoneda, K. The protective effect of hypothermia on reversibility in the neuronal function of the hippocampal slices during long lasting anoxia. Neurosci. Lett. 84:277–282; 1988. Olsen, R. W.; Wong, E. H. F.; Stauba, G. B.; Murakami, D.; King, R. G.; Fisher, J. B. Biochemical properties of the GABA/barbiturate/benzodiazepine receptor chloride ion channel complex. Adv. Exp. Med. Biol. 175:205–219; 1978. Pappas, T. N.; Mironovich, R. O. Barbiturate-induced coma to protect against cerebral ischemia and increased intracranial pressure. Am. J. Hosp. Pharm. 38:494–498; 1981. Pulsinelli, W. A.; Briely, J. B.; Plum, F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11:491–498; 1982. Sternau, L. L.; Lust, W. D.; Ricci, A. J.; Ratcheson, R. Role for gaminobutyric acid in selective vulnerability in gerbils. Stroke 20:281–287; 1989.
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Effects of MK-801 and Pentobarbital on Cholinergic Terminal Damage and Delayed Neuronal Death in the Ischemic Gerbil Hippocampus HIROHISA ISHIMARU, 1 AKIRA TAKAHASHI, YASUSHI IKARASHI AND YUJI MARUYAMA Department of Neuropsychopharmacology (Tsumura), Gunma University School of Medicine, 3-39-22 showa-machi, Maebashi-shi, Gunma 371, Japan
ABSTRACT: The present study covers both the effects of MK801, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, and pentobarbital on cholinergic terminal damage and delayed neuronal death (DND) in ischemic gerbil. To study the above effects, in vivo microdialysis, immunohistochemical, and morphological techniques were used. MK801 (3 mg/kg) or pentobarbital (50 mg/kg) were injected intraperitoneally 1 h or 30 min before 5 min ischemia, respectively. Each estimation was then carried out 4, 7, or 14 days after ischemia. Ischemia induced a significant decrease in acetylcholine (ACh) release and a disappearance of choline acetyltransferase (ChAT)-immunoreactivity in the hippocampus in addition to inducing DND. On day 4, MK-801 protected ischemia-induced DND in the hippocampal CA1 subfield. However, MK-801 had no effect against the decrease in ACh release in spite of protection of the decrease in ChAT-immunoreactivity. On day 7 and 14, no protective effect of MK801 was observed in all estimations. It became clear that the mechanism of cholinergic terminal dysfunction is different from that involved in pyramidal cell death, i.e., excitative neurotoxicity induced by overabundant extracellular glutamate. Pentobarbital also provided protection against DND. However, protective effects of pentobarbital on the decrease in ACh release and the low ChAT-immunoreactivity were incomplete. Our present study indicated a limitation on the efficacy of NMDA receptor antagonist and barbiturate against cerebral ischemia. Q 1997 Elsevier Science Inc.
the presynaptic terminals has so far been carried out. We, therefore, focused on the changes of presynaptic cholinergic function after ischemia, because the central cholinergic system may play a crucial role in learning and memory. Recently, we reported that the ability to release acetylcholine ( ACh ) and immunoreactivity for choline acetyltransferase ( ChAT ) in the hippocampal cholinergic terminals were gradually decreased prior to DND [ 9 – 11] . These findings show the importance and necessity of presynaptic neurochemical estimation in addition to postsynaptic morphological observation in the investigation of the mechanisms of cerebrovascular disease accompanied by dementia. MK-801, a noncompetitive N-methyl- D -aspartate ( NMDA ) receptor antagonist, prevents neurodegeneration induced by cerebral ischemia, hypoxic-ischemia, and carbon monoxide exposure [ 3,5,8,13 ] . Barbiturates are also known to prevent postischemic cell death in selected vulnerable regions including CA1 pyramidal cells in experimental animals [ 4,13,19 ] . Moreover, inducing coma treatment with barbiturates has been an effective therapeutic method for cerebral ischemia [17 ] . In the present study, we attempted to investigate whether MK-801 and pentobarbital, which have neuroprotective effects, protect presynaptic cholinergic terminal damage after ischemia.
KEY WORDS: Ischemia, Acetylcholine, Choline acetyltransferase, Microdialysis, Immunohistochemistry.
MATERIALS AND METHODS Animals and Experimental Schedule Male and female Mongolian gerbils weighing 50–70 g were used. The animals were housed under 12 h light/12 h dark conditions with ad lib access to food and water (except during the dialysis session) in a regulated room (23 { 17C, 55 { 5% humidity). In the neurochemical estimation, the guide cannula was installed in the brain. Two days after the guide cannula had been installed, transient cerebral ischemia was carried out. ( / ) MK801 hydrogen maleate (3 mg/kg, i.p.) or pentobarbital sodium (50 mg/kg, i.p.) were injected 1 h or 30 min before ischemia, respectively. These time periods were adopted according to previous reports that MK-801 or pentobarbital protected the isch-
INTRODUCTION Cerebral ischemic attack leads to delayed neuronal death ( DND ) in the CA1 pyramidal cell layer of hippocampus [14,18 ] . DND has received much attention in attempts to elucidate the pathogenesis of dementia in cerebrovascular disease, and there are some reports relating to the mechanism of ischemic cell death using various ischemic animal models. It has been reported, for example, that a massive release of excitatory amino acids is involved in the development of ischemic CA1 cell death [1] . However, no functional analysis of 1
To whom requests for reprints should be addressed.
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emic neuronal death [3,13]. In the vehicle-treated group, saline was injected intraperitoneally. The animals were divided into three groups for the neurochemical, morphological, and immunohistochemical estimations (five to seven animals were used for each group at each tested point). These estimations were carried out 4, 7, or 14 days after ischemia-recirculation. The experimental protocols were approved by the committee for Animal Experimentation at Gunma University, School of Medicine, and meet the guidelines of the Japanese Association for Laboratory Animal Science. Guide Cannula Installation Under pentobarbital anesthesia (50 mg/kg, i.p.), surgery was performed stereotaxically, in the right or left hemisphere, with reference to the bregma and dura, and with the incisor bar set at the level of the interaural line. The guide cannula was implanted using the following coordinates: A Å 02.6 mm, L Å 2.0 mm, and V Å 0.5 mm. Cerebral Ischemia The bilateral common carotid arteries (CCA) were exposed under 2.5% halothane anesthesia (20% O2 and 80% N2 ). Immediately after exposure of the CCA the halothane vaporizer was turned off and blood flow was completely arrested for a period of 5 min. In the vehicle- and MK-801–treated group, rectal temperature was maintained at 38.0 { 0.57C using a thermoregulated heating plate for the period of ischemia. However, in the pentobarbital-treated group, rectal temperature averaged 35.4 { 0.57C immediately after ischemia, in spite of the use of the heating plate. After surgery, the animals were placed in a heated plastic cage (377C) for 2 h. Control animals were treated in the same manner except for the absence of CCA occlusion. Neurochemistry For neurochemical estimations, changes in extracellular levels of ACh in the hippocampal formation were analyzed by in vivo microdialysis, coupled with high-performance liquid chromatography with electrochemical detection ( HPLCECD ) [ 9,10 ] . A microdialysis probe ( Carnegie Medicine, 2 mm in membrane length and 0.5 mm in diameter ) was slowly inserted via a guide cannula into the dentate gyrus-CA1 area of the hippocampal formation. This procedure was conducted on 4, 7, or 14 days after ischemia-recirculation. The tip of the probe was extended 2 mm below the end of the guide cannula ( 2.5 mm from the surface of the skull ) . The probe was continuously perfused with Ringer solution ( 147.2 mM NaCl, 4 mM KCl and 2.3 mM CaCl2 ) at a rate of 2 ml /min. Two hours after the probe was inserted, eserine sulfate ( 100 mM ) was added to the perfusion medium. According to Kametani and Kawamura [12 ] , ACh levels obtained from rats perfused with 100 mM eserine are higher by about 150% than those perfused with 10 mM eserine. Beginning with a sample 45 min after the start of eserine perfusion, 30 ml samples were collected every 15 min. For samples 1 – 20, perfusion fluid contained eserine. KCl ( 100 mM ) was added to the perfusion medium in samples 5 – 6, and atropine sulfate ( 3 mM ) was added to samples 13 – 14. Immediately after sampling, 25 pmol ethylhomocholine was added to each sample as an internal standard. Basal ACh release reflects the total amount of samples 1 – 4. The increases of ACh release induced by KCl and atropine were maintained during samples 5 – 9 and 14 – 19, respectively. Therefore, the effects of ischemia on KCl- and atropine-induced ACh release
were calculated according to the total amount of ACh in samples 5 – 9 and 14 – 19 ( with the mean basal release subtracted ) . Morphology The animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and killed by decapitation 4, 7, or 14 days after ischemiarecirculation. Coronal sections of 10 or 20 mm thickness were cut from the frozen brain in a cryostat at 0187C. A section of 2.0 mm in length of the hippocampal CA1 pyramidal cell layer was selected from the serial sections, because this area corresponded to the measuring site of the extracellular ACh. This section was then stained with hematoxylin and processed by standard procedure for light microscopic observations. Immunohistochemistry The presence of ChAT in the hippocampal cholinergic terminals was measured immnohistochemically. The animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and perfused transcardially with 0.9% saline containing heparin (500 U/100 ml), followed by 4% paraformaldehyde in 0.2 M phosphate buffer (PB), pH 7.4, at a flow rate of 10 ml/min for 30 min. The brains were removed and kept in a solution of 30% sucrose in 1/ 15 M PB containing 0.9% NaCl (PBS) for 24 h to prevent damage during the freezing procedure. They were then cut serially on a freezing microtome at 20 mm. The avidin–biotin–peroxidase complex (ABC) method [7], using Vectastain (Vector Laboratories), was adopted. The staining procedure consisted of the incubation steps described below. Free floating sections were incubated in 3% normal goat serum (NGS) in PBS that contained 0.3% Triton X-100 for 2 h (first step). Next, sections were incubated in anti-ChAT polyclonal antibody (Chemicon International Inc., diluted 1:4000) for 2 h at room temperature, and then for an additional 20–22 h at 47C (second step). Then, the sections were incubated in biotinylated secondary antibody (diluted 1:200) for 1 h (third step). After that, the sections were incubated in ABC reagent in PB (Elite ABC reagent, Vectastain) containing 0.3% Triton X-100 and 0.5 M NaCl for 1 h (fourth step). The sections were then reacted for 5 min in a solution of 0.1% diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2 in 0.1 M Tris-HCl buffer (pH 7.5, fifth step). Finally, the sections were mounted on slides in glycerin and processed by the standard procedure for light-microscopic observations. Statistical Analysis Values are presented as means { SEM. Significant differences between the two groups were evaluated using the Bonferroni test after one-way analysis of variance for multiple comparison at each day tested. RESULTS The neurochemical data are shown in Figs. 1 and 2. In the control group, depolarization through addition of KCl to the perfusion fluid produced an approximately threefold increase, and perfusion of atropine resulted in a fivefold increase from the basal release level of ACh (each value of KCl- and atropine-induced release is expressed as the total amount of ACh with the mean basal release subtracted). A concentration of basal ACh release was about 115 nM. KCl- and atropine-induced release of ACh decreased on 4, 7, and 14 days after ischemia-recirculation. MK801 had no effect against the postischemic decrease in ACh release on day 4, 7, and 14 (Fig. 1). In the group treated with pentobarbital, no significant effect on the postischemic decrease in ACh release was observed on day 7 and 14 (Fig. 2).
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FIG. 1. Release of acetylcholine (ACh) in the hippocampus in ischemic gerbil after treatment with MK-801. MK-801 (3 mg/kg) and vehicle (saline) were injected intraperitoneally 1 h before 5 min ischemia. Data are means { SEM from five animals. The control shows a nonischemic control. *p õ 0.05 vs. control (Bonferroni test).
Morphological and immunohistochemical changes are presented in Figs. 3 and 4. The morphological and immunohistochemical patterns were virtually identical among individual animals in each experimental group. In the control animals, pyramidal cells were clear and ChAT-positive structure (ChAT immunoreactivity) was seen preferentially in the stratum radiatum. In the ischemia-induced, vehicle-injected gerbils, from the fourth day, almost all pyramidal cells atrophied and disappeared, that is, DND and ChAT immunoreactivity also disappeared. MK801 protected ischemia-induced DND and disappearance in ChAT-immunoreactivity in the hippocampal CA1 subfield on day 4 (Fig. 3). On days 7 and 14, no protective effect of MK801 was observed (data not shown). In the pentobarbital-administered group, CA1 pyramidal cells survived on day 14, and a faint ChAT immunoreactivity was observed but not in the outer part of the stratum radiatum (Fig. 4). The same results were observed at 7 days after ischemia (data not shown).
DISCUSSION The change of extracellular ACh concentration indicates a dysfunction of the presynaptic terminal in the cholinergic neuron. ChAT immunoreactivity reveals a survival of the presynaptic terminal in the cholinergic neuron because ChAT is present in the cholinergic neuroterminal. We have reported that the presynaptic dysfunction and destruction of the cholinergic nerve terminal, following cerebral ischemia, precede the death of CA1 pyramidal cell [9–11]. A massive release of excitatory amino acids and an activation of NMDA receptor are involved in the development of ischemic CA1 cell death because cerebral ischemia elevates the extracellular concentration of glutamate [1] and MK-801 protects delayed neuronal death in various ischemic animals [3,5,8,13]. In the present study, MK-801 protected ischemiainduced DND and structural destruction of the cholinergic terminal on day 4. However, MK-801 could not improve the dys-
FIG. 2. Release of acetylcholine (ACh) in the hippocampus in ischemic gerbil after treatment with pentobarbital. Pentobarbital (50 mg/kg) and vehicle (saline) were injected intraperitoneally 30 min before 5 min ischemia. Data are means { SEM from seven animals. The control shows a nonischemic control. *p õ 0.05 vs. control (Bonferroni test).
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ISHIMARU ET AL. enhancement of the GABAA receptor binding [16]. In this study, rectal temperatures immediately after ischemia in the pentobarbital-treated group, were lower than those in the vehicle-treated group. Depression of brain energy metabolism, by means such as hypothermia, protects against postischemic cell damage [2,15]. It is, therefore, possible that the protective effect against CA1 cell death resulted from hypothermia induced by pentobarbital. However, pretreatment with pentobarbital improves neither dysfunction nor destruction of the cholinergic terminals. The results of our study clearly showed the inefficacy of both MK-801 and pentobarbital against postischemic dysfunction in the hippocampal cholinergic neuron in spite of the efficacy of these drugs against postischemic morphological changes. Although it
FIG. 3. Representative photomicrographs of pyramidal cells (left side) and choline acetyltransferase (ChAT)-positive structures (right side) in the hippocampal CA1 subfield in ischemic gerbil after treatment with MK-801. MK-801 (3 mg/kg) and vehicle (saline) were injected intraperitoneally 1 h before 5 min ischemia. Decapitation was carried out on the 4th day after ischemia. In the vehicle-treated animal, CA1 pyramidal cell death had occurred, and ChAT-immunoreactivity in the stratum radiatum was not seen. MK-801 protected CA1 pyramidal cell death and destruction of ChAT-positive structure. The control shows a nonischemic control. p: Stratum pyramidale. r: Stratum radiatum. Bar Å 100 mm.
function of the cholinergic terminals. From these results, we suggested that the cholinergic dysfunction may have no relation with the excitative neurotoxicity induced by overabundant extracellular glutamate. In addition, MK-801 did not protected DND on days 7 and 14. Such incomplete neuroprotective effect of MK801 may indicate a possibility that something else, which is different from the excitative neurotoxicity, that is, the postischemic terminal damage, is one factor of the mechanisms of DND. Pentobarbital resulted in complete protection against CA1 cell death in the hippocampal CA1 subfield on day 14. This result is in accordance with previous reports [4,13,19]. The neuroprotective mechanism of pentobarbital is generally considered to operate through the central nervous system depression or through
FIG. 4. Representative photomicrographs of pyramidal cells (left side) and choline acetyltransferase (ChAT)-positive structures (right side) in the hippocampal CA1 subfield in ischemic gerbil after treatment with pentobarbital. Pentobarbital (50 mg/kg) and vehicle (saline) were injected intraperitoneally 30 min before 5 min ischemia. Decapitation was carried out on the 14th day after ischemia. In the vehicle-treated animal, CA1 pyramidal cell death had occurred, and ChAT-immunoreactivity in the stratum radiatum was not seen. Pentobarbital protected CA1 pyramidal cell death, but did not quite protect destruction of ChAT-positive structure. The control shows a nonischemic control. p: Stratum pyramidale. r: Stratum radiatum. Bar Å 100 mm.
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MK-801, PENTOBARBITAL, AND CHOLINERGIC DAMAGE seems that the mechanism of cholinergic dysfunction may be different from that involved in postsynaptic cell death, it remains to explicate essentially the mechanism of postischemic cholinergic dysfunction. Recently, it has become clear that the survival of cholinergic neurons projecting to the hippocampus is maintained by nerve growth factor (NGF) [6]. The therapeutic effects of some neurotrophic factors on presynaptic dysfunction in the hippocampal cholinergic neurons following ischemia are to be expected because the damage of presynaptic terminals may be caused by a deficit of neurotrophic factors. In conclusion, our study indicates a limitation on the efficacy of NMDA receptor antagonist and barbiturate therapy, in that the hippocampal cholinergic system plays an important role in learning and memory. The development of neuroprotectants that have beneficial effects against cholinergic dysfunction is necessary for therapeutics of ischemic disease. REFERENCES 1. Benveniste, H.; Drejer, J.; Schousboe, A.; Diemer, N. H. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43:1369–1374; 1984. 2. Busto, R.; Dietrich, W. D.; Globus, M. Y.-T.; Valdes, I.; Scheinberg, P.; Ginsberg, M. D. Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J. Cereb. Blood Flow Metab. 7:729–738; 1987. 3. Gill, R.; Foster, A. C.; Woodruff, G. N. Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil. J. Neurosci. 7:3343–3349; 1987. 4. Hallmayer, J.; Hossmann, K. A.; Mies, G. Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes. Acta Neuropathol. 68:27–31; 1985. 5. Hattori, H.; Morin, A. M.; Schwartz, P. H.; Fujikawa, D. G.; Wasterlain, C. G. Posthypoxic treatment with MK-801 reduces hypoxicischemic damage in the neonatal rat. Neurology 39:713–718; 1989. 6. Hefti, F. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transection. J. Neurosci. 6:2155–2162; 1986. 7. Hsu, S. M.; Raine, L.; Fanger, H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: A comparison
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