Journal of Cerebral Blood Flow and Metabolism 12:2-11 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Postischemic Blockade of AMPA but not NMDA Receptors Mitigates Neuronal Damage in the Rat Brain Following Transient Severe Cerebral Ischemia B. N ellgard
and T. Wieloch
Laboratory for Experimental Brain Research, University Hospital, Lund University, Lund, Sweden
Summary: Olutamatergic transmission is an important factor in the development of neuronal death following transient cerebral ischemia. In this investigation the ef fects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists on neuronal damage were studied in rats exposed to 10 min of transient cerebral ischemia in duced by bilateral common carotid occlusion combined with hypotension. The animals were treated with a blocker of the ionotropic quisqualate or a-amino-3hydroxy-5-methyl-4-isoxazole (AMPA) receptor, 2.3dihydroxy-6-nitro-7 -sulfamoyl-benzo(F)q uinoxaline (NBQX), given postischemia as an intraperitoneal bolus dose of 30 mg kg-I followed by an intravenous infusion of 75 Il-g min -I for 6 h, or with the noncompetitive NMDA receptor blocker dizocilpine (MK-801) given 1 mg kg-I i.p. at recirculation and 3 h postischemia, or with the competitive NMDA receptor antagonist DL-(E)-2amino-4-methyl-5-phosphono-3-pentenoic acid (COP
40116), 5 mg kg-I, given intraperitoneally at recircula tion. Treatment with NBQX provided a significant reduc tion of neuronal damage in the hippocampal CAl area by 44-69%, with the largest relative decrease in the temporal part of the hippocampus. In neocortex a significant de crease in the number of necrotic neurons was also noted. No protection could be seen following postischemic treat ment with dizocilpine or COP 40116. Our data demon strate that AMPA but not NMDA receptor antagonists decrease neuronal damage following transient severe ce rebral ischemia in the rat and that the protection by NBQX may be dependent on the severity of the ischemic insult. We propose that the AMPA receptor-mediated neurotoxicity could be due to ischemia-induced changes in the control mechanisms of AMPA receptor-coupled processes or to changes of AMPA receptor characteris tics. Key Words: Brain-Glutamate-Ischemia-Neuro nal death-Receptors.
The synaptic release of the excitatory amino acid glutamate following an ischemic insult is considered to be an important factor in the development of neuronal death. This notion is supported by exper iments where surgical transection of glutamate containing afferents to the hippocampus was per formed, thereby inhibiting glutamatergic neuro transmission and leading to a decrease in ischemic cell damage ipsilateral to the transection (Wieloch et aI., 1985; Onodera et aI., 1986; J�rgensen et aI.,
1987). However, it is not fully understood which of the glutamate receptors activate the deleterious in tracellular processes 'leading to cell death. Follow ing the first report on neuronal protection by an N-methyl-o-aspartate (NMDA) receptor antagonist (Simon et aI., 1984), research has mainly been fo cused on blockade of this receptor complex. In a vast number of investigations, significant neuronal protection by competitive or noncompetitive NMDA receptor antagonists has been achieved in in vitro models of hypoxia, ischemia, and hypogly cemia (Weiss et aI., 1986; Goldberg et aI., 1987; Monyer et aI., 1989). Furthermore, protection by NMDA antagonists has also been demonstrated in in vivo models of transient cerebral ischemia (Boast et aI., 1988; Gill et aI., 1988; Rod et aI., 1990; Swan and Meldrum, 1990), focal ischemia (Kochhar et aI., 1988; Park et aI., 1988; Steinberg et aI., 1988), and hypoglycemic coma (Wieloch, 1985; Westerberg et
Received April 30, 1991; revised June 18, 1991; accepted June 27, 1991. Address correspondence and reprint requests to Dr. B. Nel\gard at Laboratory for Experimental Brain Research, Lund University, Lund Hospital, S-221 85 Lund, Sweden. Abbreviations used: AMPA, 0l-amino-3-hydroxy-5-methyl-4isoxazole; BBB, blood-brain barrier; COP 40116, DL-(E)-2amino-4-methyl-5-phosphono-3-pentenoic acid; NBQX, 2.3dihydroxy-6-nitro-7-sulfamoyl-benzo(E)quinoxaline; NMOA, N-methyl-D-aspartate; 2-VO, two-vessel occlusion.
2
AMPA RECEPTORS AND ISCHEMIC BRAIN DAMAGE ai., 1988; Papagapiou and Auer, 1990). Neuronal protection by NMDA antagonists in models of tran sient severe ischemia has recently been questioned (Fleischer et aI., 1989; Michenfelder et ai., 1989; Sterz et ai., 1989; Lanier et ai., 1990; Buchan et ai., 199 1; Nellgard et aI., 1991). For example, in a model of cardiac arrest ischemia, dizocilpine, a noncompetitive NMDA antagonist, did not mitigate neuronal damage in the hippocampus (Nellgard et ai., 199 1) despite the protective effects of surgical lesions to the glutamate system in a similar experi mental paradigm (Wieloch et ai., 1985). The discovery of a new class of compounds, the quinoxalinediones (Honore et ai., 1988), that block non-NMDA glutamate receptors, including the ion otropic quisqualate receptor or a-amino-3-hydroxy5-methyl-4-isoxazole (AMPA) receptor (Watkins and Evans, 198 1), has greatly facilitated the study of these glutamate receptor subclasses. The quinox alinedione 2.3-dihydroxy-6-nitro-7-sulfamoyl benzo(F) quinoxaline (NBQX) is an AMPA recep tor antagonists that passes the blood-brain barrier (BBB) and diminishes neuronal damage following transient cerebral ischemia in the gerbil (Sheardown et ai., 1990). The aim of this study was to investigate and com pare the effects of NMDA and AMPA receptor an tagonists, administered in the postischemic phase, on neuronal damage in the hippocampal CAl region and cerebral neocortex, following 10 min of two vessel occlusion (2-YO) ischemia (Smith et ai., 1984). The following receptor blockers that pass the BBB were chosen: the noncompetitive NMDA re ceptor antagonist dizocilpine (MK-80 1) (Cline schmidt et al., 1982), the competitive NMDA recep tor a n t a g o n i s t DL-(E) - 2 - a m i n o - 4 - m e t h y l - 5 phosphono-3-pentenoic acid (CGP 40116) (Fagg et aI. , 1989), and the AMPA receptor antagonist NBQX (Sheardown et ai., 1990). MATERIALS AND METHODS
The surgical procedures were approved by the local ethical committee at Lund University. Male Wistar rats (MjiSllegaard A/S, Denmark), with an average weight of 322 g, were used in this study. Prior to surgery the ani mals were fasted overnight with free access to tap water. Anesthesia was induced by placing the rat in a jar with 3% isoflurane in a mixture of oxygen/nitrous oxide (30:70%). Following intubation with a plastic tube, they were arti ficially ventilated by a rodent respirator (7025 Rodent Ventilator; Ugo Basile Biological Research Appartus, Comeno, Italy). Anesthesia was maintained with 1-2% isoflurane in the oxygen/nitrous oxide (30:70%) gas mix ture. An external jugular vein catheter was then inserted and positioned in the superior caval vein. A tail artery catheter and one vein catheter were also inserted to allow blood sampling, arterial blood pressure recording, and
3
infusions of drugs. The arterial blood pressure was mea sured and recorded continuously until the rats could be extubated. In all animals both common carotid arteries were exposed and encircled by loose ligatures. Arterial blood samples (200 ILl) were collected 15 min prior to ischemia and 15 min postischemia to measure blood gases and blood glucose. If blood gases could not be corrected (Pao2> 90 mm Hg, Paco2 35-45 mm Hg, pH 7.35-7.45) by adjustments of tidal volume or frequency (normally f 85) of the rodent ventilator, or if the blood glucose was 0
a &> 0 0
60
a;
S3
0
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-8(
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Postischemic Blockade of AMPA but not NMDA Receptors Mitigates Neuronal Damage in the Rat Brain Following Transient Severe Cerebral Ischemia B. N ellgard
and T. Wieloch
Laboratory for Experimental Brain Research, University Hospital, Lund University, Lund, Sweden
Summary: Olutamatergic transmission is an important factor in the development of neuronal death following transient cerebral ischemia. In this investigation the ef fects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists on neuronal damage were studied in rats exposed to 10 min of transient cerebral ischemia in duced by bilateral common carotid occlusion combined with hypotension. The animals were treated with a blocker of the ionotropic quisqualate or a-amino-3hydroxy-5-methyl-4-isoxazole (AMPA) receptor, 2.3dihydroxy-6-nitro-7 -sulfamoyl-benzo(F)q uinoxaline (NBQX), given postischemia as an intraperitoneal bolus dose of 30 mg kg-I followed by an intravenous infusion of 75 Il-g min -I for 6 h, or with the noncompetitive NMDA receptor blocker dizocilpine (MK-801) given 1 mg kg-I i.p. at recirculation and 3 h postischemia, or with the competitive NMDA receptor antagonist DL-(E)-2amino-4-methyl-5-phosphono-3-pentenoic acid (COP
40116), 5 mg kg-I, given intraperitoneally at recircula tion. Treatment with NBQX provided a significant reduc tion of neuronal damage in the hippocampal CAl area by 44-69%, with the largest relative decrease in the temporal part of the hippocampus. In neocortex a significant de crease in the number of necrotic neurons was also noted. No protection could be seen following postischemic treat ment with dizocilpine or COP 40116. Our data demon strate that AMPA but not NMDA receptor antagonists decrease neuronal damage following transient severe ce rebral ischemia in the rat and that the protection by NBQX may be dependent on the severity of the ischemic insult. We propose that the AMPA receptor-mediated neurotoxicity could be due to ischemia-induced changes in the control mechanisms of AMPA receptor-coupled processes or to changes of AMPA receptor characteris tics. Key Words: Brain-Glutamate-Ischemia-Neuro nal death-Receptors.
The synaptic release of the excitatory amino acid glutamate following an ischemic insult is considered to be an important factor in the development of neuronal death. This notion is supported by exper iments where surgical transection of glutamate containing afferents to the hippocampus was per formed, thereby inhibiting glutamatergic neuro transmission and leading to a decrease in ischemic cell damage ipsilateral to the transection (Wieloch et aI., 1985; Onodera et aI., 1986; J�rgensen et aI.,
1987). However, it is not fully understood which of the glutamate receptors activate the deleterious in tracellular processes 'leading to cell death. Follow ing the first report on neuronal protection by an N-methyl-o-aspartate (NMDA) receptor antagonist (Simon et aI., 1984), research has mainly been fo cused on blockade of this receptor complex. In a vast number of investigations, significant neuronal protection by competitive or noncompetitive NMDA receptor antagonists has been achieved in in vitro models of hypoxia, ischemia, and hypogly cemia (Weiss et aI., 1986; Goldberg et aI., 1987; Monyer et aI., 1989). Furthermore, protection by NMDA antagonists has also been demonstrated in in vivo models of transient cerebral ischemia (Boast et aI., 1988; Gill et aI., 1988; Rod et aI., 1990; Swan and Meldrum, 1990), focal ischemia (Kochhar et aI., 1988; Park et aI., 1988; Steinberg et aI., 1988), and hypoglycemic coma (Wieloch, 1985; Westerberg et
Received April 30, 1991; revised June 18, 1991; accepted June 27, 1991. Address correspondence and reprint requests to Dr. B. Nel\gard at Laboratory for Experimental Brain Research, Lund University, Lund Hospital, S-221 85 Lund, Sweden. Abbreviations used: AMPA, 0l-amino-3-hydroxy-5-methyl-4isoxazole; BBB, blood-brain barrier; COP 40116, DL-(E)-2amino-4-methyl-5-phosphono-3-pentenoic acid; NBQX, 2.3dihydroxy-6-nitro-7-sulfamoyl-benzo(E)quinoxaline; NMOA, N-methyl-D-aspartate; 2-VO, two-vessel occlusion.
2
AMPA RECEPTORS AND ISCHEMIC BRAIN DAMAGE ai., 1988; Papagapiou and Auer, 1990). Neuronal protection by NMDA antagonists in models of tran sient severe ischemia has recently been questioned (Fleischer et aI., 1989; Michenfelder et ai., 1989; Sterz et ai., 1989; Lanier et ai., 1990; Buchan et ai., 199 1; Nellgard et aI., 1991). For example, in a model of cardiac arrest ischemia, dizocilpine, a noncompetitive NMDA antagonist, did not mitigate neuronal damage in the hippocampus (Nellgard et ai., 199 1) despite the protective effects of surgical lesions to the glutamate system in a similar experi mental paradigm (Wieloch et ai., 1985). The discovery of a new class of compounds, the quinoxalinediones (Honore et ai., 1988), that block non-NMDA glutamate receptors, including the ion otropic quisqualate receptor or a-amino-3-hydroxy5-methyl-4-isoxazole (AMPA) receptor (Watkins and Evans, 198 1), has greatly facilitated the study of these glutamate receptor subclasses. The quinox alinedione 2.3-dihydroxy-6-nitro-7-sulfamoyl benzo(F) quinoxaline (NBQX) is an AMPA recep tor antagonists that passes the blood-brain barrier (BBB) and diminishes neuronal damage following transient cerebral ischemia in the gerbil (Sheardown et ai., 1990). The aim of this study was to investigate and com pare the effects of NMDA and AMPA receptor an tagonists, administered in the postischemic phase, on neuronal damage in the hippocampal CAl region and cerebral neocortex, following 10 min of two vessel occlusion (2-YO) ischemia (Smith et ai., 1984). The following receptor blockers that pass the BBB were chosen: the noncompetitive NMDA re ceptor antagonist dizocilpine (MK-80 1) (Cline schmidt et al., 1982), the competitive NMDA recep tor a n t a g o n i s t DL-(E) - 2 - a m i n o - 4 - m e t h y l - 5 phosphono-3-pentenoic acid (CGP 40116) (Fagg et aI. , 1989), and the AMPA receptor antagonist NBQX (Sheardown et ai., 1990). MATERIALS AND METHODS
The surgical procedures were approved by the local ethical committee at Lund University. Male Wistar rats (MjiSllegaard A/S, Denmark), with an average weight of 322 g, were used in this study. Prior to surgery the ani mals were fasted overnight with free access to tap water. Anesthesia was induced by placing the rat in a jar with 3% isoflurane in a mixture of oxygen/nitrous oxide (30:70%). Following intubation with a plastic tube, they were arti ficially ventilated by a rodent respirator (7025 Rodent Ventilator; Ugo Basile Biological Research Appartus, Comeno, Italy). Anesthesia was maintained with 1-2% isoflurane in the oxygen/nitrous oxide (30:70%) gas mix ture. An external jugular vein catheter was then inserted and positioned in the superior caval vein. A tail artery catheter and one vein catheter were also inserted to allow blood sampling, arterial blood pressure recording, and
3
infusions of drugs. The arterial blood pressure was mea sured and recorded continuously until the rats could be extubated. In all animals both common carotid arteries were exposed and encircled by loose ligatures. Arterial blood samples (200 ILl) were collected 15 min prior to ischemia and 15 min postischemia to measure blood gases and blood glucose. If blood gases could not be corrected (Pao2> 90 mm Hg, Paco2 35-45 mm Hg, pH 7.35-7.45) by adjustments of tidal volume or frequency (normally f 85) of the rodent ventilator, or if the blood glucose was 0
a &> 0 0
60
a;
S3
0
6
-8(
P
