Neuroprotective effects of an AMPA receptor antagonist YM872 in a rat transient middle cerebral artery occlusion model

Neuropharmacology 39 (2000) 211–217 www.elsevier.com/locate/neuropharm

Neuroprotective effects of an AMPA receptor antagonist YM872 in a rat transient middle cerebral artery occlusion model Sachiko Kawasaki-Yatsugi a,*, Chikako Ichiki a, Shin-ichi Yatsugi a, Masayasu Takahashi a, Masao Shimizu-Sasamata a, Tokio Yamaguchi a, Kazuo Minematsu b a

Neuroscience Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan b Cerebrovascular Division, Department of Medicine, National Cardiovascular Center, Osaka, Japan Accepted 19 May 1999

Abstract The neuroprotective effects of YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate), a novel α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist with high water solubility, were examined in rats with transient middle cerebral artery (MCA) occlusion. The right MCA of male SD rats was occluded for 3 h using the intraluminal suture occlusion method. YM872 significantly reduced the infarct volume 24 hours after occlusion, at dosages of 20 and 40 mg/kg/h (iv infusion) when given for 4 h immediately after occlusion. Furthermore, delayed administration of YM872 (20 mg/kg/h iv infusion for 4 h, starting 2 or 3 h after the occlusion) also reduced the infarct volume and the neurological deficits measured at 24 h. Additionally, the therapeutic efficacy of YM872 persisted for at least seven days after MCA occlusion in animals treated with YM872 for 4 h starting 2 h after MCA occlusion. These data demonstrate that AMPA receptors contribute to the development of neuronal damage after reperfusion as well as during ischemia in the focal ischemia models and that the acute effect of the blockade of AMPA receptors persists over a long time period. YM872 shows promise as an effective treatment for patients suffering from acute stroke.  2000 Elsevier Science Ltd. All rights reserved. Keywords: YM872; α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA); receptor; Focal cerebral ischemia

1. Introduction The involvement of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors, as well as N-methyl-D-aspartate (NMDA) receptors, in the development of post-ischemic neuronal damage has been previously demonstrated (Sheardown et al., 1990; Buchan et al., 1991). Quinoxalinedione derivatives possessing AMPA receptor antagonistic activity such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX) and 6-(1H-imidazol-1-yl)7-nitro-2,3-(1H,4H)-quinoxalinedione (YM90K) show neuroprotective effects in several cerebral ischemia * Corresponding author. Tel.: +81-298-52-5111; fax: +81-298-562515. E-mail address: [email protected] (S. KawasakiYatsugi)

models when administered during the post-ischemic period (Sheardown et al., 1990; Xue et al., 1994; Graham et al., 1996; Shimizu-Sasamata et al., 1996). However, the low water solubility of these compounds may limit their clinical use despite their marked neuroprotective effects in many experimental cerebral ischemia models. Recently, the novel AMPA receptor antagonist YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4tetrahydro-1-quinoxalinyl] acetic acid monohydrate), which is an analogue of YM90K but has high water solubility (Kohara et al., 1998), was discovered. YM872 shows neuroprotective effects against neuronal damage similar to YM90K following permanent middle cerebral artery (MCA) occlusion in rats (Kawasaki-Yatsugi et al., 1998a; Shimizu-Sasamata et al., 1998) and cats (Takahashi et al., 1998). However, unlike NBQX (Xue et al., 1994) or YM90K, YM872 has no nephrotoxicity at a dose eight times higher than the neuroprotective dos-

0028-3908/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 1 1 7 - 3

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ing rats (unpublished data). These findings suggest YM872 is a good therapeutic candidate for the treatment of acute stroke. In the present study, the neuroprotective effects of YM872 in rat transient MCA occlusions were investigated. Both the dose–response relationship and the therapeutic time window for efficacy against the ischemic damage 24 h after MCA occlusion were examined. Additionally, protective effects of YM872 against both cerebral infarct and neurological deficits at seven days were also evaluated. Although AMPA antagonists are reported to be neuroprotective when evaluated in acute phase (24 to 72 h), there have been no studies which demonstrate whether the beneficial effects including functional recovery of the blockade of AMPA receptors persists or not.

2. Materials and methods All experiments were performed in accordance with the guidelines of the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd. 2.1. Induction of ischemia Male Sprague–Dawley rats (Charles River Japan, Yokohama, Japan) weighing 250–300 g were anesthetized with chloral hydrate (350 mg/kg i.p.). A polyethylene tube 50 (PE-50) was inserted into the left jugular vein to continuously administer treatment. One to two days after this surgery, the animals were anesthetized with 0.5–1.5% halothane in 25% O2/75% N2, and the right MCA was occluded using the intraluminal suture occlusion method described by Longa et al. (1989). In this study, a 3-0 poly-l-lysine-coated suture (Belayev et al., 1996) was used as an occluder. A 3-0 (diameter: 0.23 mm) nylon monofilament suture (Nicho Kogyo Co., Inc., Tokyo, Japan) with its tip rounded by heating near a flame was coated with a 1% poly-l-lysine solution and dried at 60°C for 1 h. This prepared suture was introduced through the right external carotid artery (ECA) into the internal carotid artery and advanced approximately 17 mm intracranially from the common carotid artery (CCA) bifurcation to block the origin of the MCA. After surgery, the halothane anesthesia was turned off. Three hours after MCA occlusion, animals were reanesthetized with halothane and the MCA was reopened by withdrawal of the inserted suture. Rectal temperature was maintained at 37–38°C using a heated blanket and an overhead lamp during both the surgery and recovery from anesthesia, and monitored throughout administration period. In separate experiments, brain temperature and physiological parameters were also monitored throughout the administration period (n=5). A guide cannula was implanted stereotaxically into the left striatum to monitor brain temperature 3–5 days before ischemia

and a polyethylene tube (PE-50) was inserted into the tail artery before MCA occlusion to monitor blood pressure and obtain blood samples. Brain temperature was monitored with a needle-type thermoprobe (BAT-12, Physitemp Instrument Inc., Clifton, NJ, USA) inserted into the left striatum (anterior: 0.0 mm; lateral: 2.5 mm from bregma; depth: 3.5 mm from skull surface) through a guide cannula. 2.2. Drug administration YM872 (10, 20 and 40 mg/3 ml) was dissolved in physiological saline and the solution was adjusted to pH 7.4 with a few drops of 1 N NaOH. In a first experiment, YM872 (10, 20 or 40 mg/kg/h) or saline was administered for 4 h by continuous i.v. infusion starting 5 min after MCA occlusion (n=12 to 13 per group). In a second experiment, YM872 (20 mg/kg/h) or saline was administered for 4 h starting 2, 3 or 4 h after MCA occlusion (n=11 to 15 per group) to determine the therapeutic time window of efficacy. In a third experiment in which histological outcome seven days after MCA occlusion was evaluated, YM872 (20 mg/kg/h) or saline was administered for 4 h starting 2 h after MCA occlusion (n=9 to 12 per group). During administration, animals were placed in Bollman cages (Natsume Seisakusho Tokyo, Japan) for continuous infusion. After administration, animals were allowed to move freely and, after observation of their spontaneous behavior, they were allowed free access to water and food. 2.3. Measurement of neurological deficit Neurological deficit was measured at 2 h after occlusion (before administration) and 24 h after occlusion in the second experiment. In the third experiment, neurological deficit was also measured three days and seven days after occlusion. Neurological deficit was assessed by observers blind to the treatment using the following behaviors according to the method described by Garcia et al. (1995) with some modification: (a) symmetry in the movement of four limbs while held in the air by the tail, (b) forepaw outstretching while held by tail, (c) climbing the wall of a wire cage, (d) reaction to a touch on the side of trunk, (e) response to vibrissae touch, (f) circling to the leftward. Functions (a) and (b) were graded on a scale of 0 to 3, the other functions were graded on a scale of 1 to 3. The minimum score is 4 representing severe deficit and the maximum score is 18 for normal animals. To standardise the severity of ischemia, animals showing only mild neurological deficit (score ⱖ13) 2 h after occlusion were excluded. 2.4. Measurement of infarct size 24 h after the occlusion Twenty four hours after MCA occlusion, animals were reanesthetized with chloral hydrate (300 mg/kg i.p.) after

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neurological deficit evaluation and 2% Evans blue dye was injected intravenously 10 minutes before decapitation to confirm that the suture passed the origin of the MCA. Evans blue stains the vascular wall along the path of the suture (Chopp et al., 1994). Brains were removed and inspected to confirm both the position where the suture was lodged and the absence of subarachnoid hemorrhage, arterial penetration or other defects. The brains were sectioned with a tissue chopper (McIlwain Tissue Chopper, Mickle Laboratory Engineering Co. Inc., USA) at 1-mm intervals, then stained with a 2% solution of 2,3,5-triphenyltetrazolium hydrochloride (TTC, Tokyo Kasei Co., Ltd., Tokyo, Japan) and fixed by immersion in 10% phosphate-buffered formalin. Images of these 13 brain sections were recorded with a CCD color video camera (ICD-740, Ikegami, Tokyo, Japan). Areas not stained with TTC were classified as lesioned tissue by an observer unaware of the drug treatment for each rat. The lesioned, right hemispheric and left hemispheric areas were measured by a video image analysing system (NIH Image, version 1.56). The infarct, right hemispheric and left hemispheric volume were calculated by multiplying the each area by slice thickness. To compensate for the effect of brain edema, the corrected infarct volume was calculated by the following equation: VC=VI×VLH/VRH, VC is corrected infarct volume, VI is the infarct volume obtained by direct measurement, VLH is the volume of the left hemisphere, and VRH is the volume of the right hemisphere (Golanov and Reis, 1995). 2.5. Measurement of infarct size seven days after MCA occlusion Seven days after MCA occlusion, rats were anesthetized with chloral hydrate (300 mg/kg i.p.) following neurological deficit evaluation and their brains were fixed by transcardiac perfusion with 10% formalin neutral buffered solution. The animals were decapitated and the heads left in 10% formalin neutral buffered solution for 24 h. The brains were removed, embedded in paraffin, and 5 µm of eight coronal slices were cut and stained with hematoxylin and eosin. Regions of cerebral damage in each slice were delineated under light microscopy by an observer unaware of the drug treatment for each rat. The areas of striatal and cortical damage were quantitatively assessed using an image analyzer system (Luzex III, Nireco, Tokyo, Japan). The volume of ischemic damage was calculated by integrating these areas using the distance between each stereotaxic level according to the coordinates of Ko¨nig and Klippel (1963). The endpoints for integration were anterior 10.5 mm to anterior 0.35 mm from the interaural line. Parametric values are represented as the means±S.D. Student’s t-test was used to test infarct volume between two groups and one-way ANOVA followed by Dun-

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nett’s multiple-range test was used to test infarct volume among more than two groups. To compare neurological deficit score in the second experiment and in the third experiment, Wilcoxson’s rank sum test and (two-factor, repeated-measure ANOVA were used. To compare physiological parameters, a two-factor, repeated-measure ANOVA was used. In all cases P⬍0.05 was considered significant.

3. Results In all experiments there was no significant difference between rectal temperatures in each of the treatment groups during administration (Table 1) or throughout the post-ischemic period (data not shown). There were also no significant differences in brain temperatures, or other physiological parameters (Table 1). The animals treated with a high dose (40 mg/kg/h) of YM872 exhibited mild sedation during administration. In the first experiment, YM872 reduced corrected infarct volume when administered immediately following occlusion (Fig. 1). Doses of 20 and 40 mg/kg/h resulted in significant reductions in the corrected infarct volume (55% and 56%, respectively; P⬍0.05, Fig. 1). In the 20 and 40 mg/kg/h treated groups, several animals had only a small infarction in the striatum. At 10 mg/kg/h, there was no significant reduction in infarct volume. In the second experiment, the corrected infarct volume in the control animals was larger and less varied than that in the first experiment because animals which did not exhibit obvious neurological deficits before administration were excluded. In the YM872-treated animals, a significant reduction in infarct volume was observed when YM872 was given 2 h after the occlusion or immediately after reperfusion (3 h after occlusion). The grades of protection were 45% for a 2 h delay and 35% for a 3 h delay (P⬍0.01, P⬍0.05, respectively, Fig. 2a). Neurological scores before administration (2 h after the occlusion) were not significantly different between saline- and YM872-treated groups. However, in the 2 h or 3 h delayed treatment group, neurological deficits 24 h after occlusion were significantly reduced in the YM872treated group compared with the saline-treated group (P⬍0.05, Fig. 2b). The administration starting 4 h after MCA occlusion failed to reduce infarct volume and neurological deficits (Fig. 2a and b). Fig. 3a and b show the histological and neurological outcome seven days after MCA occlusion in the third experiment. In saline-treated animals, the percentage of infarct to the total hemispheric volume at seven days (24.64±1.08%) was similar in degree compared to that at 24 h (22.65±2.32%) in the second experiment (it must be noted that absolute values of the infarct size at seven days appears to be smaller due to the difference in the

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Table 1 Physiological parameter in rats with MCA occlusions Parameter

Dose (mg/kg/h)

Rectal temp. (°C)

Saline YM872

Brain temp. (°C)

MABP (mmHg)

pH

PaCO2 (mmHg)

PaO2 (mmHg)

Saline YM872

Saline YM872

Saline YM872

Saline YM872

Saline YM872

na

Before

2 h after

4 h after

24 h after

(10) (20) (40)

13 12 13 13

37.5±0.4 37.3±0.3 37.5±0.4 37.1±0.6

38.3±0.7 37.9±0.9 37.7±0.7 37.7±0.7

37.9±0.7 37.8±0.8 37.5±0.4 37.3±0.7

37.9±0.6 37.2±1.2 37.0±1.3 37.9±0.7

(10) (20) (40)

5 5 5 5

36.8±0.6 36.4±0.4 36.7±1.1 36.6±0.7

36.9±0.6 37.7±0.7 36.8±0.8 37.2±0.5

36.5±0.3 37.7±0.8 36.9±1.0 36.9±0.7

(10) (20) (40)

5 5 5 5

73.0±7.1 81.0±15.5 87.2±22.2 76.2±3.8

83.2±16.7 90.0±26.7 84.0±23.5 80.4±14.2

68.8±7.1 86.4±23.2 77.6±24.9 74.4±11.4

(10) (20) (40)

5 5 5 5

7.45±0.02 7.45±0.03 7.45±0.04 7.46±0.03

7.45±0.02 7.45±0.02 7.43±0.03 7.41±0.04

7.43±0.03 7.42±0.03 7.42±0.02 7.41±0.02

(10) (20) (40)

5 5 5 5

37.7±7.1 43.4±1.9 41.8±5.2 40.7±2.7

36.0±4.2 36.6±3.0 41.6±4.7 44.1±8.1

38.5±5.9 41.2±4.3 44.0±3.3 46.6±4.6

(10) (20) (40)

5 5 5 5

121.5±27.7 129.9±25.4 115.1±25.3 110.7±31.2

132.3±22.8 129.8±28.0 119.1±31.7 111.3±31.9

131.2±27.5 130.8±27.0 129.3±26.3 101.7±37.4

a Rectal temperature in rats in the first experiment was represented. Brain temperature and other physiological parameters were monitored in a separate experiment.

histological evaluation techniques between both experiments). 20 mg/kg/h of YM872 also reduced the infarct volume seven days after MCA occlusion (Fig. 3a). Neurological deficits in the YM872-treated animals were improved earlier than that in the saline-treated animals (P⬍0.05 when overall difference was tested, Fig. 3b), although deficits gradually improved in both groups during the seven days after occlusion. 4. Discussion

Fig. 1. The corrected infarct volume for individual rats from the first experiment are depicted. Boxes and bars indicate the mean±S.D for each group. 10, 20, or 40 mg/kg/h YM872 or saline was intravenously infused for 4 h starting 5 min after occlusion. *P⬍0.05 vs. saline treated-group (one-way ANOVA followed by Dunnett’s multiple range test).

YM872 is a novel AMPA antagonist with a high water solubility. Prior studies (Kawasaki-Yatsugi et al., 1998a; Shimizu-Sasamata et al., 1998; Takahashi et al., 1998) have demonstrated the neuroprotective effects of YM872 by administration immediately after the onset of ischemia in permanent MCA occlusion models without accompanying nephrotoxicity. In the present study, we have evaluated 1) the dose response relationship for YM872, 2) the effect of delayed treatment with YM872, and 3) the protective effect of YM872 during the subacute phase (seven day end point) using the 3 h transient focal cerebral ischemia model. YM872 showed marked

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Fig. 2. The therapeutic time window of YM872 (the second experiment). YM872 (20 mg/kg/h for 4 h, hatched bar or closed circle) or saline (open bar or open circle) was intravenously infused starting 2, 3, or 4 h after occlusion. (a): The corrected infarct volume for individual rats, the mean±S.D and (n) for each group are represented. (b): Neurological scores for individual rats, the median value and (n) for each group are represented. *P⬍0.05, **P⬍0.01 vs. respective saline treated-group (Student’s t-test in (a), Wilcoxon rank sum test in (b)).

neuroprotective effects, even when given immediately after reperfusion, indicating that the therapeutic time window of this compound was 3 h after MCA occlusion. In addition to protecting tissue damage, YM872 also ameliorated behavioral deficits following MCA occlusion. In the first experiment, striatal infarction was also reduced in the several animals in the 20 or 40 mg/kg/h-treated group. Unlike the electrocoagulation method (Tamura’s method), in this intraluminal suture method the main trunk of the MCA is left intact and drugs can reach into the striatal region from collateral blood flow (Minematsu et al., 1993a) and via the MCA following reperfusion. In our reversible MCA occlusion technique, incomplete infarction in the striatal region is sometimes observed. Therefore, animals which did not exhibit obvious neurological deficit before administration were excluded in the second and third experiments. One of the most important findings of this study is

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Fig. 3. The effects of YM872 on the ischemic damage 7 days after MCA occlusion (the third experiment). YM872 (20 mg/kg/h for 4 h, hatched bar or closed circle) or saline (open bar or open circle) was intravenously infused starting 2 h after occlusion. (a): Infarct volume for individual rats, the means±S.D and (n) for each group are represented. **P⬍0.01 vs. saline treated-group (Student’s t-test). (b): Neurological deficits following MCA occlusion. Each value represents the mean ±S.D. P⬍0.05 when overall difference was tested (two-factor, repeated-measure ANOVA).

that the therapeutic efficacy of YM872 persisted for at least seven days after MCA occlusion. There are many reports demonstrating the effects of AMPA antagonists in the focal cerebral ischemia models, but no studies have been carried out when the evaluation is delayed. NBQX causes renal toxicity at neuroprotective doses (Xue et al., 1994) due to low solubility at neutral pH. In this respect there is also a problem with the solubility of YM90K. Therefore, evaluation of the neuroprotective effects of these compounds over a long time period has been difficult, particularly effect on behavioral function. The current results strongly suggests that YM872 does not delay the development of neuronal damage, but the reduction of infarction 24 h after MCA occlusion, evaluated by TTC staining, is due to its neuroprotective effect in this model. The brain concentration at the effective dose (20 mg/kg/h) obtained from our preliminary study is estimated to be approximately 100 ng/g (苲0.3 µM, unpublished data). This is roughly comparable to the concentration which protects against cell death induced by AMPA exposure in cortical neuronal cell cultures and

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oxygen/glucose deprivation in hippocampal slices (Small et al., 1998). As brain temperature and the other physiological parameters were not affected at the effective dose, the protective effects of YM872 are thought to be due to direct action on AMPA receptors, and not due to non-specific effects such as hypothermia. In addition, a study using intact rats found cerebral blood flow was not affected by the same administration regimen which reduced infarct volume in this study (Shimizu-Sasamata et al., 1998). Therefore, hemodynamic effects may not significantly contribute to the protective effects of YM872 either. We have previously reported the neuroprotective effect of YM872 in a rat permanent MCA occlusion model using this suture occlusion technique (KawasakiYatsugi et al., 1998a). With respect to the extent of ischemic damage in that study and transient occlusion model in this study, 20 mg/kg/h of YM872 for 4 h was enough to show significant neuroprotection in the transient occlusion model, whereas 40 mg/kg/h for 4 h was required in the permanent occlusion model. A similar phenomenon was found in studies of YM90K: a 4 h infusion of YM90K at 10 mg/kg/h for 4 h, which did not show significant neuroprotection in a permanent occlusion model using this suture occlusion technique, showed significant neuroprotection in a transient occlusion model (Kawasaki-Yatsugi et al., 1998b). These findings suggest that the neuroprotective effects both of these AMPA antagonists are enhanced by reperfusion. Minematsu et al. (1993a,b) have studied the time course of the lesion size development in rats with permanent and transient (3 h) MCA occlusion using diffusion magnetic resonance imaging and have evaluated the protective effect of CNS1102, a non-competitive NMDA antagonist, in the both models. According to their report, the lesion size was extended after termination of drug administration (3 h after the occlusion) in rats with permanent MCA occlusion. In contrast, in the transient occlusion model, they observed significant reduction in the lesioned area after reperfusion compared with before reperfusion in the drug-treated animals. Their observations and the results from our studies support concept that the combination of neuroprotectants such as glutamate antagonists and reperfusion is a potential therapy in acute stroke patients. In the transient occlusion model, the brain concentration of drugs in the ischemic region is thought to be higher than that in the permanent occlusion at the same dosing regimen because reperfusion allows the transport of drug into the ischemic region. This may also contribute to the difference between effective doses of YM872 in permanent and transient occlusion models, although the brain concentration of YM872 was not directly measured in these experiments. AMPA antagonists are reported to exhibit neuroprotection even if administration is delayed in several cer-

ebral ischemia models (Smith et al., 1992; Xue et al., 1994; Graham et al., 1996; Umemura et al., 1997). Unlike NMDA antagonists such as MK-801 (Xue et al., 1994; Margaill et al., 1996), delayed administration of NBQX also provided neuroprotection in the transient MCA occlusion model (Xue et al., 1994) as well as in the permanent MCA occlusion model (Graham et al., 1996). In the present study, treatment with YM872 after reperfusion (3 h after occlusion) also reduced the ischemic damage. These results suggest that AMPA receptors play an important role in the development of neuronal damage after reperfusion, as well as during ischemia, although extracellular glutamate is rapidly restored to normal levels following reperfusion (Margaill et al., 1996). It is known that the sustained Ca++ accumulation in the ischemic tissue occurs after reperfusion as well as during ischemia in the focal ischemia model (Nagasawa et al., 1990). Neuronal Ca++ channel antagonists such as SNX-111, a synthetic ω-conopeptide (Valentino et al., 1993), were also reported to protect against cerebral infarction when administration started concurrently with reopening of the MCA in transient focal ischemia model (Buchan et al., 1994). This protective effect is thought to be due to the block of postsynaptic Ca++ entry though N-type Ca++ channels rather than the inhibition of glutamate release via presynaptic Ca++ channels. AMPA antagonists are also thought to exert their neuroprotective action by preventing Ca++ influx into postsynaptic neuronal cells via several pathways (Gill, 1994). Therefore, the reduction of Ca++ influx by blockade of AMPA receptors after ischemia, as well as during ischemia, may contribute to the post-treatment efficacy of AMPA antagonists such as YM872 even if they are administered after reperfusion in focal ischemia models. In conclusion, the present study, using a transient MCA occlusion model, supports the beneficial effect of AMPA receptor blockade combined with reperfusion following cerebral ischemia. Furthermore, our results indicate that the effect of the blockade of AMPA receptors persists over a long time period. YM872 has great potential clinical utility as a therapeutic agent in treating acute stroke in humans, without accompanying renal toxicity.

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