Profiles of glutamate and GABA efflux in core versus peripheral zones of focal cerebral ischemia in mice.

Efflux of glutamate during cerebral ischemia is known to contribute to brain cell death via processes of excitotoxicity. However, gamma-aminobutyric acid (GABA) is also released during ischemia, and may be protective. In this study, we used in vivo microdialysis to map the efflux of glutamate and GABA from central core and peripheral zones of focal ischemia in mouse brain. We show that the temporal profiles of glutamate and GABA efflux are significantly different in core versus peripheral zones. Calculation of glutamate/GABA ratios demonstrate that, in the core, there is a significant increase above baseline ratios during the first 30 mm of ischemia, which then rapidly renormalizes. In contrast, no significant changes in glutamate/GABA ratios were seen in the ischemic periphery. These data suggest that imbalances in glutamate versus GABA efflux may be an initial trigger of excitotoxic brain damage in the core but not the peripheral zones of focal cerebral ischemia.

Protective effects of pamiteplase, a modified t-PA, in a rat model of embolic stroke.

The effects of alteplase (tissue plasminogen activator, t-PA) and pamiteplase (a modified t-PA with longer half-life and increased potency) were compared in a clinically relevant model of embolic stroke. Rats were treated with pamiteplase (0.5 mg/kg or 1 mg/kg bolus), alteplase (10 mg/kg infusion) or normal saline. Pamiteplase (1 mg/kg) was as effective as alteplase in reducing 24 h brain infarct volumes, neurological deficit scores and residual clot grades. Cerebral blood flow recovery at 30 min after thrombolytic treatment was partial and did not correlate with 24 h infarct volumes or neurological deficits. However, there was good correlation between 24 h residual clot grades and infarct volumes, suggesting a delayed timeframe for pamiteplase- and alteplase-induced reperfusion.

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

The neuroprotective effects of YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl]acetic acid monohydrate), a novel alpha-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.

Impaired neurotransmitter release and elevated threshold for cortical spreading depression in mice with mutations in the alpha1A subunit of P/Q type calcium channels.

The P/Q type voltage-gated Ca2+ channels are involved in membrane excitability and Ca2+-dependent neurotransmitter release within the CNS. Mutations in the CacnalA gene encoding the alpha1A subunit of the P/Q type Ca2+ channel have recently been reported in tottering mice and a more severely affected allele, leaner. Here we show using in vivo cortical microdialysis that evoked increases of extracellular glutamate levels are markedly attenuated in both mutants upon KCl-induced depolarization compared with wild-type mice. Tottering and leaner mice also show a 10-fold resistance to cortical spreading depression induced by cortical electrical stimulation or KCl application to the pial surface. A slower transcortical propagation speed and failure to sustain regenerative spread of the depolarizing wave were more pronounced in leaner neocortex. Both signaling defects appeared unrelated to the developmental history of repeated cortical spike-wave discharges, since neither were observed in the stargazer mouse, a Ca2+ channel gamma2 subunit mutant with a similar seizure phenotype. These data demonstrate two cortical excitability defects revealed by prolonged depolarization in cerebral networks expressing mutant P/Q type Ca2+ channels, and are the first to identify a gene linked to a spreading depression phenotype.

YM90K, an AMPA receptor antagonist, protects against ischemic damage caused by permanent and transient middle cerebral artery occlusion in rats.

The neuroprotective effect of YM90K, a potent AMPA receptor antagonist, was examined in rats with permanent and transient occlusion of middle cerebral artery (MCA) using intraluminal suture occlusion method. In rats with permanent MCA occlusions, two types of occluders were used to compare the efficacy of YM90K. When a 4-0 (diameter: 0.19 mm) suture was used, YM90K (20 mg kg(-1) h(-1) i.v. infusion for 4 h) significantly reduced infarct volume (P<0.05) and neurologic deficits (P<0.05) 24 h after MCA occlusion. Infarct volume was also reduced by YM90K at the same dose (P<0.01) when severe ischemia was induced by a 3-0 (diameter: 0.23 mm) suture. In rats with transient (3 h) MCA occlusions, a 10-mg kg(-1) h(-1) dose of YM90K that did not show significant protection in rats with permanent MCA occlusion offered neuroprotective effects. These data demonstrate that YM90K provides cerebral neuroprotection against a wide range of ischemic insults.

Effects of YM872 on atrophy of substantia nigra reticulata after focal ischemia in rats.

Middle cerebral artery (MCA) occlusion causes atrophy in the ipsilateral substantia nigra reticulata (SNR). The effects of glutamate AMPA receptor antagonism on SNR atrophy, which is supposed to inhibit excitatory inputs from the subthalamic nucleus to the SNR, was investigated in rats with permanent MCA occlusions. Histological examination revealed marked atrophy two weeks after MCA occlusion in the saline-treated control group. However, constant i.v. infusion of YM872, a selective AMPA receptor antagonist, for 2 weeks significantly reduced SNR atrophy; neurological deficits also decreased. These results suggest that the AMPA receptor may be involved in the pathogenesis of SNR atrophy during the subacute phase of focal cerebral ischemia.

Neuroprotective efficacy of YM872, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, after permanent middle cerebral artery occlusion in rats.

The neuroprotective efficacy of YM872, a novel, highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, was investigated in rats subjected to permanent occlusion of the left middle cerebral artery. The rats were assessed either histologically or neurologically 24 hr or 1 wk after ischemia. YM872 was intravenously infused for either 4 or 24 hr at dose rates of 0 to 20 mg/kg/hr starting 5 min after ischemia to examine the effect of prolonged treatment. YM872 was then infused at 20 mg/kg/hr beginning 0 to 4 hr after ischemia to determine the efficacy time window. Additionally, a 20 mg/kg/hr dose rate of YM872 was infused for 4 hr in single day- or 5-day repetitive-administrations to evaluate long-term benefits of the drug. YM872 significantly reduced infarct volume in both 4- and 24-hr treatment groups measured 24 hr after ischemia. No difference was observed in the degree of protection between length of infusion. Significant neuroprotection was maintained even when drug administration was delayed up to 2 hr after ischemia. A single YM872-administration significantly improved neurological deficit and reduced infarct volume (30%, P <.01) measured 1 wk after ischemia. YM872 treatment did not induce such adverse effects as physiological changes, serious behavioral abnormalities or nephrotoxicity. These data suggest that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor plays a crucial role in the progression of neuronal damage in the early phase of ischemia and that YM872 may be useful in treating acute ischemic stroke.

Attenuated neurotransmitter release and spreading depression-like depolarizations after focal ischemia in mutant mice with disrupted type I nitric oxide synthase gene.

Nitric oxide (NO) plays a complex role in the pathophysiology of cerebral ischemia. In this study, mutant mice with disrupted type I (neuronal) NO synthase (nNOS) were compared with wild-type littermates after permanent focal ischemia. Cerebral blood flow in the central and peripheral zones of the ischemic distribution were measured with laser doppler flowmetry. Simultaneously, microdialysis electrodes were used to measure extracellular amino acid concentrations and DC potential in these same locations. Blood flow was reduced to <25 and 60% of baseline levels in the central and peripheral zones, respectively; there were no differences in nNOS mutants versus wild-type mice. Within the central ischemic zone, DC potentials rapidly shifted to -20 mV in all mice. In the ischemic periphery, spreading depression (SD)-like waves of depolarization were observed. SD-like events were significantly fewer in the nNOS mutant mice. Concurrent with these hemodynamic and electrophysiological perturbations, extracellular elevations in amino acids occurred after ischemia. There were no detectable differences between wild-type and mutant mice in the ischemic periphery. However, in the central zone of ischemia, elevations in glutamate and GABA were significantly lower in the nNOS mutants. Twenty-four hour infarct volumes in the nNOS mutant mice were significantly smaller than in their wild-type littermates. Overall, the number of SD-like depolarizations and the integrated efflux of glutamate were significantly correlated with infarct size. These results suggest that NO derived from the nNOS isoform contributes to tissue damage after focal ischemia by amplifying excitotoxic amino acid release in the core and deleterious waves of SD-like depolarizations in the periphery.

Prolonged therapeutic window for ischemic brain damage caused by delayed caspase activation.

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimer's disease and Huntington's disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.

Continuous assessment of perfusion by tagging including volume and water extraction (CAPTIVE): a steady-state contrast agent technique for measuring blood flow, relative blood volume fraction, and the water extraction fraction.

A new technique, CAPTIVE, that is a synthesis of arterial spin labeling (ASL) blood flow and steady-state susceptibility contrast relative blood volume imaging is described. Using a single injection of a novel, long half-life intravascular magnetopharmaceutical with a high tissue:blood susceptibility difference (deltachi) to deltaR1 ratio, changes in tissue transverse relaxivity (deltaR2 or deltaR2*) that arise from changes in blood volume were measured, while preserving the ability to measure blood flow using traditional T1-based ASL techniques. This modification permits the continuous measurement of both blood flow and blood volume. Also, because the contrast agent can be used to remove the signal from intravascular spins, it is possible to measure the first-pass water extraction fraction. Contrast-to-noise is easily traded off with repetition rate, allowing the use of non-EPI scanners and more flexible imaging paradigms. The basic theory of these measurements, several experimental scenarios, and validating results are presented. Specifically, the PaCO2-reactivity of microvascular and total relative cerebral blood volume (rCBV), cerebral blood flow (CBF), and the water extraction-flow product (EF) in rats with the new contrast agent MPEG-PL-DyDTPA is measured, and the values are concordant with those of previous literature. As an example of one possible application, continuous flow and volume measurements during transient focal ischemia are presented. It is believed that CAPTIVE imaging will yield a more complete picture of the hemodynamic state of an organ, and has further application for understanding the origins of the BOLD effect.

YM872, a highly water-soluble AMPA receptor antagonist, preserves the hemodynamic penumbra and reduces brain injury after permanent focal ischemia in rats.

BACKGROUND AND PURPOSE: We recently described an image analysis technique based on the temporal correlation mapping (TCM) of injected contrast agents that can be used to distinguish the hemodynamic core and hemodynamic penumbra after focal ischemia. In this study we used this technique for the first time to investigate the effects of the water-soluble AMPA receptor antagonist YM872 in permanent focal ischemia. METHODS: Fischer 344 rats were subjected to permanent occlusion of the middle cerebral artery. Approximately 30 minutes after ischemia, functional CT images were collected with the use of a dynamic scanning protocol with bolus injections of nonionic contrast agent iohexol (1 mL/kg). TCM analysis defined the distributions of hemodynamic core and hemodynamic penumbra. Cerebral perfusion indices were calculated on the basis of the area under the first-pass transit curves. One hour after ischemia, animals were randomly treated with YM872 (n=8, 20 mg/kg per hour over 4 hours) or normal saline (n=10). Twenty-four hours later, neurological deficits were evaluated, and conventional CT and triphenyltetrazolium chloride staining were used to define volumes of ischemic damage. RESULTS: At 24 hours after ischemia, hypodense lesions were visible on conventional CT scans that were highly correlated with triphenyltetrazolium chloride lesion volumes. YM872 improved neurological deficits and reduced volumes of ischemic damage in cortex (90+/-14 versus 170+/-16 mm3 in controls) but not striatum (57+/-14 versus 79+/-6 mm3 in controls). Comparison of early TCM images with conventional CT scans of ischemic injury showed that the hemodynamic core was always damaged in all rats. In controls, 54% of the tissue within the hemodynamic penumbra evolved into ischemic damage compared with 24% in YM872-treated rats. Furthermore, the perfusion index corresponding to the ischemic damage threshold was significantly reduced by YM872 (28+/-2% versus 37+/-2% in controls). CONCLUSIONS: These results indicate that YM872 is a neuroprotective compound that ameliorates the deterioration of the hemodynamic penumbra after focal ischemia.

Delayed treatment with YM90K, an AMPA receptor antagonist, protects against ischaemic damage after middle cerebral artery occlusion in rats.

The neuroprotective effect of an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride] has been examined in a rat middle cerebral artery occlusion model. Intravenous infusion of YM90K (2.5-20mgkg(-l)h(-l) for 4h) starting immediately after occlusion of the middle cerebral artery significantly reduced the cortical infarct volume 24h after occlusion compared with the control group. The protection at the highest dose was 39% (P < 0.05). Similar protective effects were observed when YM90K (20mgkg(-1)h(-1) for 4h) was delayed up to 2h after middle cerebral artery occlusion (45% reduction, P < 0.05). CNS1102 [N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine hydrochloride], a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the cortical infarct volume when 1.13mgkg(-1) was administered by intravenous bolus injection immediately after middle cerebral artery occlusion, followed by intravenous infusion at 0.785mgkg(-l)h(-1) for 4h (35% reduction, P<0.05). This neuroprotective effect was not observed when administration was delayed lh after middle cerebral artery occlusion. These results suggest that AMPA receptors might play a more important role than NMDA receptors in the late development of neuronal cell damage after focal cerebral ischaemia and that AMPA receptor blockade would be one beneficial strategy in treating acute stroke.

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist.

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.

Effects of nitric oxide synthase gene knockout on neurotransmitter release in vivo.

Nitric oxide serves as a diffusible messenger within the neuronal networks of the brain. Recent studies have suggested that nitric oxide may amplify neurotransmitter release via its ability to diffuse in a retrograde manner from postsynaptic to presynaptic neurons. Two isoforms of nitric oxide synthase may be present in neurons: Type I nitric oxide synthase (neuronal isoform) and Type III nitric oxide synthase (endothelial isoform). In this study, we examined the role of nitric oxide as an amplifier of neurotransmitter release by using K+ and N-methyl-D-aspartate stimulations via microdialysis probes located in cortex, striatum, and hippocampus. We compared responses obtained in wild-type mice versus knockout mice deficient in either neuronal isoform of nitric oxide synthase or endothelial isoform of nitric oxide synthase gene expression. No significant differences in glutamate and GABA release were observed between knockout mice and wild-type mice after K+ stimulations. In contrast, N-methyl-D-aspartate-stimulated glutamate release in cortex was significantly reduced in the neuronal NOS knockout mice, and N-methyl-D-aspartate-stimulated GABA release was significantly reduced in all brain regions of endothelial NOS knockout mice. These data suggest that the two nitric oxide synthase isoforms, most likely due to their specific neuronal localizations, may serve different roles in the modulation of excitatory versus inhibitory neurotransmission in mammalian brain.

A novel AMPA receptor antagonist, YM872, reduces infarct size after middle cerebral artery occlusion in rats.

The neuroprotective effect of YM872 ([2.3-dioxo-7-(1H-imidazol-1-yl) 6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate), a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist with improved water solubility, was examined in a rat focal cerebral ischemia model. Rats were subjected to permanent middle cerebral artery (MCA) occlusion using the intraluminal suture occlusion method for 24 h. YM872 was intravenously infused for 4 h (20 and 40 mg/kg/h) or 24 h (10 and 20 mg/kg/h), starting 5 min after the MCA occlusion, to investigate the effect of prolonged duration of the treatment on infarct volume. In the 4 h infusion study, YM872 reduced the cortical infarct volume by 48% at a dose of 40 mg/kg/h. YM872 did not significantly reduce the infarct at 20 mg/kg/h for 4 h. In the 24 h infusion study, however, YM872 markedly reduced the cortical infarct volume by 62%, even at 20 mg/kg/h. The present study indicates that the neuroprotective effect of YM872 is enhanced by extending the duration of treatment, and demonstrates the benefit of the prolonged treatment with AMPA antagonists following focal cerebral ischemia. YM872, a highly water soluble compound, is applicable to investigate the role of AMPA receptors in ischemic models without concern about nephrotoxicity and could be useful in the treatment of human stroke.

Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family.

Inhibitors of apoptosis and of excitotoxic cell death reduce brain damage after transient and permanent middle cerebral artery occlusion. We compared the neuroprotective effects of two caspase family inhibitors with the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in a newly characterized cycloheximide-sensitive murine model of transient middle cerebral artery occlusion (30 minutes) in which apoptotic cell death is prominent. Ischemic infarction, undetected by 2,3,5-triphenyltetrazolium chloride staining at 24-hour reperfusion, featured prominently in the striatum at 72 hours and 7 days on hematoxylin-eosin-stained sections. Markers of apoptosis, such as oligonucleosomal DNA damage (laddering) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive cells first appeared at 24 hours and increased significantly at 72 hours and 7 days after reperfusion. The TUNEL-labeled cells were mostly neurons and stained negative for glial (GFAP, glial fibrillary acid protein) and leukocyte specific markers (CD-45). The caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK; 120 ng intracerebroventricularly) or N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD.FMK; 480 ng intracerebroventricularly) decreased infarct size and neurologic deficits when administered 6 hours after reperfusion. The extent of protection was greater than in models of more prolonged ischemia or after permanent occlusion, and the therapeutic window was extended from 0 to 1 hours after 2-hour middle cerebral artery occlusion to at least 6 hours after brief ischemia. Also, z-VAD.FMK and z-DEVD.FMK treatment decreased oligonucleosomal DNA damage (DNA laddering) as assessed by quantitative autoradiography after gel electrophoresis. By contrast, MK-801 protected brain tissue only when given before ischemia (3 mg/kg intraperitoneally), but not at 3 or 6 hours after reperfusion. Despite a decrease in infarct size after MK-801 pretreatment, the amount of DNA laddering did not decrease 72 hours after reperfusion, thereby suggesting a mechanism distinct from inhibition of apoptosis. Hence, 30 minutes of reversible ischemia augments apoptotic cell death, which can be attenuated by delayed z-VAD.FMK and z-DEVD.FMK administration with preservation of neurologic function. By contrast, the therapeutic window for MK-801 does not extend beyond the time of occlusion, probably because its primary mechanism of action does not block the development of apoptotic cell death.

YM872, a novel selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, reduces brain damage after permanent focal cerebral ischemia in cats.

YM872 ([2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3, 4-tetrahydro-1-quinoxalinyl]-acetic acid monohydrate), a selective, potent and highly water-soluble competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, was investigated for its neuroprotective effect against focal cerebral ischemia in halothane-anesthetized cats. Cats were subjected to permanent occlusion of the left middle cerebral artery for 6 h, then sacrificed and examined histologically. The electroencephalogram and cerebral blood flow were monitored. Intravenous infusion of YM872 starting 10 min after the onset of ischemia at a rate of 2 mg/kg/h for 6 h markedly reduced the volume of ischemic damage by 61% (from 2604 +/- 202 mm3 of the cerebral hemisphere in saline-treated cats to 1025 +/- 277 mm3 in YM872-treated cats; P < .01), as assessed in 12 stereotaxically determined coronal sections. No significant differences were observed between YM872- and saline-treated cats concerning physiological variables including brain temperature. No precipitation of YM872 in the kidney was seen in any YM872-treated animal. The present data further support the notion that the AMPA receptor plays an important role in the progression of focal ischemic damage in a gyrencephalic model. This evidence for the neuroprotective efficacy of YM872 suggests its therapeutic potential in the treatment of acute stroke in humans.

Neuroprotective effect of YM90K, an AMPA-receptor antagonist, against delayed neuronal death induced by transient global cerebral ischemia in gerbils and rats.

We investigated the neuroprotective effect of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist YM90K in transient global ischemia models. In a gerbil model, transient ischemia was induced by bilateral common carotid artery (CCA) occlusion for 5 min. On administration at 1 hr after ischemia, the AMPA antagonists NBQX (30 mg/kg, i.p. x 3) and YM90K (15 mg/kg, i.p. x 3 or 30 mg/kg, i.p. x 3) significantly reduced the delayed neuronal death in the hippocampal CA1 region from 4 days after bilateral CCA occlusion. Furthermore, YM90K (30 mg/kg, i.p. x 3) showed a neuroprotective effect even when given at 6 hr after ischemia. In contrast, the N-methyl-D-aspartate receptor antagonists CGS19755, MNQX (30 mg/kg, i.p. x 3, each) and (+/-)MK-801 (10 mg/kg, i.p.) were not effective on injection at 1 hr after ischemia in this model. In a rat model, ischemia was induced by 4-vessel occlusion (4-VO) for 10 min. YM90K was administered 60 min after reperfusion. Rectal and temporal muscle temperatures were maintained at the same level as in the control group for 6 hr. YM90K markedly prevented the development of delayed neuronal death from 7 days after 4-VO at doses of 15 or 30 mg/kg, i.p. x 3, with neuroprotective efficacy similar to that in the gerbil model. These results suggest that the AMPA receptor plays a critical role in the development of the delayed neuronal death induced by transient global cerebral ischemia. They also suggest that the neuroprotective effect of YM90K is not related to its hypothermic effect.

8-(1H-imidazol-1-yl)-7-nitro-4(5H)-imidazo[1,2-alpha]quinoxalinone and related compounds: synthesis and structure-activity relationships for the AMPA-type non-NMDA receptor.

As a part of our program to discover novel antagonists for the AMPA subtype of EAA receptors, we designed and synthesized a series of heterocyclic-fused imidazolylquinoxalinones 5a-c, 9, 11, 14a-e, and 18 which led from 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (1a.HCl, YM90K) by replacement of its amide with the imidazole and triazole rings. Their activity was evaluated by inhibiting [3H]AMPA binding from rat whole brain. As a result, it appeared that 8-(1H-imidazol-1-yl)-7-nitro-4(5H)-imidazo[1,2-alpha]quinoxalinone (5a) and its [1,2,4]triazolo[4,3-alpha] analogue 14a possessed high affinity for AMPA receptors with Ki values of 0.057 and 0.19 microM, respectively, similar to the activity of 1a and NBQX (2) (1a, Ki = 0.084 microM; 2, Ki = 0.060 microM). In contrast, 8-(1H-imidazol-1-yl)-7-nitro-4(5H)-imidazo[1,5-alpha]quinoxalinone (5b) and 7-(1H-imidazol-1-yl)-8-nitro-4(5H)-[1,2,4]triazolo[4,3-alpha]quinoxalino ne (18) showed no or weak affinity for the receptors. Hence, we deduced that the nitrogen atom of the fused heterocycles at the 3-position of 5a and 14a plays an essential role as hydrogen bond acceptors in binding to AMPA receptors, whereas their amides act as proton donors. From the SAR on 1-alkyl derivatives of 5a and 14a, it was indicated that introduction of suitable 1-alkyl substituents led to a severalfold improved AMPA affinity. A computational study on a model of water-quinoxaline complexes, a mimic of the putative hydrogen-bonding interaction between the receptors and quinoxalines, indicated that the different affinities of 5a, 14a, 1a, and 19 for the AMPA receptor may depend on, at least in part, each stabilization energy for the interaction. On this basis, we propose a pharmacophore model of AMPA receptors for the binding of the imidazolylquinoxaline derivatives. The heterocyclic-fused quinoxalinones 5a,c and 9 showed potent inhibitory activity in KA-induced toxicity for hippocampal cell culture with IC50 values of 0.30, 0.32, and 0.30 microM, respectively (1a, 0.81 microM; 2, 0.38 microM). Moreover 5a possesses over 5000-fold AMPA selectivity against both the NMDA receptor and the glycine site on the NMDA receptor.

Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage.

The interleukin 1beta converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1beta (IL-1beta) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1beta levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1beta levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-D-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.