PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by up-regulating Bcl-2 expression.

Neurotoxicity in primary neurons was induced using hypoxia/hypoglycemia (H/H), veratridine (10microM), staurosporine (1microM) or glutamate (100microM), which resulted in 72%, 67%, 75% and 66% neuronal injury, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811; 10microM; Panacea Pharmaceuticals, Gaithersburg, MD) pretreatment for 24 h provided maximal neuroprotection of 89%, 42%, 47% and 89% against these toxicities, respectively. Glutamate or H/H treatment of cells increased cytosolic cytochrome c levels, which was blocked by pretreatment of cells with PAN-811. Pretreatment of neurons with PAN-811 produced a time-dependent increase in the protein level of Bcl-2, which was evident even after glutamate or H/H treatments. An up-regulation in the expression of the p53 and Bax genes was also observed following exposure to these neurotoxic insults; however, this increase was not suppressed by PAN-811 pretreatment. Functional inhibition of Bcl-2 by HA14-1 reduced the neuroprotective efficacy of PAN-811. PAN-811 treatment also abolished glutamate or H/H-mediated internucleosomal DNA fragmentation.

Down-regulation of the sodium channel Na(v)1.1 alpha-subunit following focal ischemic brain injury in rats: in situ hybridization and immunohistochemical analysis.

Change in sodium channel (NaCh) activity can play a role in reorganization, recovery, or possibly excitotoxic damage after CNS injury. Alteration of sodium channel function has been reported to occur in a variety of neuropathological states including epilepsy and brain injury. Previously we reported that out of five NaCh alpha subunit genes that were down-regulated, Na(v)1.1 exhibited the most dramatic and sustained alterations following focal cerebral ischemia in the rat. In the present study, we evaluated the acute spatial and temporal time course distribution of Na(v)1.1 mRNA (in situ hybridization) and protein (immunohistochemistry) following ischemic brain injury. Male rats were subjected to 2 h of middle cerebral artery occlusion (MCAo) followed by reperfusion and brain tissue was collected at 2, 6, 24, and 48 h post-MCAo. Analysis of brain tissue revealed a qualitative drop in both mRNA and protein levels of Na(v)1.1 throughout ischemic regions, beginning at the early stage of injury (6 h) with dramatic losses at later stages (24 and 48 h). Quantitative cell counts and optical density measurements indicated significant decreases in the percent of brain cells immunoreactive for Na(v)1.1 as well as a loss of signal in those cells positive for Na(v)1.1 in the injured cortex and striatum as compared to the contralateral hemisphere. Double labeling with NeuN and Na(v)1.1 immunoflouresence confirmed that the predominate loss of Na(v)1.1 immunoreactivity was in neurons. In conclusion, these data map the time-dependent loss of Na(v)1.1 mRNA and protein following focal ischemic brain injury in the rat out to 48 h post-injury.

Antiepileptic drug treatment of nonconvulsive seizures induced by experimental focal brain ischemia.

Nonconvulsive seizures (NCSs) after traumatic and ischemic brain injury are often refractory to antiepileptic drug therapy and are associated with a decline in patient outcome. We recently characterized an in vivo rat model of focal brain ischemia-induced NCS and here sought to evaluate potential pharmacological treatments. Electroencephalographic activity was recorded continuously for 24 h in freely behaving rats subjected to permanent middle cerebral artery occlusion (MCAo). Rats were treated with an antiepileptic drug from one of seven different drug classes at ED(50) and 2x ED(50) doses (as reported in other rat seizure models), delivered as a single i.v. injection 20 min post-MCAo. Vehicle-treated rats (n = 9) had an 89% incidence of NCS with an average number of NCS of 8.6 +/- 1.9. The latency to onset of NCS was 32.5 +/- 3.4 min post-MCAo with an average duration of 49.1 +/- 8.2 s/event. The high doses of ethosuximide, gabapentin, fos-phenytoin, and valproate significantly reduced the incidence of NCS (11, 14, 14, and 38%, respectively), whereas midazolam, phenobarbital, and dextromethorphan had no significant effect at either dose. Across treatment groups, there was a low but significant correlation between the number of NCS events per animal and volume of brain infarction (r = 0.352). Antiepileptic drug therapy that prevented the occurrence of NCS also reduced mortality from 26 to 7%. Based on combined effects on NCS, infarction, neurological recovery, and mortality, ethosuximide and gabapentin were identified as having the best therapeutic profile.

DNA fragmentation in leukocytes following subacute low-dose nerve agent exposure.

The objective of the present study was to determine levels of DNA fragmentation in blood leukocytes from guinea pigs by single-cell gel electrophoresis (comet assay) after exposure to the chemical warfare nerve agent (CWNA), soman, at doses ranging from 0.1 LD50 to 0.4 LD50, once per day for either 5 or 10 days. Post-exposure recovery periods ranged from 0 to 17 days. Leukocytes were imaged from each animal, and the images analyzed by computer. Data obtained for exposure to soman demonstrated significant increases in DNA fragmentation in circulating leukocytes in CWNA-treated guinea pigs compared with saline-injected control animals at all doses and time points examined. Notably, significantly increased DNA fragmentation was observed in leukocytes 17 days after cessation of soman exposure. Our findings demonstrate that leukocyte DNA fragmentation assays may provide a sensitive biomarker for low-dose CWNA exposure.

Treatment with the snail peptide CGX-1007 reduces DNA damage and alters gene expression of c-fos and bcl-2 following focal ischemic brain injury in rats.

Delayed cell death following ischemic brain injury has been linked to alterations in gene expression. In this study we have evaluated the upregulation of several genes associated with delayed cell death (c-fos, bax, and bcl-2) during the initial 24 h of transient middle cerebral artery occlusion (MCAo) in the rat and the effects of postinjury treatment with the NR2B subunit specific NMDA receptor antagonist CGX-1007 (Conantokin-G, Con-G). C-fos mRNA levels peaked at 1 h postinjury in both cortical and subcortical ischemic brain regions (30-fold increase), remained elevated at 4 h and returned to within normal, preinjury levels 24 h postinjury. The increase in mRNA levels correlated to increased protein expression in the entire ipsilateral hemisphere at 1 h. Regions of necrosis at 4 h were void of C-Fos immunoreactivity with continued upregulation in surrounding regions. At 24 h, loss of C-Fos staining was observed in the injured hemisphere except for sustained increases along the border of the infarct and in the cingulate cortex of vehicle treated rats. CGX-1007 treatment reduced c-fos expression throughout the infarct region by up to 50%. No significant differences were measured in either bcl-2 or bax mRNA expression between treatment groups. However, at 24 h postinjury CGX-1007 treatment was associated with an increase in Bcl-2 immunoreactivity that correlated to a reduction in DNA fragmentation. In conclusion, CGX-1007 effectively attenuated gene expression associated with delayed cell death as related to a neuroprotective relief of cerebral ischemia.

Central neuro-inflammatory gene response following soman exposure in the rat.

Effective treatments to improve survivability following exposure to the nerve agent soman have been established and are currently available. Unfortunately, electrographic brain seizures, neuroinflammation and brain cell death are still a potential problem even with treatment. In the present study we have characterized the time course of the central neuro-inflammatory gene response using quantitative real time-PCR (TaqMan). Male Sprague-Dawley rats were pre-treated with HI-6 (1-2-hydroxy-iminomethyl-1-pyridino-3-(4-carbamoyl-1-pyridino-2-oxapropane dichloride); 125 mg/kg, i.p.) and exposed 30 min later to 1.6 x LD(50) of soman (pinacolyl methyl-phosphonofluoridate, 180 microg/kg, s.c.) followed at 1 min by atropine methyl nitrate (4 mg/kg, i.m.). Initially, a significant and dramatic upregulation of tumor necrosis factor-alpha and vascular cell adhesion molecule-1 mRNA levels was measured 2 h post-exposure followed at 6 h by upregulation of interleukin-1beta, interleukin-6, E-selectin, and intercellular adhesion molecule-1 with eventual resolution by 24-48 h. In conclusion, an acute and transient upregulation of the inflammatory gene response is activated following soman exposure that may be involved in the soman-induced brain injury process.

The sodium channel blocker RS100642 reverses down-regulation of the sodium channel alpha-subunit Na(v) 1.1 expression caused by transient ischemic brain injury in rats.

In this study we evaluated the expression of five sodium channel (NaCh) Alpha-subunit genes after transient middle cerebral artery occlusion (MCAo) in the rat and the effects of treatment with the NaCh blocker and experimental neuroprotective agent RS100642 as compared to the prototype NaCh blocker mexiletine. The expression of Na(v) 1.1, Na(v) 1.2, Na(v) 1.3, Na(v) 1.7, Na(v) 1.8 and the housekeeping gene beta-actin were studied in vehicle or drug-treated rats at 6, 24 and 48 h post-MCAo using real-time quantitative RT-PCR. RS100642 (1 mg/kg), mexiletine (10 mg/kg), or vehicle (1 ml/kg) was injected (i.v.) at 30 min, 2, 4, and 6 h post-injury. Following MCAo only the Na(v) 1.1 and Na(v) 1.2 genes were significantly down-regulated in the ipsilateral hemisphere of the injured brains. RS100642 treatment significantly reversed the down-regulation of Na(v) 1.1 (but not Na(v) 1.2) at 24-48 h post-injury. Mexiletine treatment, on the other hand, had no significant effect on the down-regulation of either gene. These findings demonstrate that treatment with a neuroprotective dose of RS100642 significantly reverses the down-regulation of Na(v) 1.1 caused by ischemic brain injury and suggests that RS100642 selectively targets the Na(v) 1.1 Alpha-subunit of the NaCh. Furthermore, our findings strengthen the hypothesis that ischemic injury may produce selective depletion of voltage-gated NaChs, and suggest that the Na(v) 1.1 NaCh Alpha-subunit may play a key role in the neuronal injury/recovery process.

Effect of the proteasome inhibitor MLN519 on the expression of inflammatory molecules following middle cerebral artery occlusion and reperfusion in the rat.

Anti-inflammatory treatment with the proteasome inhibitor MLN519 has been previously reported to be neuroprotective against ischemic brain injury in rats. These effects have been related to inhibition of the transcription factor NF-kappaB, which is activated through ubiquitin-proteasomal degradation. The aim of this study was to evaluate the effects of MLN519 to alter the expression of several inflammatory genes under the control of NF-kappaB. Male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAo) followed by vehicle or MLN519 (1.0 g/kg, i.v.) treatment immediately after reperfusion of blood to the brain at 2h. Gene expression was evaluated 3-72 h post-MCAo. The most striking effects of intravenous treatment with MLN519 were associated with reductions in ICAM-1 expression at 3 h followed by reductions in E-selectin (12-72 h). Less dramatic reductions were observed in IL-1Beta (3-24 h) and TNF-Alpha (24 h) with no apparent effects on IL-6 and VCAM-1 mRNA levels. Immunohistochemical analysis revealed that the genes most dramatically affected by MLN519 had highest expression in endothelial cells and leukocytes (E-selectin, ICAM-1),indicating that these cell types may be the primary targets of intravenously delivered MLN519 treatment.

Differential pattern of expression of voltage-gated sodium channel genes following ischemic brain injury in rats.

This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in rats. Using quantitative RT-PCR, our findings demonstrated the expression ratio of NaCh genes in normal rat brain to be Na(v)1.1 > Na(v)1.8 > Na(v)1.3 > Na(v)1.7 (rBI > PN3 > rBIII > PN1). In contrast, brain injury caused by middle cerebral artery occlusion (MCAo) for 2 h followed by reperfusion significantly down-regulated Na(v)1.3 and Na(v)1.7 genes in both injured and contralateral hemispheres; whereas the Na(v)1.8 gene was down regulated in only the injured hemisphere (though only acutely at 2 or 2-6 h post-MCAo). However, the time-course of NaCh gene expression revealed a significant down-regulation of Na(v)1.1 only in the ischemic hemisphere beginning 6 h post-MCAo and measured out to 48 h post-MCAo. In a separate preliminary study Na(v)1.2 (rBII) gene was found to be expressed at levels greater than that of Na(v)1.1 in normal rats and was significantly down regulated at 24 h post-MCAo). Our findings document, for the first time, quantitative and relative changes in the expression of various NaCh genes following ischemic brain injury and suggest that the Na(v)1.1 sodium channel gene may play a key role in ischemic injury/recovery.

Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia.

Activation of group I metabotropic glutamate receptors (mGluR) has been implicated in the pathophysiology of acute central nervous system injury. However, the relative roles of the two group I subtypes, mGluR1 or mGluR5, in such injury has not been well examined. We compared the effects of treatment with the newly developed, selective mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) and the selective mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) in a rat intraluminal filament model of temporary middle cerebral artery occlusion (MCAo). Rats were administered MPEP or CHPG i.c.v. beginning 15 or 135 min after induction of ischemia for 2 h. Infarct size was measured after either 22 or 70 h of reperfusion, and neurological function was quantified at 2, 24, 48 and 72 h. Treatment with MPEP or CHPG at 15 min reduced 24 h infarct volume by 61 and 44%, respectively. The neuroprotective effects were dose dependent. Delaying MPEP treatment until 135 min eliminated the neuroprotective effects. In other studies, using early MPEP treatment (15 min) at optimal doses, infarct volume was reduced by 44% at 72 h and this was correlated with significant neurological recovery. These data suggest that both MPEP and CHPG are neuroprotective when administered after focal cerebral ischemia. In separate, recent studies we found that although MPEP does act as an mGluR5 antagonist and blocks agonist induced phosphoinositide hydrolysis, it also serves as a non-competitive NMDA antagonist; in contrast, other results indicate that CHPG mediated neuroprotection may reflect anti-apoptotic activity. Therefore, both types of compounds may prove to have therapeutic potential for the treatment of stroke.

Electroencephalogram analysis and neuroprotective profile of the N-acetylated-alpha-linked acidic dipeptidase inhibitor, GPI5232, in normal and brain-injured rats.

We have evaluated the effects of the N-acetylated-alpha-linked acidic dipeptidase (NAALADase) inhibitor, GPI5232 [2-[(pentafluorophenylmethyl)hydroxyphosphinyl]methyl)-pentanedioic acid], to not only decrease brain injury but also to alter the inherent electroencephalographic (EEG) changes observed in a rat model of transient middle cerebral artery occlusion (MCAo). Continuous i.v. infusion of GPI5232 starting 1 h after injury resulted in more than a 50% reduction in brain infarct volume caused by 2 h of MCAo. This effect was dose-dependent and significant even when first treatment was delayed for 2 h post-MCAo. At 24 h post-MCAo, EEG spectral analysis of the injured hemisphere revealed functional improvement in GPI5232-treated rats. Significant recovery in high-frequency EEG power (8-30 Hz) was measured in GPI5232-treated animals in both parietal and temporal brain regions but not in vehicle-treated animals. MCAo-injured rats were also predisposed to developing cortical brain seizures, and GPI5232-treated rats had significantly fewer brain seizures than vehicle-treated animals. In separate experiments, acute high doses of GPI5232 in normal rats did not significantly alter EEG brain activity as evaluated by spectral analysis and did not produce any signs of seizure activity or behavioral abnormalities. These results show GPI5232 to be an effective neuroprotective treatment when given postinjury by reducing brain infarction and ameliorating the pathological EEG associated with focal brain ischemia.

Quantitative electroencephalography spectral analysis and topographic mapping in a rat model of middle cerebral artery occlusion.

Electroencephalography (EEG) has a long history in clinical evaluations of cerebrovascular disease. Distinct EEG abnormalities, such as increased slow delta activity, voltage depression and epileptiform discharge, have been identified in stroke patients. However, preclinical use of EEG analysis of cerebral ischaemia is less documented. We report a new rat model of EEG topographic mapping during permanent and transient middle cerebral artery occlusion. Ten EEG electrodes were implanted on the rat skull, symmetrically covering the cortical regions of two hemispheres. Monopolar EEG recordings were acquired from each animal at multiple time points during the initial 24 h, and again once daily for 7 days. Traditional EEG examinations, quantitative EEG (qEEG) spectral analysis and topographic EEG mapping were employed for comprehensive data analyses. Several distinct spatiotemporal EEG abnormalities were identified in the ischaemic rat brain. In the ipsilateral hemisphere, pronounced increase in delta activity was observed in each recorded area within 24 h of injury. While sharp waves and spike complexes dominated the parietal region, a nearly isoelectric EEG state was seen in the temporal region. After 48 h, spontaneous, albeit incomplete, recovery of EEG activities developed in all rats. Reperfusion appeared to promote delta and alpha recovery more efficiently. The contralateral EEG changes were also recorded in two phases: an acute moderate increase in delta activities with intermittent rhythmic activities, followed by a delayed and significant increase in beta activities across the hemisphere. The similarities of rat qEEG profiles identified in this study to that of stroke patients and the application of topographic mapping broaden our research technology for preclinical experimental studies of brain injury.

RS-100642-198, a novel sodium channel blocker, provides differential neuroprotection against hypoxia/hypoglycemia, veratridine or glutamate-mediated neurotoxicity in primary cultures of rat cerebellar neurons.

The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), in in vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 micro M. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS-100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium [Ca(2+)]i) mobilization revealed that glutamate caused immediate and sustained increases in [Ca(2+)]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca(2+)]i mobilization produced by veratridine or KCl depolarization. QX-314 produced similar effects on glutamate-, veratridine- or KCl-induced [Ca(2+)]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca(2+)]i, and attenuating the sustained increase in [Ca(2+)]i mobilization. By using different in vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na(+) channels.

Neuroprotection produced by the NAALADase inhibitor 2-PMPA in rat cerebellar neurons.

The present study examined the neuroprotective actions of the N-acetylated-alpha-linked-acidic dipeptidase (NAALADase) inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) in four in vitro models of neurotoxicity. Using neuron-enriched primary cultures derived from rat embryo (E15) cerebellum, 2-PMPA afforded 100% neuroprotection from injuries induced by hypoxia (EC(50)=8.4 microM). In contrast, against glutamate or N-methyl-D-aspartate (NMDA) injury, 2-PMPA was less potent and its efficacy limited to a maximum of 46% and 16%, respectively. 2-PMPA was not effective against veratridine-induced injury. Also, the less potent analog of 2-PMPA, 2-[phosphonomethyl]succinic acid (2-PMSA), was ineffective. Unlike 2-PMPA, the endogenous NAALADase substrate and mGlu(3) receptor agonist N-acetyl-aspartyl-glutamate (NAAG) was neuroprotective against all four injury mechanisms and compared to 2-PMPA, exhibited a different "phosphate effect" on neuroprotection. These results confirm the superior efficacy of 2-PMPA to protect against injury caused by cellular anoxia, and are discussed relative to upstream modulation of hyperglutamatergic activity vs. downstream modulation of metabotropic receptors as possible targets for ischemia/stroke therapy.

Proteasome inhibitor PS519 reduces infarction and attenuates leukocyte infiltration in a rat model of focal cerebral ischemia.

BACKGROUND AND PURPOSE: Reperfusion brain injury after cerebral ischemia is associated with a developing inflammatory response at the site of infarction. Proteasome inhibitors block nuclear factor-kappaB activation and provide anti-inflammatory effects in several animal models of peripheral inflammation. We tested the novel proteasome inhibitor PS519 in a rat model of transient focal ischemia to establish its pharmacodynamics as a neuroprotection treatment and related effects on leukocyte infiltration. METHODS: Rats were subjected to 2 hours of focal cerebral ischemia by means of the filament method of middle cerebral artery occlusion (MCAo). After either 22 or 70 hours of reperfusion, infarct size was measured and neurological function, electroencephalographic (EEG) activity, and/or neutrophil and macrophage infiltration was quantified. PS519 was administered in a single intravenous bolus at 2 hours after MCAo. In addition, the therapeutic window for PS519 was estimated by delaying treatment for 4 or 6 hours after MCAo. RESULTS: Dose-response analysis of infarct volume at 24 hours revealed that PS519 neuroprotection approached 60%, and clinical evaluations showed significant improvements in neurological function and EEG activity. Neutrophil infiltration at 24 hours was also significantly decreased in cortical and striatal infarcted tissue of PS519-treated rats. Delaying the PS519 treatment up to 4 hours continued to result in significant neuroprotection. In the 72-hour injury model, infarction was reduced 40% by PS519, and significant improvements in neurological function and EEG recovery were again measured. Considerable reductions in both neutrophil and macrophage infiltration were evident. CONCLUSIONS: PS519 mitigates infarction and improves neurological recovery in brain-injured rats, an effect in part caused by a reduction in the leukocyte inflammatory response.

Neuroprotective efficacy and therapeutic window of the high-affinity N-methyl-D-aspartate antagonist conantokin-G: in vitro (primary cerebellar neurons) and in vivo (rat model of transient focal brain ischemia) studies.

Conantokin-G (Con-G), a 17-amino-acid peptide derived from marine snails and a potent N-methyl-D-aspartate (NMDA) antagonist, was evaluated for its neuroprotective properties in vitro and in vivo. In primary cerebellar neurons, Con-G was shown to decrease excitotoxic calcium responses to NMDA and to exhibit differential neuroprotection potencies against hypoxia/hypoglycemia-, NMDA-, glutamate-, or veratridine-induced injury. Using the intraluminal filament method of middle cerebral artery occlusion as an in vivo rat model of transient focal brain ischemia, the neuroprotective dose-response effect of Con-G administration beginning 30 min postocclusion was evaluated after 2 h of ischemia and 22 h of reperfusion. In the core region of injury, an 89% reduction in brain infarction was measured with significant neurological and electroencephalographic recovery at the maximal dose tested (2 nmol), although mild sedation was noted. Lower doses of Con-G (0.001-0.5 nmol) were significantly neuroprotective without causing sedation. Postinjury time course experiments demonstrated a therapeutic window out to at least 4 to 8 h from the start of the injury, providing a 47% reduction in core injury. The neuroprotective effect of Con-G (0. 5 nmol) was also evaluated after 72 h of injury, where a 54% reduction in core brain infarction was measured. Critically, in both recovery models (i.e., 24 and 72 h), the reduction in brain infarction was associated with significant improvements in neurological and electroencephalographic recovery. These data provide evidence for the potent and highly efficacious effect of Con-G as a neuroprotective agent, with an excellent therapeutic window for the potential intervention against ischemic/excitotoxic brain injury.

Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

Neuroprotection (focal ischemia) and neurotoxicity (electroencephalographic) studies in rats with AHN649, a 3-amino analog of dextromethorphan and low-affinity N-methyl-D-aspartate antagonist.

AHN649, an analog of dextromethorphan (DM) and a relatively selective low-affinity N-methyl-D-aspartate antagonist, was evaluated for neuroprotective effects using the rat intraluminal filament model of temporary middle cerebral artery occlusion. Rats were subjected to 2 h of focal ischemia followed by 72 h of reperfusion. In vehicle-treated rats, middle cerebral artery occlusion resulted in neurological deficits and severe infarction measuring 232 +/- 25 mm(3), representing approximately 25% contralateral hemispheric infarction. Post-treatment with AHN649 (0.156-20 mg/kg i.v.) or DM (0.156-10 mg/kg i.v.) significantly reduced cortical infarct volume by 40 to 60% compared with vehicle-control treatments. AHN649 neuroprotection was linear and dose dependent (ED(50) = 0.80 mg/kg), whereas DM neuroprotection (ED(50) = 1.25 mg/kg) was nonlinear and less effective at the higher doses (2.5-10 mg/kg). Although impaired neurological function scores improved in all groups by 24 to 72 h, the most dramatic improvement was associated with AHN649 treatments. In a rat electroencephalographic model of brain function, separate neurotoxicity experiments revealed that acute i.v. doses of DM caused seizures (ED(50) = 19 mg/kg) and death (LD(50) = 27 mg/kg). In contrast, AHN649 failed to induce seizure activity at doses up to 100 mg/kg (LD(50) = 79 mg/kg). Collectively, AHN649 is described as a potent, efficacious neuroprotective agent devoid of serious central nervous system neurotoxicity and possessing potential therapeutic value as antistroke treatment. Furthermore, the feasibility of targeting low-affinity N-methyl-D-aspartate-site ligands as postinjury therapy for ischemic brain injury has been confirmed.

Anticonvulsant efficacy of N-methyl-D-aspartate antagonists against convulsions induced by cocaine.

Convulsions associated with cocaine abuse can be life threatening and resistant to standard emergency treatment. Cocaine (75 mg/kg, i. p.) produced clonic convulsions in approximately 90% of male, Swiss-Webster mice. A variety of clinically used antiepileptic agents did not significantly protect against cocaine convulsions (e. g., diazepam and phenobarbital). Anticonvulsants in clinical practice that did significantly protect against convulsion did so only at doses with significant sedative/ataxic effects (e.g., clonazepam and valproic acid). In contrast, functional N-methyl-D-aspartate (NMDA) antagonists all produced dose-dependent and significant protection against the convulsant effects of cocaine. Anticonvulsant efficacy was achieved by blockade of both competitive and noncompetitive modulatory sites on the NMDA receptor complex. Thus, competitive antagonists, ion-channel blockers, polyamine antagonists, and functional blockers of the strychnine-insensitive glycine modulatory site all prevented cocaine seizures. The role of NMDA receptors in the control of cocaine-induced convulsions was further strengthened by the positive correlation between the potencies of noncompetititve antagonists or competitive antagonists to block convulsions and their respective affinities for their specific binding sites on the NMDA receptor complex. Although some NMDA blockers produced profound behavioral side effects at efficacious doses (e.g., noncompetitive antagonists), others (e.g., some low-affinity channel blockers, some competitive antagonists, and glycine antagonists) demonstrated significant and favorable separation between their anticonvulsant and side effect profiles. The present results provide the most extensive evidence to date identifying NMDA receptor blockade as a potential strategy for the discovery of agents for clinical use in averting toxic sequelae from cocaine overdose. Given the literature suggesting a role for these drugs in other areas of drug abuse treatments, NMDA receptor antagonists sit in a unique position as potential therapeutic candidates.

Neuroprotective role of c-fos antisense oligonucleotide: in vitro and in vivo studies.

We investigated the dose-response and time-course of c-fos antisense oligodeoxynucleotide (ASO) treatment against excitatory amino acid (EAA)-induced neurotoxicity in rat hippocampal neurons. Glutamate (in vitro) or NMDA (in vivo) produced significant neuronal degeneration. Neuroprotection produced by 30 min or 4 h pretreatment with c-fos ASO in cultured hippocampal neurons was dose-dependent. In vivo, bilateral intrahippocampal injections of c-fos ASO (0.025 nmol/site) was neuroprotective when administered 30 min before or after NMDA treatment. However, 4 h pretreatment was ineffective. A higher dose (0.125 nmol) of c-fos ASO was neurotoxic and failed to afford neuroprotection regardless of the treatment schedule. Collectively, these results demonstrate a neuroprotective effect of c-fos ASO against EAA-induced neuronal injury supporting a causative role of c-fos expression in EAA neurotoxicity.