Effective Neuroprotective Agent Research Articles

動物実験における症候評価の問題点

Recently, several clinical trials in acute stroke have failed to produce efficacy. This suggests that there is a large gap between results in human trials and in animal. Animal models may present on aspect of pathophysiology in patients. We have to know how similar and what aspect of animal models can be extrapolated to patients. It is a big question whether or not therapeutic time window between animal models and patients is similar. In some animal models, cerebral damage establishes within 6 hours of the MCA occlusion, and this indicates that it is impossible to cure cerebral damage when drug injection is administrered over a 6 hour pe-riod. Recently, some investigators demonstrated that in human penumbra disappears within 6 hours after ischemia and therapeutic time window is as narrow as that in animal models. Finally, in animal studies, effects of neuroprotective agents are evaluated by the size of cerebral damage and neurological deficits. In human trials, effects of these agents are evaluated by stroke scores in including Japan stroke scores. In stroke scores, consciousness, language ana paralysis are examined, but in animal models, only paralysis is examined. Data of neurological symptoms of animal studies do not accord with those in human trials.

Review of clinical trials of neuroprotection in acute spinal cord injury

In this paper the authors review the clinical trials of neuroprotection that have been performed for the treatment of acute spinal cord injury (SCI). The biological rationale for the selection of each treatment modality is discussed with reference to current knowledge of the principles in the management of acute SCI as well as the primary and secondary injury mechanisms identified by experimental and clinical studies of the pathophysiology of acute SCI. The trials are evaluated with regard to the availability and use of accurate clinical outcome measures, and the methodologies of the trials are critically evaluated with an emphasis on prospective randomized controlled studies. A detailed description and critical analysis are provided of the results of the 10 clinical trials conducted to date in which a randomized prospective controlled design has been used. The issue of the therapeutic time window in acute SCI is discussed. To date, methylprednisolone is the only effective neuroprotective agent that has been established for use in human SCI, and the only therapeutic time window established in human SCI is a maximum trauma-to-treatment time of 8 hours.

Progesterone facilitates the acquisition of avoidance learning and protects against subcortical neuronal death following prefrontal cortex ablation in the rat

Following a cortical injury, neurons in areas near and connected to the site of injury begin to degenerate. The observed neuronal death may contribute to the severity of the observed behavioral impairments. The purpose of the present study was to examine if progesterone, a hormone known for its effectiveness at reducing cerebral edema, could protect against secondary neuronal death and facilitate the acquisition of an avoidance learning task in an ablation model of cortical injury. Rats served as sham controls or received bilateral ablation of the medial prefrontal cortex followed by a 10-day regimen of progesterone (4 mg/kg) or oil vehicle (1 ml/kg) beginning 1 h after cortical lesions. Progesterone-treated lesion rats showed a significant facilitation of avoidance learning compared to oil-treated lesion controls. In addition, progesterone-treated lesion animals did not differ from either progesterone- or oil-treated sham controls in avoidance learning. Anatomical analysis revealed that progesterone treatment decreased the amount of neuronal death seen in the striatum and the mediodorsal nucleus of the thalamus. The findings are consistent with the notion that progesterone is an effective neuroprotective agent and suggest that the hormone can reduce the behavioral impairments associated with frontal cortical ablation injury.

MDL 101,002, a free radical spin trap, is efficacious in permanent and transient focal ischemia models

The present work describes the neuroprotective effects of the free radical spin trap, MDL 101,002, in models of permanent and transient focal ischemia. Permanent focal ischemia was carried out by occlusion of the distal segment of the middle cerebral artery (MCA) and CCA's in Spontaneously Hypertensive (SH) and Wistar rats. Transient focal ischemia was undertaken by occluding the origin of the MCA for 180 min by the intraluminar monofilament method in Wistar rats. With permanent distal MCA occlusion in SH rats, 100 mg kg i.v. at 30 min post-ischemia resulted in a significant 40% reduction in infarct volume. Similarly, a 75 mg kg bolus + 45 mg kg-h dose of MDL 101,002 given i.v. at 5 min post-ischemia resulted in a 90% or 60% decrease in infarct volume in the mixed permanent/transient distal MCA model with Wistar rats using 120 or 180 min of CCA occlusion, respectively. When full reperfusion was established, after 180 min of occlusion in the proximal MCA model, a dose of 40 mg kg + infusion and 75 mg kg + infusion resulted in a significant 50% and 70% decrease in ischemic damage, respectively. MDL 101,002 is clearly an effective neuroprotective agent in all models examined. This work would suggest that this novel cyclic nitrone spin trap affords effective neuroprotection and is useful for the treatment of ischemic stroke.

State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in a mammalian cell line expressing rat brain type IIA Na+ channels.

The compound 619C89 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-tricholorophenyl)-pyr imidine] is an effective neuroprotective agent in in vivo models of cerebral ischaemia. It has been suggested to act by inhibiting voltage-gated Na+ channels. To test this hypothesis, the action of 619C89 on recombinant rat brain type IIA Na+ channels expressed in Chinese hamster ovary cells and on native Na+ channels in acutely dissociated rat hippocampal neurons has been studied using whole-cell voltage-clamp recording techniques. In the cell line expressing type IIA Na+ channels, 619C89 caused a reversible inhibition of Na+ currents in a concentration- and voltage-dependent manner. A half-maximal inhibitory concentration (IC50) of approximately 50 microM was obtained at a holding potential of -90 mV whereas, with a conditioning prepulse to -60 mV for 30 s, the IC50 was reduced to 8 microM. Furthermore, the inhibition was markedly enhanced by a use-dependent action, which was dependent not only on the frequency of stimulation, but also on the duration (3.5-40 ms) of the pulses. Trains (10-50 Hz) of up to 60 depolarizing pulses of 0.7 ms duration did not evoke any use-dependent inhibition in the presence of 619C89, suggesting that this compound is not an open channel blocker. The voltage- and use-dependent inhibition by 619C89 was also observed on native Na+ channels in hippocampal neurons. 619C89 (10 microM) produced a small hyperpolarizing shift in the fast inactivation curve and a substantial (13 mV) hyperpolarizing shift in slow inactivation. The compound dramatically delayed the recovery from inactivation without affecting the development of inactivation. Moreover, 619C89 has no effect on the shape of the current-voltage relationship or on the voltage activation curve. These data indicate that 619C89 interacts selectively with the inactivated state of the Na+ channel with an estimated affinity of 3 microM. This primary action of 619C89 may underlie its neuroprotective effects.

Differences in striatal extracellular amino acid concentrations between wistar and fischer 344 rats after middle cerebral artery occlusion

We hypothesized that the interstrain difference between Wistar and Fischer-344 (F344) rats in cerebral infarction volume after proximal middle cerebral artery (MCA) occlusion might be explained by differences in excitotoxicity between both rat strains. Using microdialysis we measured during a 5 h period after MCA occlusion the release of aspartate, glutamate and taurine in the cerebral cortex and the striatum. The volume of striatal infarction was comparable in Wistar and F344 rats. We found, however, in Wistar rats a significantly higher striatal efflux of aspartate and glutamate than in F344 rats, whereas the striatal taurine efflux was of a similar magnitude in the two strains. Because of the (variably) smaller volume of cortical infarction in Wistar rats (than that in F344), the location of the microdialysis probe-membrane with respect to the area of cortical infarction differed between Wistar rats. Hence, a reliable comparison between the quantitative amount of amino acids in the dialysate from the cortical probes of both rat strains could not be made. These results, demonstrating differences in striatal excitotoxicity between Wistar and F344 rats after MCA occlusion, are the first to show interstrain differences in striatal pathophysiology of focal ischemia between these normotensive rat strains. They do however not explain why MCA occlusion results in a significantly different volume of cortical infarction between Wistar and F344 rats. The F344 strain will probably show in a more sensitive way, as compared to Wistar rats, neuroprotective effects of agents that diminish excitotoxic damage during focal cerebral ischemia.

ARL-15896, a NovelN-Methyl-D-aspartate Receptor Ion Channel Antagonist: Neuroprotection against Mitochondrial Metabolic Toxicity and Regional Pharmacology

ARL-15896 [(+)-α-phenyl-2-pyridineethanamine], formerly known as FPL-15896, is a novelN-methyl-D-aspartate (NMDA) receptor ion channel antagonist. Using quantitative receptor autoradiography, we examined the regional binding characteristics of ARL-15896 and compared them to those of MK-801, the prototypical NMDA receptor channel blocker. The affinity of ARL-15896 was much lower (3000-fold) than that of MK-801 in all brain regions examined. In addition, in contrast to MK-801, which has a higher affinity in the forebrain than in the cerebellum (IC50of 10 nMvs 24 nM), ARL-15896 had a higher affinity in the cerebellum than in the forebrain (IC50of 17 μMvs 45 μM). The neuroprotective potential of ARL-15896 was investigated in a rat model of excitotoxicity, the intrastriatal injection of malonate. Malonate is a competitive inhibitor of succinate dehydrogenase, and its toxicity has been shown to be mediated largely by the NMDA receptor. Administration of ARL-15896 either intrastriatally (200 nmol) or subcutaneously (9.0 mg/kg) reduced the volume of the lesion produced by 1 μmol of malonate by 80%, a degree similar to that reported for MK-801. ARL-15896 was also protective when administered after the malonate injection. Furthermore, in contrast to MK-801, administration of ARL-15896 was not associated with any apparent behavioral side effects. This report is consistent with previous studies suggesting that drugs with regional pharmacological profiles similar to that of ARL-15896 have better clinical tolerability; it also indicates that ARL-15896 is an effective neuroprotective agent.

Therapeutic Potential of AMPA Receptor Ligands in Neurological Disorders

Summary Neurodegeneration produced by toxic levels of glutamate is now suggested to be a causative factor in the pathologies found in a number of neurological diseases. This glutamate-induced toxicity is mainly due to activation of both the N-methyl-D-aspartate (NMDA) and non-NMDA classes of glutamate receptors. Hence, drugs that act as antagonists at these receptors are potentially neuroprotective in many diseases. Noncompetitive antagonists appear to be the preferred type of ligand because their action is not diminished by the levels of glutamate reached during a trauma. In addition, they may have reduced adverse effects compared with competitive antagonists. The group of non-NMDA receptors consists of the a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, which can be defined on the basis of their pharmacology or by recombinant gene techniques. The latter methods demonstrate a far more complex picture for the receptors. Studies show the presence of at least 9 different protein subunits, which, when linked in groups of 5, comprise the AMPA and kainate subclasses of glutamate receptors. The native non-NMDA receptor has at least 3 separate binding sites at which non-NMDA receptor antagonists can act: glutamate, desensitisation and intra-ion channel binding sites. The glutamate binding site is the site for competitive antagonists such as 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX). Non-NMDA receptors show rapid desensitisation, which limits the duration of activation of the receptor. One group of noncompetitive antagonists (e.g. GYKI 52466) binds at this desensitisation site. Another group of noncompetitive antagonists, the spider and wasp toxins, bind at the third site within the ion channel. Protection against ischaemic damage is the most well researched indication for the application of non-NMDA receptor antagonists and the one that shows the most promise. However, almost all studies in any indication so far have only been carried out in rodents. Recent evidence suggests that antagonists at non-NMDA receptors are more effective neuroprotective agents than NMDA receptor antagonists after ischaemic attacks, and that their administration can be delayed for up to 12 hours without seriously compromising the extent of neuroprotection. Protection against neuronal loss caused by physical injury to the brain or motor neuron disease are other potential uses for non-NMDA receptor antagonists. The antagonists are less effective than the NMDA receptor antagonists against neuronal loss caused by hypoglycaemia or status epilepticus. Non-NMDA receptor antagonists are also effective as anticonvulsants and as antiemetics during cancer chemotherapy. As antiparkinsonian drugs, they show marked synergistic effects when given in combination with levodopa, but are unlikely to be useful as a monotherapy for this disorder. Despite evidence for potential in a number of disorders, prolonged use of non-NMDA receptor antagonists may be contraindicated due to their adverse effects on memory, cognition, motor activity and autonomic functions. Nephrotoxicity due to poor solubility and a short duration of action are also limitations of at least some of the current generation of non-NMDA receptor antagonists. Minimising these adverse effects, particularly with drugs that are selective for the receptor subunits, and translating the results of animal studies to human conditions will be awaited with interest.

The effects of (-)- and (+)-β-cyclazocine on NMDA-evoked responses and NMDA-mediated cell damage in cultured rat hippocampal neurons

Microspectrofluorimetric measurements of excitatory amino acid-evoked rises in intracellular free calcium concentration ([Ca 2+] i), electrophysiological measurements of currents through single NMDA receptor-operated ion channels and estimates of cellular viability following NMDA challenge were employed to examine the interactions of (−)- and (+)-β-cyclazocine with the NMDA receptor-channel complex in cultured rat hippocampal neurons. Rises in [Ca 2+] i evoked by NMDA, but not those evoked by kainate, AMPA or 50 mM K +, were reduced by (−)-β-cyclazocine in a concentration- and use-dependent manner with an estimated IC 50 value of 272 nM. In outside-out pathces, (−)-β-cyclazocine did not change the magnitudes of unitary NMDA-evoked currents but diminished both the frequency of channel openings and their mean open time. The IC 50 for (−)-β-cyclazocine against NMDA channel open state probability was estimated at 84 nM. The actions of (−)-β-cyclazocine were consistent with a voltage-dependent open channel block of the NMDA channel with a blocking rate constant of 7.03·10 7 M −1·s −1 at −40 mV. Neurons exposed to a high concentration of NMDA in vitro were protected from death by 1 and 10 μM (−)-β-cyclazocine. In all of the above assays, (+)-β-cyclazocine was considerably less potent an NMDA antagonist and neuroprotective agent than (−)-β-cyclazocine; the IC 50 for (+)-β-cyclazocine against channel open state probability was estimated at 14 μM. The results demonstrate that (−)-β-cyclazocine is a potent and selective inhibitor of NMDA-evoked responses in cultured rat hippocampal neurons and an effective neuroprotective agent in vitro.

The neuroprotective effect of chlormethiazole on ischaemic neuronal damage following permanent middle cerebral artery ischaemia in the rat

The ability of chloremethiazole to protect against ischaemic cell damage in a rat model of permanent focal ischaemia has been examined. Chlormethiazole (1 mmol/kg) was administered intraperitoneally either 1 or 3 h after occlusion of the middle cerebral artery with an intraluminal filament. Twenty four hours after the start of occlusion there was histological evidence for ischaemic damage in both cortex and striatum. The volume of ischaemic damage in control (saline injected) animals was 310 ± 25 mm 3 (mean ± SEM; n = 6). Chlormethiazole administered 1 h after occlusion reduced this damage by 58% (128 ± 40 mm 3; n = 6; P < 0.01), protection being observed in both brain regions. The drug was ineffective when given 3 h after occlusion (304 ± 25 mm 3; n = 5). Chlormethiazole had no effect on body temperature, mean arterial blood pressure, blood pH, pO 2 or pCO 2, but did induce mild bradycardia. Chlormethiazole therefore appears to be an effective neuroprotective agent in this model of permanent ischaemia, complementing previous data on the efficacy of this drug in other focal and global models of cerebral ischaemia.

The effect of chlormethiazole on neuronal damage in a model of transient focal ischaemia.

1. The effect of chlormethiazole has been studied in a transient middle cerebral artery (MCA) occlusion model of cerebral ischaemia in the rat. The MCA was occluded for 1 h by use of an intraluminal suture technique, with reperfusion for 24 h following removal of the occluding filament. Neuronal damage was determined by measurement of the area of necrosis following Cresyl Violet staining of sections taken through the ischaemic region. 2. In the initial experiment, occlusion of the MCA produced a large volume of ischaemic damage in both cortex and striatum, characterized by necrosis and pyknosis (total volume of damage, 287 +/- 13 mm3, n = 9). Rats injected with chlormethiazole (1000 mumol kg-1, i.p.) 60 min before occlusion had a reduced volume of damage in both regions (104 +/- 11 mm3; n = 9; P < 0.001). 3. In a subsequent study systemic physiological parameters (heart rate, blood pressure, blood pH, blood gases and rectal temperature) were measured throughout the ischaemic period. 4. Chlormethiazole (1000 mumol kg-1) pretreatment produced little change in systemic physiology and the neuroprotective effect of the drug when given 60 min prior to the MCA occlusion was confirmed. Chlormethiazole was also neuroprotective when given 10 min following the start of reperfusion (control group: 244 +/- 52 mm3, n = 10; chlormethiazole pretreatment group: 102 +/- 23 mm3, n = 10; P < 0.001; chlormethiazole post-ischaemia group: 122 +/- 16 mm3; P < 0.001, n = 10). 5. It is concluded that chlormethiazole is an effective neuroprotective agent in this model of transient focal ischaemia. The observation that chlormethiazole is protective when given after reperfusion indicates that the effect of the drug is unlikely to be due to an alteration of intra-ischaemic cerebral blood flow, but is more probably a direct effect on the development of ischaemic damage.

The neuroprotective efficacy of ebselen (a glutathione peroxidase mimic) on brain damage induced by transient focal cerebral ischaemia in the rat

The neuroprotective efficacy of the hydroperoxide scavenger ebselen was assessed in a model of transient focal ischaemia that utilises the potent vasoconstrictor peptide endothelin-1 to induce temporary occlusion of the middle cerebral artery (MCA). Pretreatment with ebselen (10 or 30 mg/kg p.o., 40 min pre-MCA occlusion) dose dependently reduced the volume of ischaemic damage assessed 4 h post-endothelin-1 application in the anesthetised rat. The lower dose of ebselen (10 mg/kg) resulted in a non-significant 35% reduction in the total volume of ischaemic damage compared with the vehicle control. In contrast the higher dose of ebselen (30 mg/kg) significantly reduced the volume of ischaemic damage in the cerebral hemisphere and cerebral cortex by 48% and 53%, respectively. The marked reduction in brain damage achieved with ebselen cannot be attributed to drug-induced alterations in blood pressure, body temperature or arterial blood gases since these physiological variables were closely monitored and were not significantly altered by ebselen treatment. Thus ebselen is an effective neuroprotective agent against acute focal ischaemic-reperfusion injury.

Solution structure of omega-conotoxin GVIA using 2-D NMR spectroscopy and relaxation matrix analysis.

We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.

Limiting ischemic injury by inhibition of excitatory amino acid release.

Excitatory amino acids (EAAs) are important mediators of ischemic injury in stroke. N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to be very effective neuroprotective agents in animal models of stroke, but may have unacceptable toxicity for human use. An alternative approach is to inhibit the release of EAAs during stroke. BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine], a drug that inhibits veratrine-induced release of the EAA glutamate in vitro, was tested in a rat model of proximal middle cerebral artery (MCA) occlusion. BW1003C87 significantly decreased ischemia-induced glutamate release in brain when given either 5 min before or 15 min following permanent MCA occlusion. Pretreated and posttreated rats had smaller infarct volumes and preserved glucose metabolism in the ischemic cortex at 24 h after MCA occlusion. BW1003C87 did not induce heat shock protein in the cingulate or retrosplenial cortex, suggesting that it does not injure neurons in these regions as do NMDA antagonists. These results demonstrate that drugs that inhibit glutamate release in ischemia may be nontoxic and show promise for the treatment of stroke.

The neurotoxic effects of methamphetamine on 5-hydroxytryptamine and dopamine in brain: Evidence for the protective effect of chlormethiazole

Studies were undertaken in mice and rats on the neurotoxic effects of methamphetamine on dopaminergic and 5-hydroxytryptaminergic neurones in the brain and the neuroprotective action of chlormethiazole. In initial studies, mice were injected with methamphetamine (5 mg/kg, i.p.) at 2 hr intervals, to a total of 4 times. This procedure produced a 66% loss of striatal dopamine and a 50% loss of tyrosine hydroxylase activity 3 days later. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each dose of methamphetamine, totally prevented the methamphetamine-induced loss of tyrosine hydroxylase activity and partly prevented the loss of dopamine. Phencyclidine (20 mg/kg, i.p.), given in place of chlormethiazole, also prevented the loss of tyrosine hydroxylase. Administration to rats of 4 doses of methamphetamine (15 mg/kg, i.p.) at 3 hr intervals resulted in a 75% loss of striatal dopamine 3 days later and a similar loss of 5-HT and 5-HIAA in cortex and hippocampus. Chlormethiazole (50 mg/kg, i.p.), given 15 min before each injection of methamphetamine, protected against the loss of dopamine and indoleamine content, in the respective regions. Pentobarbital (25 mg/kg, i.p.) also provided substantial protection but diazepam (2.5 mg/kg, i.p.) was without effect. Confirming earlier studies, dizocilpine (1 mg/kg) also provided substantial protection against the methamphetamine-induced neurotoxicity. Preliminary data indicated that chlormethiazole was not neuroprotective because of a hypothermie action. These data therefore demonstrate that chlormethiazole is an effective neuroprotective agent against methamphetamine-induced neurotoxicity and extend the evidence for the possible value of this drug in preventing neurodegeneration.

Temporal changes in edema, Na+, K+, and Ca++ in focal cortical stroke: GM1 ganglioside reduces ischemic injury

Cortical focal ischemia in the rat was induced by middle cerebral artery occlusion (MCAo) together with permanent occlusion of the ipsilateral common carotid artery (CCAo) and a temporary (1 hr) occlusion of the contralateral CCA. By using a defined cortical tissue sampling procedure at 3, 6, 24, 72, 96, and 120 hr after the MCAo + CCAo, patterns of edema and ion (Na+, K+, and Ca++) changes in a primary and three peri-ischemic cortical areas are described. Ionic imbalances and edema formation have distinct patterns, are time dependent, and are different when comparing primary and peri-ischemic areas. Calcium increases to "neurotoxic" levels appear temporally independent of edema formation, reaching magnitudes 20 times greater than basal levels in the primary infarct area. Na+ increases correlate with increases in water, while K+ losses do not appear to be directly related to edema formation of Na+ and Ca++ increases. K+ losses are only significant in the primary infarct area. Rats treated with GM1 ganglioside (10 mg/kg, i.m.) daily showed significant reductions in edema, Na+ and Ca++ increases. These ganglioside effects were evident as early as 24 hr after the ischemic injury. Ca++ increases, which was maximal at 72 hr after the ischemic injury, was reduced by greater than 50% in GM1-treated animals. The mechanism by which GM1 is an effective neuroprotective agent may be evidenced by its effects on Ca++ influx/efflux processes in injury.