Gabor Gigler

Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats

BACKGROUND AND PURPOSEn2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue during the early phases of ischemia-reperfusion assessed either by perfusion of the brain with TTC solution transcardially, in vivo, or by staining brain slices, in vitro.nnnMETHODSnThe middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining, in vivo and in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy).nnnRESULTSnCore volume of tissue injury measured in vivo was large at 0 h and steadily decreased by 50% (p<0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only, in vitro, and gradually increased up to 24 h. Core volume was larger in vivo than in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h.nnnCONCLUSIONSnFormation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.

Synthesis and pharmacological study of new 3,4-dihydro-2H,6H-pyrimido-[2,1-b][1,3]thiazines

Summary A series of racemic pyrimido-thiazine derivatives was synthesized and many of their in vivo activities found to be comparable to acetylsalicylic acid and aminophenazone in an antiinflammatory model and an antipyretic test. Analogues 7a and 7e are the most potent in rat carrageenin and yeast fever assays. These compounds did not inhibit prostaglandin biosynthesis in vitro.

Comparison of anticonvulsive and acute neuroprotective activity of three 2,3-benzodiazepine compounds, GYKI 52466, GYKI 53405, and GYKI 53655

GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive AMPA [alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate] and kainate receptor antagonist and its two analogues, GYKI 53405 [1-(4-aminophenyl)-3-acetyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] and GYKI 53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine] were investigated in two seizure models and in MgCl2 induced global cerebral ischaemia, as an acute neuroprotective model. The ED(50) values of GYKI 52466 for suppression of the tonic and clonic phases of sound-induced seizures were 3.6 and 4.3 mg/kg, respectively. The corresponding data for GYKI 53405 were 1.1 and 3.1 mg/kg, while ED(50) values of GYKI 53655 were 1.3 and 2.0 mg/kg, respectively. The inhibition of seizure evoked by maximal electroshock was also found to be remarkable: the ED(50) values of GYKI 52466 and its two analogues were 6.9, 2.6, and 2.2 mg/kg, respectively. All compounds prolonged the survival times in MgCl2 induced global cerebral ischaemia test in a dose-dependent fashion, with PD(50) (dose of 50% prolongation) values of 24.1, 8.3, and 8.2 mg/kg intraperitoneal, respectively. In audiogenic seizure model the duration of anticonvulsant action of 10 mg/kg GYKI 52466 and 5 mg/kg GYKI 53405, GYKI 53655 were examined, too. The effect of GYKI 52466 decreased to 50% after 2 h, while the analogues showed more than 80% seizure suppression 3 h after treatment. After 6 h the effect of GYKI 53655 decreased to zero, while the effect of GYKI 52466, remained on the 50% level.

Kynurenine diminishes the ischemia-induced histological and electrophysiological deficits in the rat hippocampus

The neuroprotective effect of L-kynurenine sulfate (KYN), a precursor of kynurenic acid (KYNA, a selective N-methyl-D-aspartate receptor antagonist), was studied. KYN (300 mg/kg i.p., applied daily for 5 days) appreciably decreased the number of injured pyramidal cells from 1850+/-100/mm(2) to 1000+/-300/mm(2) (p<0.001) in the CA1 region of the hippocampus in the four-vessel occlusion (4VO)-induced ischemic adult rat brain. A parallel increase in the number of intact, surviving neurons was demonstrated. Post-treatment with KYN (applied immediately right after reperfusion) proved to be much less effective. In parallel with the histology, a protective effect of KYN on the functioning of the CA1 region was observed: long-term potentiation was abolished in the 4VO animals, but its level and duration were restored by pretreatment with KYN. It is concluded that the administration of KYN elevates the KYNA concentration in the brain to neuroprotective levels, suggesting its potential clinical usefulness for the prevention of neuronal loss in neurodegenerative diseases.

Neuroprotection achieved in the ischaemic rat cortex with l-kynurenine sulphate

L-kynurenine is a metabolic precursor of kynurenic acid, which is one of the few known endogenous N-methyl-D-aspartate receptor inhibitors. In contrast with kynurenic acid, L-kynurenine is transported across the blood-brain barrier, and it may therefore come into consideration as a therapeutic agent in certain neurobiological disorders, e.g. ischaemia-induced events. The present study evaluated the effect of L-kynurenine administration (300 mg/kg i.p.) on the global ischaemic brain cortex both pre- and post-ischemic intervention. The statistical evaluation revealed that L-kynurenine administration beneficially decreased the number of neurones injured per mm(2) in the cortex, not only in the pre-treated animals, but also in those which received L-kynurenine after the ischaemic insult. It is concluded that even the post-traumatic administration of L-kynurenine may be of substantial therapeutic benefit in the treatment of global brain ischaemia. This is the first histological proof of the neuroprotective effect achieved by the post-traumatic administration of L-kynurenine in the global ischaemic cortex.

Reduction of cerebral infarct size by non-competitive AMPA antagonists in rats subjected to permanent and transient focal ischemia

Antagonists of 2-amino-3(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptors can considerably reduce brain damage after cerebral ischemia, but effectiveness of selective AMPA antagonists has been questioned recently. Therefore, we evaluated the antiischemic efficacy of [+/-]-7-acetyl-5-[4-aminophenyl]-7,8-dihydro-8-cyano-8-methyl-9H-1,3-dioxolo-[4,5-h]-2,3-benzodiazepine (EGIS-8332) and GYKI 53405, two selective, non-competitive AMPA antagonists in two rat models of focal cerebral ischemia. Permanent focal ischemia was produced by electrocoagulation of the middle cerebral artery (MCA). EGIS-8332 and GYKI 53405 were administered 30 min after MCA occlusion at doses of 1, 3 or 10 mg/kg i.p. In transient focal ischemia, MCA was occluded for 1 h and reperfused for 24 h using the intraluminal filament technique and the compounds were given at 3x10 mg/kg i.p. 60, 120 and 180 min following occlusion. In permanent focal ischemia, EGIS-8332 decreased the volume of cerebral infarction both at 10 mg/kg i.p. (36.4%, p<0.01) and at 3 mg/kg i.p. (26.4%, p<0.05) in a dose-dependent manner. GYKI 53405 produced a similar antiischemic effect at 10 mg/kg i.p. (36.4%, p<0.01), but it was ineffective at 3 mg/kg i.p. (6.5%, p=0.57). In transient focal ischemia, EGIS-8332 reduced the volume of necrotic brain tissue (38.7%, p<0.01) and GYKI 53405 was similarly effective (32.6%, p<0.05). Both compounds afforded neuroprotection in the cortical and subcortical regions of the MCA territory. Selective, non-competitive AMPA antagonists administered after the ischemic insult can produce effective neuroprotective action in experimental models of focal cerebral ischemia; therefore, these compounds may be useful as therapeutic agents for the treatment of stroke and neurodegenerative disorders.

The neuroprotective and hypothermic effect of GYKI-52466, a non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-antagonist on histological and behavioural variables in the gerbil global ischemia model

The neuroprotective activity of the non-competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist GYKI-52466 (1-[4-aminophenyl]-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodia zep ine HCI; EGIS-8159) was studied in the gerbil bilateral carotid occlusion (BCO) model of global ischemia. Drug effect on hippocampal CA1 neuronal loss, hypermotility, and cognitive deficit (decrease in spontaneous alternation (SA) behaviour in the Y-maze) induced by 5-min or 3-min BCO were measured. GYKI-52466 was administered at 4 x 15 mg/kg intraperitoneal (i.p.) doses 30, 45, 60, and 75 min following surgery. The competitive AMPA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline) applied at 3 x 30 mg/kg i.p. doses 60, 70, and 85 min after reperfusion was also tested for comparison. Both compounds showed weak and non-significant effects on 5-min BCO-induced changes in all the three variables. However, following 3-min ischemia GYKI-52466 and NBQX produced significant inhibition (49% and 48%, respectively) on CA1 cell loss. Moreover, GYKI-52466, but not NBQX, significantly inhibited the 3-min ischemia induced hypermotility and decrease in SA. At their neuroprotective doses, both compounds caused long-lasting (min. 8 h) hypothermia in gerbils. GYKI-52466 induced much higher decrease in body temperature (6 degrees C at peak level) than NBQX did (2 degrees C at peak level). Administration of 4 x 10 mg/kg i.p. chlorpromazine to gerbils 15 min before and 0, 15, and 30 min after 3-min BCO resulted in considerable hypothermia (5.5 degrees C peak effect, 8 h duration), but no protective action of the compound on CA1 cell loss and hypermotility was observed. However, chlorpromazine inhibited the ischemia-induced cognitive impairment. The results suggest that drug-induced hypothermia may differentially influence the histological and the behavioural outcomes of ischemic intervention.

Neuroprotective and anticonvulsant effects of EGIS-8332, a non-competitive AMPA receptor antagonist, in a range of animal models

Blockade of AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) receptors is a good treatment option for a variety of central nervous system disorders. The present study evaluated the neuroprotective and anticonvulsant effects of EGIS‐8332, a non‐competitive AMPA receptor antagonist, as a potential drug candidate.

Therapeutic time window of neuroprotection by non-competitive AMPA antagonists in transient and permanent focal cerebral ischemia in rats.

EGIS-8332 and GYKI 53405 are selective, non-competitive AMPA (2-amino-3[3-hydroxy-5-methyl-4-isoxazolyl] propionic acid) antagonists that effectively protected against tissue injury caused by global and focal cerebral ischemia in laboratory animals. This study evaluated the therapeutic time window of neuroprotection by EGIS-8332 and GYKI 53405 in permanent and transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Infarct size was measured by TTC staining 48 h after permanent MCAO (electrocoagulation), and 24 h after reperfusion following a 1-h transient MCAO carried out using the intraluminal filament technique. Treatment with EGIS-8332 (10 mg/kg, i.p.) 60 or 120 min after permanent MCAO, decreased infarct size by 30% and 36%, respectively, and the effect of GYKI 53405 (10 mg/kg, i.p.) was similar (30% and 33%, respectively; p<0.01 all). Neither compound was effective if administered 180 or 240 min after permanent MCAO. Both EGIS-8332 and GYKI 53405 (20 mg/kg, i.p.) reduced the core and total (core plus penumbra) volumes of tissue injury in the whole brain and the cerebral cortex when administered 120 or 180 min after transient MCAO. The compounds did not alter tissue damage in the striatum. No neuroprotective effect was obtained at 240 min after transient MCAO. In conclusion, the therapeutic time window of neuroprotection by EGIS-8332 and GYKI 53405 was 2 h in permanent and 3 h in transient focal cerebral ischemia in rats. The results suggest that treatment with non-competitive AMPA antagonists can only be expected to produce a neuroprotective action in humans if administered shortly after the appearance of stroke symptoms.

Egis-11150: a candidate antipsychotic compound with procognitive efficacy in rodents.

Classical antipsychotics, e.g. haloperidol, chlorpromazine, are potent at controlling the positive symptoms of schizophrenia but frequently elicit extrapyramidal motor side-effects. The introduction of atypical antipsychotics such as risperidone, olanzapine and clozapine has obviated this problem, but none of the current drugs seem to improve the cognitive deficits accompanying schizophrenia. Thus there is an unmet need for agents that not only suppress the psychotic symptoms but also ameliorate the impairment of cognition. Here, we report the preclinical properties of a candidate antipsychotic, Egis-11150, that shows marked pro-cognitive efficacy. Egis-11150 displayed high affinity for adrenergic α(1), α(2c), 5-HT(2A) 5-HT₇, moderate affinity for adrenergic α(2a) and D₂ receptors. It was a functional antagonist on all of the above receptors, with the exception of 5-HT₇ receptors, where it was an inverse agonist. Phencyclidine-induced hypermotility in mice and inhibition of conditioned avoidance response in rats were assessed to estimate efficacy against the positive and social withdrawal test in rats was used to predict efficacy against the negative symptoms of schizophrenia. Passive-avoidance learning, novel object recognition and radial maze tests in rats were used to assess pro-cognitive activity, while phencyclidine-induced disruption of prepulse inhibition in mice was examined to test for effects on attention. Egis-11150 (0.01-0.3 mg/kg, ip.) was effective in all of the preclinical models of schizophrenia examined. Moreover, a robust pro-cognitive profile was apparent. In summary, work in preclinical models indicates that Egis-11150 is a potential treatment for controlling the psychosis as well as the cognitive dysfunction in schizophrenia. This article is part of a Special Issue entitled Cognitive Enhancers.