Krisztina Moricz

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

BACKGROUND AND PURPOSE 2,3,5-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. METHODS The 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). RESULTS Core 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. CONCLUSIONS Formation 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.

BGP-15, a hydroximic acid derivative, protects against cisplatin- or taxol-induced peripheral neuropathy in rats

The neuroprotective effect of BGP-15 against peripheral sensory neuropathy was studied in rats that were exposed to short-term cisplatin or taxol administration. The changes of nerve conduction velocity were determined in situ after treating the Wistar rats with BGP-15 (50, 100, and 200 mg/kg po daily doses throughout the experiment), cisplatin (1.5 mg/kg ip daily dose for 5 days), or taxol (5.0 mg/kg ip daily dose every other day in a 10-day interval) alone or giving the test compound in combination with cisplatin or taxol. Electrophysiological recordings were carried out in vivo by stimulating the sciatic nerve at both sciatic notch and ankle site. Neither motor nor sensory nerve conduction velocity was altered by any dose level of BGP-15 tested. Both anticancer drugs decreased the sensory nerve conduction velocity (SNCV). BGP-15 treatment prevented the impairment of SNCV either in part or totally in the cisplatin- or taxol-treated groups. This neuroprotective potential of BGP-15 could be well correlated with its recently described poly(ADP-ribose) polymerase- inhibitory effect and its ability to protect against the damages induced by the increased level of reactive oxygen species in response to anticancer treatment.

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.

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.

Neurotransmitter Release in Experimental Stroke Models: The Role of Glutamate-Gaba Interaction

Stroke or cerebrovascular accident reduces blood flow and decreases oxygen supply (ischemia) in brain tissue. This may be resulted from vascular obstruction when a blood vessel is blocked or by hemorrhage when bleeding occurs into the brain tissue. Decrease in oxygen supply shifts pH to acidosis and increases extracellular K+ concentration, which depolarizes neural cell membrane. Anoxic depolarization leads to excessive release of glutamate, which then activates various glutamate receptors in the synapse or the extrasynaptic space. Opening of ionotropic glutamate receptors (NMDA, AMPA and kainate receptors) causes influx of Na+ through the activated glutamate-gated ion channels. In response to anoxia, Ca2+ also enters the cells in excessive amounts via activated NMDA receptors and Ca2+-permeable AMPA receptors. This will lead to activation of several Ca2+-dependent intracellular signal transduction pathways (proteases, kinases, endonucleases, lipoxygeneses and nitric oxide synthase), which ultimately leads to neural death (Vizi et al., 1996; Parsons et al., 1998).

Morphological and functional changes after benzalkonium chloride treatment of the small intestinal thiry-vella loop in rats

The aim of this work was to study the effects of benzalkonium chloride (BAC) treatment on the small intestine and its functioning in rats surgically prepared with Thiry-Vella intestinal loop. The loops were treated with either BAC, which ablated much of the myenteric plexus and extrinsic innervation, or with physiological saline (SAL). In vivo drinking experiments were performed to examine the effect on fluid intake and behavioral indices of distending the loop with a balloon. Spontaneous motility and its changes induced by acetylcholine (ACh) and histamine (His) were studied on isolated stripes in vitro. Finally, samples from the loops were examined histologically. Though reduction of the cell number was less than expected and no differences of the thickness of the muscular layer between the two groups was observed, BAC treatment altered the pattern of spontaneous activity and also the sensitivity to ACh and His in isolated stripes. In vivo distension of the SAL-treated loops reduced fluid intake and produced signs of aversivity; these effects were absent in the BAC-treated group. Our results show that despite the differences in the degree of ablation from those obtained by others, BAC treatment can be used to study the mechanisms underlying the effects of the enteral stimuli on the behavior.

Decreased vasoconstrictor responses in remote cerebral arteries after focal brain ischemia and reperfusion in the rat, in vitro

The effects of brain ischemia and reperfusion on smooth muscle function in remote cerebral and peripheral arteries are hardly known. Maximum vasoconstrictions (E(max)) caused by 120mmol/l KCl and 5-HT in endothelium-denuded ring preparations were measured in ischemic and control cerebral arteries of rats after a 1-h right middle cerebral artery occlusion followed by 0-min (I/NR) or 2-3-min (I/SR) reperfusion, and in peripheral arteries after I/SR. Surprisingly, vasoconstrictions to 5-HT and 120mmol/lK(+) were attenuated in remote brain vessels after I/SR, i.e. in the contralateral middle cerebral artery and the basilar artery, while I/NR depressed E(max) of 5-HT and high KCl only in the ischemic middle cerebral artery. Pretreatment with N-(2-mercaptopropionyl) glycine (MPG, 100mg/kg i.p.), a free radical scavenger, fully prevented the impairment of vasomotor function in the middle cerebral artery on both sides after I/SR. Moreover, vasomotor functions were normal in the coronary, renal and pulmonary arteries after I/SR. In conclusion, focal cerebral ischemia and reperfusion impaired vasoconstrictor responses in remote brain arteries of rats by a mechanism involving free radicals. The lack of similar effects in peripheral vessels indicates poor defence of brain arteries against remote injury caused by reactive oxygen species-dependent mechanisms.

Loop F of the GABA A receptor alpha subunit governs GABA potency

ABSTRACT Gamma‐amino butyric acid (GABA) is an abundant neurotransmitter in the CNS. GABAergic interneurons orchestrate pyramidal neurons in the cerebral cortex, and thus control learning and memory. Ionotropic receptors for GABA (GABAAR) are heteropentameric complexes of &agr;, &bgr; and &ggr; integral membrane‐protein subunits forming Cl− ‐channels operated by GABA, which are vital for brain function and are important drug targets. However, knowledge on how GABAAR bind GABA is controversial. Structural biology versus functional modelling combined with site‐directed mutagenesis suggest markedly different roles for loop F of the extracellular domain of the &agr;‐subunit when complexed with GABA. Here, we report that contrary to the results of structural studies, loop F of the &agr;‐subunit controls the potency of GABA on GABAAR. We examined the effect of replacing a short, variable segment of loop F of the GABAA &agr;5‐subunit with the corresponding segment of the &agr;2‐subunit (GABAA5_LF2) and vice versa (GABAA2‐LF5). When compared with their respective wild‐type counterparts, GABAA5_LF2 receptors displayed enhanced sensitivity towards GABA, whilst in GABAA2‐LF5 sensitivity was diminished. Mice homozygous for the genetic knock‐in of the GABAA5_LF2 subunit showed a marked deficit in long‐ but not short‐term object recognition memory. Working memory in place learning, spontaneous alternation and the rewarded T‐maze were all normal. The deficit in long‐term recognition memory was reversed by an &agr;5‐GABAA negative allosteric modulator compound. The data show that loop F governs GABA potency in a receptor isoform‐specific manner in vitro. Moreover, this mechanism of ligand recognition appears to be operative in vivo and impacts cognitive performance. HIGHLIGHTSWe investigated the contribution of loop F of the &agr;‐subunit of the GABAA receptor to the potency of GABA.The potency of GABA was reduced upon insertion of the variable segment of loop‐F of the &agr;5 subunit into the homologous position in the &agr;2 subunit.Conversely, the insertion of the variable segment of loop‐F of the &agr;2 subunit into &agr;5 significantly increased the potency of GABA.Mice with the genetic knock‐in of the &agr;5/&agr;2 subunit microchimeric at loop F, had normal working memory and short‐term object recognition memory.Significantly, knock‐in mice failed to show any long‐term (24 h) object recognition memory.Treatment with a negative allosteric modulator of &agr;5 GABAA receptors restored long‐term (24 h) object recognition memory to normal levels.

Persistent therapeutic effect of a novel α5-GABAA receptor antagonist in rodent preclinical models of vascular cognitive impairment

ABSTRACT This study examined the potential of the selective extra‐synaptic &agr;5‐GABAA receptor inhibitor S44819 (Egis‐13529) to improve cognitive performance in preclinical models of vascular cognitive impairment (VCI). Chronic hypoperfusion of the brain in mice was induced by permanent occlusion of the right common carotid artery (rUCO). rUCO induced impairments of cognitive function in the object recognition test (OR) and the rewarded T‐maze (RTM). In both tests, a single oral treatment with S44819 (OR – 0.1–3mg/kg, RTM – 1–3mg/kg p.o.) significantly reduced the effect of rUCO. Long‐term treatment with S44819 (1–10mg/kg twice daily p.o. for 14 days), that was initiated 24h after surgery and was followed by a 10‐ or 13‐day wash‐out period, fully prevented the decline of cognitive performance of rUCO mice. In rats, occlusion of the middle cerebral artery (MCA) for 30min caused a significantly diminished performance in the OR. This was prevented by S44819 given p.o. 15mg/kg twice daily for 8 days, starting 7 days after surgery and tested following a 7‐day wash‐out period. Taken together, S44819 markedly and stably improved reference and working memory impaired by rUCO in mice. In rats, the compound effectively suppressed the development of cognitive impairment after mild stroke. In conclusion, as longer‐term administration led to a persistent reversal of the cognitive deficits, it appears that S44819 may have symptomatic, as well as disease‐modifying effects in models of VCI. Proof of concept is therefore provided for testing S44819 in the therapy of VCI and post‐stroke dementia in humans.