Neuroprotekciós beavatkozásások globális ischemia modelleken: a stroke megelőzésének és terápiájának egy új megközelítése = Neuroprotective interventions on models of the global ischemia: a new approach for prevention and therapy of stroke

A projekt fő célkitűzése agyi ischemiás modelleken neuroprotektív stratégiák kidolgozása és új neuroprotektív mulekulák előállítása és kipróbálása volt. A kitűzött célt három különböző módon közelítettük meg: 1) az ischemiás attack-ot követő extrém magas agyi glutamát (Glu) szintet szisztémásan adott glutamát scavengerek alkalmazásával csökkentettük. 2) olyan kinurénsav (KYNA) analógokat szintetizáltunk, melyek könnyen átjutnak a vér-agy gáton, tehát szisztémásan adhatók, ugyanakkor a központi idegrendszerbe bejutva, az NMDA receptorokhoz kötődve, csökkentik az idegszövet-károsító hiperexcitabilitást. 3) a sejtek természetes védekező mechanizmusait aktiváló pre- és posztkondícionálási paradigmákat dolgoztunk ki. A fent részletezett három területen végzett kutatómunka lehetőséget biztosított a különböző neurodegenerítv betegségek pathomechanizmusában a közös biológiai momentumok felismerésére. A projekt keretében végzett munka az elméleti ismereteken túl, a jövőben gyakorlati (gyógyszerfejlesztési ill. klinikai) jelentőséggel is bírhat. | The main goal of this project was to develop new neuroprotective strategies and molecules on ischemic brain models. To approach this goal we used three different ways: 1) to reduce the excess glutamate (Glu) levels within the brain following an ischemic attack with administration of glutamate scavengers. 2) To develop such kynurenic acid (KYNA) analogues which are able to cross the blood-brain barrier (BBB) and able to reduce the hyperexcitability by blocking NMDA receptors. 3) To develop such pre- and postconditioning protocols which are able to activate the endogenous self-protective mechanisms resulting in neuroprotection after an ischemic attack. In the course of these studies, we had chance to realize the common elements in the pathomechanisms of different neurodegenerative disorders. Taken together our results, we hope that they may have pharmaceutical or clinical importance in the future

Continuous Manufacturing of Homogeneous Ultralow-Dose Granules by Twin-Screw Wet Granulation

Homogeneous ultralow-dose (30 mg) tablets were prepared from perfectly free-flowing granules manufactured by continuous Twin-Screw Wet Granulation. The gravimetrically fed mixture of lactose and potato starch of low particle size was successfully agglomerated and the size enlargement technology proved to be very robust. Since the incorporated drug was dissolved in ethanol-based granulation liquid, the resulting homogeneity largely depended on the dosing of the applied liquid administering units.A peristaltic pump generated higher deviation of the drug content in tablets (Relative Standard Deviation (RSD): 7.7 %) compared to a syringe pump (RSD: 2.3 %) or a piston pump (RSD: 4.6 %). This is due to the pulsation of the liquid flow generated by the peristaltic pump according to the real-time measured mass of the fed liquid. A good correlation was found between the RSD of the liquid mass flow (calculated from the recorded masses) and the RSD of the drug content. Based on the results, the goodness of Content Uniformity, as the most relevant critical quality attribute of low-dose products, was determined by the characteristics of the applied dosing units. The feeding characteristic of the different pumps could be easily measured by the introduced balance-based method and therefore, the applicability of the pumps could be evaluated

Spreading depression and evoked potentials recorded in the somatosensory cortex of the rat

Cortical spreading depression (CSD) is associated with changes in the caliber of surface blood vessels; others have described it as a phenomenon which arises spontaneously and repetitively following acute cortical injury in animals, including both focal ischemia and trauma, while yet other researchers consider it to be an electrophysiological substrate of migraine aura, which may trigger headache. Our group is involved in research into both migraine and ischemia-induced pathophysiological states. It therefore appeared reasonable to include the study of CSD in the methodological repertoire utilized in our laboratory. We introduced two models of CSD induction: CSD evoked during continuous topical KCl application and CSD induced through a single KCl microinjection into the cortical tissue. This paper describes details of these two methods and of basic parameters of CSDs

Kainate postconditioning restores LTP in ischemic hippocampal CA1: Onset-dependent second pathophysiological stress

Postconditioning can be induced by a broad range of stimuli within minutes to days after an ischemic cerebral insult. A special form is elicited by pharmacological intervention called second pathophysiological stress. The present study aimed to evaluate the effects of low-dose (5 mg/kg) kainate postconditioning with onsets 0, 24 and 48 h after the ischemic insult on the hippocampal synaptic plasticity in a 2-vessel occlusion model in rat. The hippocampal function was tested by LTP measurements of Schaffer collateral-CA1 pyramidal cell synapses in acute slices and the changes in density of Golgi-Cox-stained apical dendritic spines. Postconditioning 0 and 24 h after ischemia was not protective, whereas 48-h-onset postconditioning resulted in the reappearance of a normal spine density (>100,000 spines) 3 days after ischemia, in parallel with the long-term restoration of the damaged LTP function. Similar, but somewhat less effects were observed after 10 days. Our data clearly demonstrate the onset dependence of postconditioning elicited by a subconvulsant dose of kainate treatment in global ischemia, with restoration of the structural plasticity and hippocampal function. © 2011 Elsevier Ltd. All rights reserved

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 CA] 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. (C) 2008 Elsevier Inc. All rights reserved

Oxaloacetate restores the long-term potentiation impaired in rat hippocampus CA1 region by 2-vessel occlusion

Various acute brain pathological conditions are characterized by the presence of elevated glutamate concentrations in the brain interstitial fluids. It has been established that a decrease in the blood glutamate level enhances the brain-to-blood efflux of glutamate, removal of which from the brain may prevent glutamate excitotoxicity and its contribution to the long-lasting neurological deficits seen in stroke. A decrease in blood glutamate level can be achieved by exploiting the glutamate-scavenging properties of the blood-resident enzyme glutamate-oxaloacetate transaminase, which transforms glutamate into 2-ketoglutarate in the presence of the glutamate co-substrate oxaloacetate. The present study had the aim of an evaluation of the effects of the blood glutamate scavenger oxaloacetate on the impaired long-term potentiation (LTP) induced in the 2-vessel occlusion ischaemic model in rat. Transient (30-min) incomplete forebrain ischaemia was produced 72 h before LTP induction. Although the short transient brain hypoperfusion did not induce histologically identifiable injuries in the CA1 region (Fluoro-Jade B, S-100 and cresyl violet), it resulted in an impaired LTP function in the hippocampal CA1 region without damaging the basal synaptic transmission between the Schaffer collaterals and the pyramidal neurons. This impairment could be fended off in a dosedependent manner by the intravenous administration of oxaloacetate in saline (at doses between 1.5 mmol and 0.1 μmol) immediately after the transient hypoperfusion. Our results suggest that oxaloacetate-mediated blood and brain glutamate scavenging contributes to the restoration of the LTP after its impairment by brain ischaemia

Effects of Blood Glutamate Scavenging on Cortical Evoked Potentials

It is well known that traumatic or ischemic brain injury is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) in brain fluids. It has recently been demonstrated that excess Glu can be eliminated from brain into blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to intravenous OxAc administration (i.v., 12.5, 25, and 50 mg/kg, respectively), and studied its effects on somatosensory evoked cortical potentials (EPs). Against our expectation, the amplitudes of EPs did not decrease but increased in a dose- and time-dependent manner after OxAc administration. Similar effects were observed when blood Glu scavenging was enhanced by combining OxAc (12.5 mg/kgbw) with recombinant glutamate-oxaloacetate transaminase (GOT, 0.14 nmol/100 g rat). On the basis of these results, we suggest that the changes of amplitudes of the EPs involve not only a glutamatergic but also the weakening of a GABAergic component. We cannot rule out the possibility that OxAc penetrates into the brain and improves mitochondrial functions

Oxaloacetate decreases the infarct size and attenuates the reduction in evoked responses after photothrombotic focal ischemia in the rat cortex

A traumatic brain injury or a focal brain lesion is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) levels in the cerebrospinal and interstitial fluids. It has recently been demonstrated that this excess Glu in the brain can be eliminated into the blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging the blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to a photothrombotic lesion and treated them after the illumination with a single 30-min-long administration of OxAc (1.2 mg/100 g, i.v.). Following induction of the lesion, we measured the infarct size and the amplitudes of the somatosensory evoked potentials (SEPs) as recorded from the skull surface. The photothrombotic lesion resulted in appreciably decreased amplitudes of the evoked potentials, but OxAc administration significantly attenuated this reduction, and also the infarct size assessed histologically. We suggest that the neuroprotective effects of OxAc are due to its blood Glu-scavenging activity, which, by increasing the brain-to-blood Glu efflux, reduces the excess Glu responsible for the anatomical and functional correlates of the ischemia, as evaluated by electrophysiological evoked potential (EP) measurements

A novel kynurenic acid analogue: a comparison with kynurenic acid. An in vitro electrophysiological study

Kynurenic acid is an endogenous product of the tryptophan metabolism, and as a broad-spectrum antagonist of excitatory amino acid receptors may serve as a protective agent in neurological disorders. The use of kynurenic acid as a neuroprotective agent is rather limited, however, because it has only restricted ability to cross the blood-brain barrier. Accordingly, new kynurenic acid analogues which can readily cross the blood-brain barrier and exert their complex anti-excitotoxic activity are greatly needed. Such a novel analogue, 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride, has been developed and tested. In an in vitro electrophysiological study, in which its properties were compared with those of kynurenic acid, the new analogue behaved quite similarly to kynurenic acid: in the micromolar range, its administration led to a decrease in the amplitudes of the field excitatory postsynaptic potentials in the CA1 region of the hippocampus, while in nanomolar concentrations it did not give rise to inhibition, but, in fact, facilitated the field excitatory postsynaptic potentials. Moreover, the new analogue demonstrated similar protective action against PTZ-induced facilitation to that observed after kynurenic acid administration. The findings strongly suggest that the neuroactive effects of the new analogue are comparable with those of kynurenic acid, but, in contrast with kynurenic acid, it readily crosses the blood-brain barrier. The new analogue may therefore be considered a promising candidate for clinical studies