[SOME PHYSIOLOGICAL PROCESSES IN ISCHEMIC PENUMBRA OF THE BRAIN (REVIEW)].

The zone of penumbra in ischemic brain tissue lies below the local cerebral blood flow threshold for electrocortical silence but above that for anoxic depolarization and massive ionic dyshomeostasis. Conflicting data concerning the width of therapeutic window in which successful reperfusion of blood still is possible, are cited in this paper. The role of temperature in development of ischemic stroke is also considered. There are data that even small degrees of brain temperature alterations crucially affect the outcome of ischemic insults. The lowering of intraischemic brain temperature by just 2 to 30C is capable of diminishing the volume of infarction up to 70 to 80%. Hypothermia modifies a variety of intracellular processes and markedly delaying the process of cell death, hypothermia appears to widen the therapeutic window.

Ductular reaction at the early terms of common bile duct ligation in the rats.

Ductular reaction (DR) in bile duct ligated rats generally appears from 2nd day after biliary obstruction (BO). However, we show that increased amount of ductular profiles is evident already in 6 hours after BDL. The study aims to explain the origin of such an early DR in response to BO. Male Lewis rats were subjected to common bile duct ligation (CBDL) for 3, 6, 12 and 24 hours and sham operation. Liver samples were studied histologically, immunohistochemically (Ki67, pan-Cytokeratin /AE1 + AE3/ and OV-6) and by immunoblotting analyses. It appeared that number of ductular profiles increase in time-related manner after BO. These ductular profiles are formed by biliary epitheliocyte-like cells; No mitotic activity was revealed. Part of hepatocytes reveals pan-Cytokeratin positivity on 12 and 24 hours after BO. Total cytokeratins content at 24 hours after CBDL was 37% higher in comparison with control data. The significant increase was observed for the cytokeratins with molecular weights: 61, 56 and 40 KDa. Thus, early DR after BDL is mediated by widening of the existed finest biliary ramifications and is not associated with proliferation activities. This DR is accompanied by differentiation of hepatocytes toward bile duct-like cells.

Mitochondrial proteins, learning and memory: biochemical specialization of a memory system.

The enzyme cytochrome c oxidase is a mitochondrial protein complex that plays a crucial role in oxidative metabolism. In the present study we show that amounts of two of its protein subunits (cytochrome c oxidase subunit I [CO-I] and II [CO-II]) are influenced by both learning-independent and learning-dependent factors. Converging evidence has consistently implicated the left intermediate medial mesopallium (IMM) in the chick brain as a memory store for the learning process of visual imprinting. This form of learning proceeds very shortly after chicks have been hatched. In the left IMM, but not in three other brain regions studied, amounts of CO-I and CO-II co-varied: the correlation between them was highly significant. This relationship did not depend on learning. However, learning influenced the amounts of both proteins, but did so only in the left IMM. In this region, amounts of each protein increased with the strength of learning. These findings raise the possibility that the molecular mechanisms involved in the coordinated assembly of cytochrome c oxidase are precociously developed in the left IMM compared to the other regions studied. This precocious development may enable the region to respond efficiently to the oxidative demands made by the changes in synaptic connectivity that underlie memory formation and would allow the left IMM to function as a storage site within hours after hatching.

Anticonvulsant activities of myo-inositol and scyllo-inositol on pentylenetetrazol induced seizures.

Myo-inositol (MI) and its isomers are used for the treatment of various neuropathological conditions. The purpose of the present research was to study anticonvulsant properties of MI and scyllo-inositol (SCI) on pentylenetetrazol (PTZ) induced seizures in rats. Half an hour after treatment with MI (30 mg/kg) or SCI (5 mg/kg) seizures were induced in Wistar rats with PTZ (60 mg/kg). Control animals were treated either by normal saline or mannitol (control polyol of the same molecular weight, 30 mg/kg), given at the same time interval before PTZ injection, as MI/SCI groups. The anticonvulsant effects of MI/SCI treatment were assessed by the latent period (the time from PTZ-injection to the onset of first seizures), and the duration and severity (score) of seizures. The mortality rate was also assessed. Both MI and SCI treatment significantly reduced the seizure score, seizure duration and increased the latent period. These data suggest for strong potential of MI and SCI as the agents of antiepileptic therapy.

Effect of acoustic stimulation on GABAergic neurons in limbic structures of krushinskii-molodkina rats.

Quantitative study of GABAergic and main cells in the hippocampus and piriform cortex of Krushinskii-Molodkina rats was performed 1 month after the incidence of seizure activity evoked by acoustic stimulation. The number of neurons significantly decreased in both regions and, particularly, in the hippocampus and central area of the piriform cortex.

Effect of oleamide on pentylenetetrazole-induced seizures in rats.

Oleamide exhibits antiepileptic activity and significantly decreases the degree of pentylenetetrazole-induced seizures.

Effect of myo-inositol on convulsions induced by pentylenetetrazole and kainic acid in rats.

We studied the effect of myo-inositol on pentylenetetrazole and kainic acid-induced seizures in rats. Myo-inositol significantly reduced seizure activity.

Lipid peroxidation in the erythrocytes under condition of their increased aggregation.

The aim of the present study was investigation of the lipid peroxidation changes within the erythrocytes under conditions of increased RBC aggregation. This latter was produced both in the in vitro and in vivo conditions by the addition of Dextran T-500. For the in vitro studies blood samples were taken from the cubital veins of 15 healthy subjects. During the in vivo studies 10 ml of the 10 percent Dextran T-500 solution was administered intravenously in six chinchilla rabbits. Another six animals were treated with rheopolyglucyne. The RBC aggregation in blood was investigated with the "Georgian technique". The malondialdehyde (the end product of lipid peroxidation) was determined in all cases by its reaction with thiobarbituric acid. We found that in the in vitro conditions, as well as in the in vivo studies, the lipid peroxidation was significantly increased in the erythrocytes during the enhanced RBC aggregation induced by addition of Dextran T-500. Therefore we suppose that the elevated RBC aggregation cause an increase of cell's lipid peroxidation and it is possible to think that appropriate prooxidant: antioxidant balance is shifted toward the pro-oxidants in the erythrocytes.

Blood rheological properties and lipid peroxidation in cerebral and systemic circulation of neurocritical patients.

The present study was aimed at investigating the red blood cell (RBC) aggregation and the parameters of lipid peroxidation - malondialdehyde (MDA) and MDA + 4-hydroxyalkenals (HAE) - in the blood of critically deteriorated stroke patients (brain infarcts, parenchymatous and subarachnoid hemorrhages) and in the control group. Measurements were made in blood samples from the common carotid artery, the both internal jugular and cubital veins. The RBC aggregation index was found to increase by about 80 per cent in both the cerebral and systemic circulation as compared to the control blood samples. MDA content appeared also significantly higher in the blood flowing out of the damaged hemisphere of the neurocritical patients as compared to the control group. In the blood flowing out of the damaged hemisphere of the neurocritical patients MDA as well as MDA + HAE content significantly exceeded the values found in blood samples of the jugular vein of the contralateral hemisphere, carotid artery and the cubital vein. The results of this study suggest a certain interdependence between the blood plasma lipid peroxidation and the RBC aggregation in the brain vessels following its damage.

Analysis of differential gene expression supports a role for amyloid precursor protein and a protein kinase C substrate (MARCKS) in long-term memory.

Previous work has identified the intermediate and medial part of the hyperstriatum ventrale (IMHV) as a region of the chick brain storing information acquired through the learning process of imprinting. We have examined in this brain region changes in expression of candidate genes involved in memory. Chicks were exposed to a rotating red box and the strength of their preference for it, a measure of learning, determined. Brain samples were removed approximately 24 h after training. Candidate genes whose expressions were different in IMHV samples derived from strongly imprinted chicks relative to those from chicks showing little or no learning were identified using subtractive hybridization. The translation products of two candidate genes were investigated further in samples from the left and right IMHV and from two other brain regions not previously implicated in imprinting, the left and right posterior neostriatum. One of the proteins was the amyloid precursor protein (APP), the other was myristoylated alanine rich C kinase substrate (MARCKS). In the left IMHV the levels of the two proteins increased with the strength of learning. The effects in the right IMHV were not significantly different from those in the left. There were no effects of learning in the posterior neostriatum. This is the first study to relate changes in the amounts of MARCKS and APP proteins to the strength of learning in a brain region known to be a memory store and demonstrates that the systematic identification of protein molecules involved in memory formation is possible.

Neural cell adhesion molecules, CaM kinase II and long-term memory in the chick.

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain is a site of recognition memory for filial imprinting. Previous results have demonstrated learning-related changes in the amounts of the three major isoforms of neural cell adhesion molecule (NCAM) in the left IMHV. The increases were present 24 h after training. The present study enquired whether the increases persisted and were present 48 h after training. The brain regions analysed were the left and right IMHV and the left and right hyperstriatum accessorium (HA), a visual projection area. The alpha-subunit of calcium/calmodulin protein kinase II (CaMKIIalpha) was also assayed. There were significant correlations between a measure of the strength of learning and the amount of NCAM 180 in the right IMHV (r = +0.65; p = 0.012) but not in the left, and in the left HA (r = -0.61; p = 0.02), but not in the right. There were no learning-related changes for CaMKIIalpha. We conclude that in IMHV the effects of imprinting on NCAM 180 are expressed mainly in the left IMHV 24 h after training, but 48 h after training are expressed mainly in the right IMHV.

Expression of the GABA(A) receptor gamma 4-subunit gene: anatomical distribution of the corresponding mRNA in the domestic chick forebrain and the effect of imprinting training.

The learning process of imprinting involves morphological, electrophysiological and biochemical changes in a region of the chick (Gallus gallus domesticus) forebrain known as the intermediate and medial part of the hyperstriatum ventrale (IMHV). The alterations include increases in the mean length of postsynaptic density profiles of axospinous synapses and the number of N-methyl-D-aspartate (NMDA) receptor binding sites, and changes in spontaneous and evoked electrical activity. Recent immunocytochemical and behavioural studies have suggested that inhibitory GABAergic neurotransmission plays a role in learning. In this context, it has previously been reported that a novel avian gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor gene, encoding the gamma4 subunit, is highly expressed in the hyperstriatum ventrale. In this study, we have used in situ hybridization to map, in detail, the expression of the gamma4-subunit gene in the chick brain, and to assess the effect of imprinting training on the level of the corresponding transcript. Our results reveal that the gamma4-subunit mRNA has a restricted distribution, and demonstrate a highly significant, time-dependent effect of training on its steady-state level. At 10 h but not at 5 h after training there is a decrease (25-32%) in the amount of this transcript in parts of the medial hyperstriatum ventrale, including the IMHV. A decrease (28-39%) is also seen in certain visual and auditory pathway areas but no effect was observed in other forebrain regions such as the hyperstriatum intercalatus superior (HIS). These results suggest that imprinting training leads to a time-dependent down-regulation of GABAergic transmission, and raise the possibility that this down-regulation plays a role in learning.

Neural cell adhesion molecules, learning, and memory in the domestic chick.

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. The results reported here demonstrate that neural cell adhesion molecules (NCAMs) play a time-dependent role in this recognition memory. Dark-reared chicks were trained, tested, and assigned a preference score as a measure of learning. Chicks with high preference scores were designated good learners and those with lower preference scores, poor learners. Controls were untrained. Tissue was removed, 9.5 hr or 24 hr after training, from the left and right IMHV, hyperstriatum accessorium, and posterior neostriatum. Three major NCAM isoforms (180, 140, and 120 kDa) were assayed. At 24 hr only, there was in left IMHV significantly more NCAM (for each isoform) in good learners than in the other 2 groups, and also a significant correlation between the amounts of NCAM and preference scores for all isoforms; the amount predicted by each regression line at preference score 50 (no learning) did not differ significantly from the mean value for untrained controls. There were no learning-related effects in either the hyperstriatum accessorium or the posterior neostriatum.

Clathrin proteins and recognition memory.

Strong converging evidence indicates that the intermediate and medial part of the hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. Clathrin proteins have been implicated in synaptic plasticity. In the present study we demonstrate for the first time that they are involved in vertebrate learning. Chicks were trained by exposure to a conspicuous object and their preference for it versus a novel object subsequently measured as a preference score (an index of learning). Trained chicks with low preference scores were classed as "poor learners" and those with high preference scores as "good learners". An additional group of chicks was untrained ("dark-reared"). Tissue was removed from the left and right IMHV, hyperstriatum accessorium and posterior neostriatum 9.5 h or 24 h after training. Clathrin heavy chain and clathrin light chains a and b were assayed using sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting. In the IMHV, and only for clathrin heavy chain, was there a significant effect of training. The effect occurred 24 h but not 9.5 h after training, and was significant only in the left IMHV. In this region at 24 h, there was (i) significantly more clathrin heavy chain in good learners than in dark-reared chicks, and (ii) a significant positive correlation between the amount of clathrin heavy chain and preference score; the amount of protein present in the dark-reared chicks did not differ significantly from the amount predicted from the regression line for trained chicks performing at chance (preference score 50). These findings imply that for the left IMHV, visual experience per se, locomotor activity and other side effects of training did not affect the amount of clathrin heavy chain. Rather, the increase observed was a function of the amounts chick learned and, because it was delayed, is likely to be involved in long-term memory. The results for clathrin heavy chain taken together suggest that enhanced presynaptic events in the IMHV, possibly associated with an increase in synaptic vesicle release/uptake, are important in the recognition memory underlying imprinting.

Differential distribution of protein kinase C (PKC alpha beta and PKC gamma) isoenzyme immunoreactivity in the chick brain.

Protein kinase C (PKC) is involved in neural plasticity. The phosphorylation of the myristoylated alanine-rich protein kinase C substrate (MARCKS) in the left intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain has been shown previously to correlate significantly with the strength of learning in filial imprinting. The distribution of PKC alpha, beta I, beta II and PKC gamma in the brain of 1-day-old dark-reared chicks was determined immunocytochemically, using the monoclonal antibodies MC5 and 36G9, raised against purified PKC alpha beta and PKC gamma, respectively. PKC gamma-stained cells were distributed widely in the telencephalon, including all hyperstriatal structures (including the IMHV), the hippocampus, neostriatum, ectostriatum and archistriatum. There were fewer stained cells in the septum and the least cellular staining was in the paleostriatum primitivum. Fluorescent double-labelling with neuron-specific enolase (NSE) and with the glial calcium-binding protein S100 suggested that PKC gamma immunoreactivity was present in neurones but not in glia. The distribution of PKC alpha beta-stained cells was more limited, with staining in the archistriatum, hippocampus and septum but not in the hyperstriatum. However, there was PKC alpha beta-staining of some fibres in the IMHV (but little elsewhere in the hyperstriatum ventrale), in the neostriatum, paleostriatal complex and the lobus parolfactorius. Double-labelling with NSE and S100 revealed PKC alpha beta/S100-positive glial cells present in the paleostriatal region only. There was some PKC alpha beta-staining of putative neurones in the hippocampus, septum and archistriatum. The differential distribution of PKC isoenzymes suggests that in the IMHV some axonal inputs contain PKC alpha beta whereas some postsynaptic cells contain the gamma form of PKC.

Glial and neuronal opioid receptors: apparent positive cooperativity observed in intact cultured cells.

Opioid receptors were characterized in glial and neuronal homogeneous cultures of embryonic chick forebrain, using [3H]naloxone as a labelled ligand. Binding experiments were performed on intact cells. The specific binding of [3H]naloxone reached equilibrium after 1 min. The apparent dissociation constants were estimated as 0.51 nM for glial and 0.63 nM for neuronal cells. Equilibrium measurements indicated the apparent positive cooperativity of the binding, resulting in Hill coefficients of 2.61 for glial and 2.04 for neuronal cells. Competition of unlabelled naloxone for specific binding sites resulted in maximum-shape curves in glial cells if measured at low receptor occupancy. This supports the positive cooperativity of ligand binding. Opioid agonists, ethylketocyclazocine (EKC), morphine and [D-Ala2,L-Leu5]enkephalin (DALA), provoked biphasic competition curves in both cell types with a characteristic maximum at low competitor concentrations. The possible physiological role of glial opioid receptors in neuron-glia communication and the significance of cooperativity is discussed.