[Early predictive biomarkers of posttraumatic epilepsy]. / Rannie prediktivnye biomarkery posttravmaticheskoi epilepsii.

Traumatic brain injury (TBI) affects about 50 million people in the world every year. Posttraumatic epilepsy (PTE) is a significant complication of TBI of any severity. PTE occurs in 20% of patients with TBI. Treatment of patients with PTE is particularly difficult due to obvious tendency towards drug resistance. Currently, there are no validated predictive biomarkers for PTE. Development of a system of validated predictive markers would improve PTE prediction quality and therapeutic approach for these patients. This review is devoted to the current data on the most perspective predictive biomarkers of PTE for clinical practice.

[The experience of application of target sequencing in molecular diagnostic of mucoviscidosis.]

The mucoviscidosis is one of frequent monogenic diseases. In Russia, in case of mucoviscidosis carrying out of DNA-diagnostic is optional. However, its application permits shortening time of diagnosing, increasing efficiency of of therapeutic treatment and preventing secondary manifestation of disease in family. The DNA-diagnostic using panels on frequent mutations in gene CFTR is recommended in cases of uncertain clinical picture and under borderline values of specific laboratory indices. In Russia, application of such panels permit detecting up to 90% of pathological alleles in gene CFTR. To detect more rare alleles the Sanger sequencing is traditionally applied. Lately, highly productive sequencing techniques became available to detect rare mutations. The actual article presents evaluation of efficiency of application of test-system based on technology of target sequencing for detecting mutations unidentified at primary DNA-diagnostic. Besides, in two patients with mucoviscidosis the application of highly productive sequencing techniques permitted to identify previously unknown nonsense mutations Q1038X (c.3112C>T) и W1310X (c.3930G>A).

[The development of kit for hybridization extraction of DNA and RNA of agents of hemotransmissive infections from serum and blood plasma].

To decrease dependence of effectiveness of isolation of nucleic acids of composition and amount of applied sample a kit was developed for hybridization extraction of DNA HBV RNA HCV and RNA HIV from blood serum in two formats--using up to 250 mkl and up to 1 ml of sample. This kit, in complex with kits for detection using polymerase chain reaction technique in real-time, forms a test characterized by high analytical sensitivity i.e. HBV50 copies per ml, HCV37.5 copies per ml, HIV 13 copies per ml. The developed kit for extraction of target nucleic acids permits to get rid of total DNA and inhibited effect of heparin. It can be adapted for application wit factors B and automated stations of sample preparation.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

Decontamination of polymerase chain reaction reagents using DEAE-cellulose.

Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity. We also show the suitability of the proposed approach for decontamination of the Taq polymerase, monoclonal antibodies to Taq polymerase, and Moloney murine leukemia virus (M-MLV) reverse transcriptase.

[Development of uncompetitive exogenous internal amplification control for real-time PCR based on UFA method].

An uncompetitive exogenous internal amplification control method (EIAC) was developed on the basis of short synthetic DNA segment, whose amplification can be detected in real time by UFA spectroscopy principle. The EIAC was shown to be useful as internal control in diagnostic test systems based on DNA or RNA detection by multiplex real-time PCR. It can be applied to assess the quality of extracted DNA or RNA, and also to detect and study the factors causing PCR inhibition and earlier plateau effect.

Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions.

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.

[Neuronal septal activity during hippocampal seizure discharges generation in acute model of epilepsy].

The activity of the neurones of the medial septal region (MS) and the hippocampal EEG in control and during the appearance of seizure discharges provoked by electrical stimulation of the perforant path were investigated in the awake rabbit. During afterdischarge generation in the hippocampus the dense neuronal bursts separated by periods of inhibition were recorded in the MS. In one group of neurons the bursts of spikes coincided with the discharges in the hippocampus, in other group-occured during inhibitory periods. When the afterdischarge stopped, in the septal neurons with theta activity the disruption of theta pattern was recorded, which have been correlated with the occurrence of low amplitude high frequency (20-25 Hz) waves in the hippocampal EEG. As a rule, the neuronal activivity of the MS recovered much quickly than EEG of the hippocampus; in some cases the increasing of the theta regularity was observed. The definite accordance of the electrical activity of the hippocampus and MS during seizure discharges suggests that the septohippocampal system operate as integral nervous circuit in these conditions. Diverse in the temporal interrelations between the discharges of MS neurones and ictal discharges in the hippocampus in the different cells possible indicate that various groups of the septal nervous elements have different participation in the seizure development. Appearance of the high frequency bursts in the MS is a possible "precursor" of the seizure onsets.

Transplants of the embryonal rat somatosensory neocortex in the barrel field of the adult rat: responses of the grafted neurons to sensory stimulation.

The degree of participation of grafted neurons in sensory analysis was investigated in embryonal rat somatosensory neocortex transplanted into the cavity at the place of the barrel field in the neocortex (SI) of adult rats. The neurons were investigated extracellularly 3 to 6 months after grafting. In the majority of grafts the neurons had normal levels and patterns of spontaneous activity. Many of them (65%) responded to displacement of the whiskers with latencies insignificantly different (18 +/- 0.8 ms) from those for reactions in the intact barrel field (16 +/- 0.5 ms). The receptive fields of the grafted neurons were very large. None of the neurons responded to stimulation of a single vibrissa, as in intact cortex. As a rule, the same neuron responded to isolated deflections of several (up to 10-20) vibrissae. Many of them were responsive to stimulation of the small anterior vibrissae and tactile stimulation of nose, limbs and body surface. Nevertheless, there was some spatial gradient in the effectiveness of stimulation of the body surface at various distances from the vibrissal pad; among effective vibrissae, usually several adjacent ones (2-4) produced larger responses with shorter latencies than the other ones. All units responded to painful stimuli irrespective of their location. The data show that the grafted neurons receive and may transmit sensory signals. The grafts which were proved histologically to be isolated from the host's brain did not respond to sensory stimulation and were characterized by the presence of aperiodic hypersynchronous bursts in their background activity. Electrophysiological criteria may be used for intravital diagnosis on the degree of the graft morphofunctional integration.

Integration of hippocampal suspension grafts with host neocortex.

The possibility of histological and functional integration of nervous tissue heterotopically grafted into the adult host brain was investigated. Suspensions of embryonic (E17-18) rat hippocampus with dentate fascia were placed into acute cavities in the barrel field of young adult rats (n = 25). Golgi-Cox silver impregnation and Cresyl Violet stain were used for histological analysis 3-4 months postgrafting. The surviving grafts were present in 80% of the grafted animals. Only three out of 20 surviving grafts were completely isolated from the surrounding host brain; other grafts had areas of direct confluence with the host neuropil. Extracellular recording of neuronal activity revealed normal spontaneous activity typical of the hippocampus in the majority of the grafts. Electrical stimulation of the posterior nucleus of the thalamus, homolateral motor neocortex, contralateral barrel field, and sensory stimulation of the host evoked responses in 50-60% of the grafted neurons. This did not differ significantly from the responsiveness of the similarly tested neurons of homotopic neocortical suspension grafts. The latencies of the responses in the hippocampal grafts were consistently longer (by about 10 ms) than in the neocortical ones. Comparison of the hippocampal suspension grafts with other types of hippocampal and neocortical grafts suggests that under certain conditions heterotopic tissue can be successfully integrated into the host brain. Development of the host-graft interconnections depends on topical proximity, the presence of denervated synaptic loci in both tissues, elimination of the intragraft neuronal targets and disruption of the intrinsic connections between them.

Repeated changes of dendritic morphology in the hippocampus of ground squirrels in the course of hibernation.

Quantitative Golgi study of hippocampal pyramidal neurons of ground squirrels showed rapid and profound transformation of their apical dendrites in the course of hibernation. The dendrites were significantly shorter, less branched and had fewer dendritic spines in the middle of hibernation bout than in the active euthermic ground squirrels between bouts. After arousal from torpor, within 2 h dendrites completely restored their structure. During hibernation, season remodelling of the hippocampal dendrites occurs repeatedly during each torpor-activity cycle.

Behavioural, biochemical and histochemical effects of locus coeruleus transplantation in rats with neurotoxic lesions of the catecholaminergic system.

Exploratory activity in the open field and noradrenaline concentration in the neocortex and brain stem were investigated in intact rats (n = 10) and in the following groups of rats with early postnatal neurotoxic (6-hydroxydopamine) lesion of the catecholaminergic system: (i) rats with embryonal locus coeruleus grafts in the frontal neocortex (n = 5); (ii) rats with the same grafts in lateral ventricles (n = 4); (iii) a control group with intracortical hippocampal tissue grafts (n = 3); (iv) sham-operated rats (n = 3). Experiments were performed by four independent groups of investigators using a double-blind method. In sham-operated rats as well as in rats with hippocampal grafts, and with locus coeruleus grafts in the ventricles, both exploratory activity and noradrenaline content of the forebrain were significantly lowered. In the rats with intracortical locus coeruleus grafts the level of exploratory activity was much higher (almost as in intact controls). This improvement of exploratory behaviour correlated highly with increased noradrenaline concentration in the forebrain. In all animals with locus coeruleus in lateral ventricles a significant increase in noradrenaline level of the brain stem was present, but no change of exploratory behaviour was observed. The grafts with the 3-mm-thick slabs of the adjacent tissue were dissected from the brain and used for histological analysis. The presence of typical locus coeruleus cells was shown in neocortex of all animals which received the grafts of corresponding tissue. The data show the possibility of stable compensation of the forebrain noradrenaline level and exploratory behaviour by embryonic noradrenergic neurons grafted into the neocortex, but not into lateral ventricles.

Neuronal activity of the septum transplanted into the neocortical barrel field of the rat.

Embryonic (E16-17) septal solid grafts were transplanted into acute cavities in the barrel field of somatosensory neocortex of adult rats. Extracellular recording of the graft's neuronal activity was performed in lightly anesthetized rats, or in brain slices in vitro 8-10 months after grafting. Analysis of Nissl and Golgi-Cox stained preparations showed that 81% of the grafts survived. Judging by combined histological and electrophysiological criteria, 69% of surviving grafts were integrated with the host brain. All septal grafts contained neurons with high spontaneous activity (mean 14.9 ± 8.3 spikes · s -1). Irregular bursts or rhythmic theta-bursts were present in background activity. The frequency of theta-bursts varied in parallel with the state of the animal from 3 Hz (deep barbiturate sleep) up to 7-8 Hz (arousal). Somatosensory stimulation evoked initial bursts or suppression of activity, often followed by a period of rhythmic theta-bursts. Though a high level of convergence for stimulation of vibrissae and body surface was typical of the grafted neurons, a certain spatial gradient of body representation was present. Many neurons responded also by initial burst or prolonged suppression of activity to electrical stimulation of thalamus and homolateral motor cortex. In the frontal slices of neocortex the neurons of septal grafts were highly reactive to stimulation of adjacent neocortex, but usually when the distance between the stimulating electrodes and the graft border did not exceed 1-1.5 mm. The data obtained in the septal slices are compared to the results described earlier in homotopic neocortical and heterotopic hippocampal grafts to the barrel field. It is concluded that heterotopic embryonic septal grafts can successfully develop and establish functional afferent connections with the host brain. Their neurons can participate in processing of sensory information, appropriate to the substituted cortical area, though the characteristics of responses are to a great extent determined by intrinsic properties of the septal neurons.

Sensory deprivation prevents integration of neocortical grafts with the host brain.

The histological and electrophysiological properties of embryonic neocortical grafts transplanted into the barrel field of adult rats were compared between the side receiving normal vibrissae input vs. chronic deprivation of input produced by clipping of the recipients vibrissae. Each animal received ablation of the neocortex and transplantation of neocortical tissue bilaterally. Vibrissae were clipped unilaterally immediately after surgery and were trimmed for up to 4 months. Significant differences were found between volume of the grafts as well as the number of grafts showing contact with the wall of the lesion cavity, indicating that the tissue growth was directly influenced by sensory deprivation. Decrements in functional integration with the host brain measured electrophysiologically were also observed.

Comparison of neuronal activity in septal and hippocampal grafts developing in the anterior eye chamber of the rat.

Septal and hippocampal tissue of the rat embryos (17-18 days of gestation) was transplanted into the anterior eye chamber of adult rats. Four to six months later the grafts with the piece of iris were transferred into an incubating chamber, and extracellular registration of neuronal activity was performed. Septal grafts (SG) were characterised by the presence of a greater number (70%) of spontaneously-active units, than the hippocampal grafts (HG, 41%). The level of activity in SG units was higher (mean 2.2 spikes X s-1, maximal 24 spikes X s-1), than in HG (mean 1.0 spikes X s-1, maximal 3.0 spikes X s-1). Among spontaneously active HG units, the cells with 'complex spikes' constituted the majority; in SG, besides the units with irregular single spike activity, the neurons with regular pacemaker-like and rhythmic burst (0.5-5 per s) activity were present. Stimulation of interface between iris and a graft evoked a single-spike or short burst responses in HG units. In SG, responses were tonic with gradual increase of discharge frequency or with repetitive bursts; in some cases suppression of the activity was observed. Responses of HG units followed repetitive stimulation up to 30-50 Hz and were characterized by prominent frequency potentiation. This was not observed in the SG units. These data show that embryonal nervous tissue differentiating in the anterior eye chamber develops some organotypic features of spontaneous and evoked activity similar to the activity of corresponding structures in normal conditions and in slices of adult brain.

Effects of norepinephrine and serotonin upon spontaneous activity and responses to mossy fiber stimulation of CA3 neurons in hippocampal slices.

Effects of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) upon spontaneous activity and responses to mossy fiber stimulation (mfs) were tested in 192 units of the field CA3 in the guinea pig and rat hippocampal slices. The drugs were added to the incubating medium or ejected by pressure from a micropipette. After NE superfusion firing rate increased in 52% of the reactive units, while activity of 48% was suppressed. The direction of the effect strongly correlated with pattern of spontaneous activity: only the cells with 'complex discharges' (short bursts of 2-4 spikes with attenuation of amplitude) were suppressed by NE; the cells with single spikes increased the level of activity. Similar excitatory effects of NE were observed in all units (n = 14) with single spike activity recorded in the field CA1. 5-HT increased activity in 30% of the reactive units and suppressed it in 70% of them. Some of the cells which were suppressed by 5-HT, were excited by NE. In more than a half of the units tested, 5-HT led to prolonged (up to 30-40 min) increase of the level of background activity irrespective of the initial excitatory or suppressive action; periodic grouped discharges appeared in some units under the influence of 5-HT. The response to mfs usually changed in the same direction as the level of background activity during application of NE and 5-HT, though some exceptions were observed in both cases. Prolonged (up to 30-40 min) facilitation of responses to mfs was present after application of 5-HT.

Neuronal activity in suspension transplants of the neocortex.

The characteristics of suspension (ST) and tissue (TT) transplants of the embryonal neocortex, transplanted into adult rats into the neocortical region of the representation of the vibrissae, were compared. The degree of taking of the ST and the TT did not differ significantly (89.5 and 95%, respectively). Transplants completely isolated from the brain were not found in the ST on the basis of histological and electrophysiological indices. The reactivity of ST neurons during electrical stimulation of the brain structures of the recipient and sensory stimulation, like the latent periods of the on-responses, did not differ significantly in the ST and the TT; however, the per cent of neurons responding with on-responses, was nearly twice as low in the ST as in the TT. At the same time, there were substantially more neurons in the ST responding to tactile stimulation with inhibition of discharges. It is hypothesized that the disruption in the primary cytoarchitectonics of the ST which takes place inevitably in the preparation of the suspensions is a cause of the differences indicated between the ST and the TT.

[Ultrastructure of the cells of surviving hippocampal slices during the incubation process]. / Izuchenie ul'trastruktury kletok perezhivaiushchikh srezov gippokampa v protsesse inkubatsii.

The dynamics of ultrastructural changes in the pyramidal neurons from the CA3 area of hippocampal slices was studied. It was shown that after 5 hours' incubation changes in the reticulum, nuclear envelope and mitochondria took place. No visible changes were seen in different synapses after incubation longer than 5 hours. It might be seen that the glial cells had their structural integrity after 30 hours. The data obtained were in a good agreement with our electrophysiological observations of neurons of the same area. The present data suggests that the brain slices in vitro are convenient objects for a parallel ultrastructural and electrophysiological study of the properties of neurons within a 1-5 hours incubation.

[Ultrastructural study of the bases of postsynaptic potentiation in hippocampal sections by the freeze-substitution method]. / Ul'trastrukturnoe izuchenie osnov posttetanicheskoi potentsiatsii v srezakh gippokampa metodom zamorazhivaniia-zameshcheniia.

Some morphological features of the pyramidal neurons depending on their physiological state were studied in the CA3 area of the guinea pig hippocampal slices by the freeze-substitution method. This method provided a better possibility to observe certain visible structural changes in the majority of fiber synapses in stratum lucidum after the rhythmic stimulation of dentate girus, compared to the conventional electron microscopy. The width of the spine stalks during the long-lasting potentiation in the tissue processed by the freeze-substitution method increased by 60%, being below 50% in conventional preparations. A correlation between changes in the post-synaptic densities and the physiological state of synapses was checked. A two-fold increase in the width of post-synaptic densities without changes in their length was found.

[Thermal activity of the isolated brain fragment]. / Teplovaia aktivnost' izolirovannogo fragmenta mozga.

The paper deals with thermovisional studies of thermal activity of hippocampus transplants placed in the eye anterior chamber of the rat. The method permits investigation into the dynamics of temperature field in the nervous tissue directly. In the transplants with synchronous spontaneous activity of large neuron groups there were found zones of high thermoactivity with mean amplitude 0.1 degree C, as well as thermopassive zones with temperature variations not above 0.03 degree C. Under certain conditions transfer of heat-up regions along the transplant surface, synchronous thermoactivation of single zones and chaotic regime of thermoactivation were observed. Causes of the observed effects are analysed. The method applied opens a new approach to the studies of space and time organization of the nervous tissue functioning in the course of its living activity.