[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.

[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.

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.

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.

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.

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.

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.

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.

[The comparative characteristics of the neuronal activity in surviving slices of transplanted and intact neocortex]. / Sravnitel'nye kharakteristiki aktivnosti neironov v perezhivaiushchikh srezakh transplantirovannogo i intaktnogo neokorteksa.

The background single unit activity and single or multiunit responses to electrical stimulation of the rat's embryonic cortex, grafted into the somatosensory cortex of adult rats were investigated in a slice preparation. The same characteristics of the intact cortex neurons were observed as a control. The percentage of neurons with background activity within grafts was higher, than that within intact neocortex slices (23% and 6%, respectively). However the percentage of neurons responding to electrical stimulation of the recipient neocortex was lower in grafts. The mean latency of multiunit responses of graft neurons was longer, than that of neurons of the intact neocortex recorded at the same distance from the stimulating electrode (19.4 +/- 5.0 and 5.8 +/- 1.1 ms, respectively). Duration of the evoked population discharges was approximately ten times longer in grafts. It is concluded that there are local functional connections between a graft and the host brain and that inhibitory processes within grafts are weak. Functional integration of grafts with the host brain of the recipients after cranial trauma is shown.

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.

Growth of the hippocampal grafts in the rat anterior eye chamber: effects of age and strain of the recipients.

Hippocampal tissue (1 mm3) was taken from embryonal (E17-18) rats of Wistar stock in population breeding and grafted into anterior eye chamber of the four groups of recipients: young (3 weeks) and old (18 months) males of the same stock and of inbred strain WAG. Morphometric analysis of the grafts developing up to 12 weeks in oculo showed rapid initial growth in both groups of the young hosts during the first three weeks, and limited increase of the graft volume during next three weeks. The start of growth was significantly retarded in the old hosts, but this was partly compensated by prolonged increase of the graft volume during the later stages. Both mean and maximal finite volume were much smaller in both WAG groups. The hippocampal grafts in these groups had rounded (not elongated, as in Wistar groups) shape and showed tendency to fragmentation and resorbtion at the late stages. Histological analysis revealed well organized layer of the pyramidal cells in the both Wistar groups and nearly complete absence of neuronal organization into layer in both WAG groups. Possible role of trophic and immune factors in development of intraocular grafts is discussed.

[Compensation in behavioral disorders in rats receiving 5,7-DHT neonatally and by transplantation of embryonic raphe nucleus tissue]. / Kompensatsiia narushenii povedeniia krys, neonatal'no poluchavshikh 5,7-DOT, putem transplantatsii émbrional'noi tkani iader shva.

In 62 male Wistar rats the influence was studied of the transplanted embryonal tissue of raphe nuclei (NR) on the mechanisms of compensation of disturbances of exploratory activity, sensory attention, learning and emotional reactivity induced by neonatal injection of 5,7-DHT. In histochemical studies by Falk-Hillarp method the presence of yellow fluorescence confirmed the specificity of transplanted 5-HT neurones. It is found that NR transplantation causes in animals after 3 months recovery of orienting reaction to sensory stimuli, reduces rats reactivity in the open field, restores the ability to discrimination of emotionally positive influence, disturbed by neonatal injection of 5,7-DHT. The obtained data show the possibility of compensation of behaviour disturbances caused by chronic deprivation of 5-HT system activity by transplantation in the neocortex parenchyma of the embryonal tissue, containing serotoninergic neurones.