[APOΕ gene polymorphism and markers of brain damage in the outcomes of severe traumatic brain injury in children]. / Polimorfizm gena APOΕ i markery povrezhdeniya mozga v iskhodakh tyazheloi cherepno-mozgovoi travmy u detei.

OBJECTIVE: To compare apolipoprotein E (APOE) genotypes with outcomes and levels of neuromarkers in children with severe traumatic brain injury (TBI). MATERIAL AND METHODS: APOE polymorphisms were genotyped in 69 children with severe TBI. The following markers of brain damage were identified: neuron-specific enolase (NSE), glial protein S100b, content of autoantibodies (aAB) to glutamate receptors (to the NR2 subunit of NMDA receptors), aAB to S100b and brain-derived neurotrophic factor (BDNF). RESULTS AND CONCLUSION: There was no association between APOE 3/3, 3/4, 3/2 genotypes and outcomes assessed by the Glasgow Outcome Scale (GOS). The greatest number of favorable outcomes was noted in the group of APOE 3/3 genotype carriers (60%). The ratio of favorable outcomes to unfavorable outcomes was equal (50%:50%) in groups with APOE 3/4 and APOE 3/2 genotypes. An association between APOE polymorphism and BDNF was found: there were normal BDNF levels in the APOE 3/3 group and reduced levels in the APOE 3/2 group. The correlation between neuromarkers and GOS scores was shown for BDNF and aAB to S100b. In children with favorable TBI outcomes, normal BDNF levels and a lower level of aAB to S100b were observed. Regardless of APOE genotypes, almost all children with severe TBI (95%) showed a significant increase in aAB to glutamate receptors in the remote period and most children had an increase in aAB to S100b in the blood. This fact can be explained by the presence of cerebral hypoxia, activation of autoimmune processes and increased BBB permeability, which may be enhanced by increased NO content and intensification of oxidative processes in children with severe TBI.

Neuroprotective Potential of Peptides HFRWPGP (ACTH6-9PGP), KKRRPGP, and PyrRP in Cultured Cortical Neurons at Glutamate Excitotoxicity.

Glutamate (Glu) excitotoxicity, which accompanies brain ischemia or traumatic brain injury, is the leading mechanism of neuronal death. In the present work, we studied the effects of the peptides HFRWPGP (ACTH6-9PGP), KKRRPG, and PyrRP on the survival of cultured cortical neurons on the background of excitotoxic effect of Glu (100 µM). Biochemical (MTT/WST) and morphometric analyzes showed that, depending on the dose, ACTH6-9PGP and KKRRPGP protect neurons from the cells death, while PyrRP, conversely, enhances it. The neuroprotective effect of ACTH6-9PGP is accompanied by a slowdown in the development of delayed calcium dysregulation and synchronous mitochondrial depolarization. Among the studied peptides, only ACTH6-9PGP significantly increased the number of neurons that restored Ca2+ homeostasis after Glu was abolished. The influence of KKRRPGP was less pronounced, whereas PyrRP, on the contrary, reduced the number of neurons with low [Ca2+]i. Thus, this study revealed the high therapeutic significance of ACTH6-9PGP and allowed assessing the prospects for its possible clinical use.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 µM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

[Estimation of Time-Dependent microRNA Expression Patterns in Brain Tissue, Leukocytes, and Blood Plasma of Rats under Photochemically Induced Focal Cerebral Ischemia].

miRNA expression over different time periods (24 and 48 h) using the quantitative RT-PCR and deep sequencing has been evaluated in a model of photochemically induced thrombosis. A combination of two approaches allowed us to determine the miRNA expression patterns caused by ischemia. Nine miRNAs, including let-7f-5p, miR-221-3p, miR-21-5p, miR-30c-5p, miR-30a-3p, miR-223-3p, miR-23a-3p, miR-22-5p, and miR-99a-5p, were differentially expressed in brain tissue and leukocytes of rats 48 h after onset of ischemia. In addition, six miRNAs were differentially expressed in the brain tissue and blood plasma of rats 24 h after exposure, among which miR-145-3p and miR-375-3p were downregulated and miR-19a-3p, miR-92a-3p, miR-188-5p, and miR-532-5p were upregulated. In our opinion, miR-188-5p and miR-532-5p may be considered to be new potential markers of ischemic injury. The level of miRNA expression tended to increase 48 h after the onset of ischemia in brain tissue and leukocytes, which reflects not only the local response in brain tissue due to inflammation, vascular endothelial dysfunction, and disorders of the permeability of the blood-brain barrier, but also the systemic response of the organism to multifactor molecular processes induced by ischemic injury.

Disruption of Functional Activity of Mitochondria during MTT Assay of Viability of Cultured Neurons.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

[Biomarkers in children with traumatic brain injury].

OBJECTIVE: to study the content of biomarkers of diagnostic and prognostic value in the assessment of traumatic brain injury (TBI) severity in children. MATERIAL AND METHODS: Authors determined the levels of glial protein S100B, neuronspecific enolase (NSE), autoantibodies (aAb) to glutamate receptors and natural autoantibodies (nAb) to S100B and brain-derived neurotrophic factor (BDNF) in serum/plasma of children with different outcomes of TBI. All parameters were analyzed in the 1-3rd, 7-8th, 14-15th and 20-23rd days after TBI, and, in some cases of severe brain injury and long stay patients in hospital, in 11-12 months after TBI. The severity and outcome of TBI were assessed according the Glasgow coma scale (GCS) and the Glasgow outcome scale (GOS), respectively. RESULTS AND CONCLUSION: The content of NSE and S100B increased immediately after TBI regardless of TBI severity, but in cases with favorable outcome it dropped to a normal level in the first 3 days. The maximum levels of S100B protein and NSE were observed in children with fatal TBI, and higher values were observed throughout the post-traumatic period. The low levels of aAb to NR2-subtype of glutamate receptors that were similar to controls and the high level of nAb to S100B in the first days after severe TBI indicate the failure of compensatory-adaptive immunological mechanisms and the high permeability of the brain-blood barrier, which were poor prognostic signs for children with severe TBI. Mild and severe TBI with full recovery were accompanied by higher values of ВDNF in the 1st day followed by a decline to the 3rd day. The level of BDNF in the 1st day of TBI was the lowest and subsequently continued to decline in patients with severe TBI with fatal outcome.

[Peripheral Blood Lymphocytes Mitochondrial Function in Children with Traumatic Brain Injury].

BACKGRAUND: It is known that mitochondria play an important role in the mechanisms of brain cells damage and death following traumatic brain injury (TBI). However, the relationship between the severity of brain damage following TBI and mitochondrial dysfunction are not well defined. AIM: to study activities of NADN- and succinate dehydrogenases, a key enzyme of mitochondrial oxidative phosphorylation in children with TBI of varying severity and different outcomes; to detect ATP content in lymphocytes; the level of NOx and 3-nitrotyrosine in serum and plasma. Methods: all parameters were determined in the dynamics of one month following TBI, and in some cases up to the death ofpatients. The severity of TBI was scored by Glasgow Coma Scale (GCS), the outcome of TBI-Glasgow Outcome Scale (GOS). Based on the clinical examination children with TBI were divided into 3 groups: (1) mild TBI; (2) severe TBI and (3) severe TBI with fatal outcome. RESULTS: we found that activity of dehydrogenases is significantly reduced only in patients with the poor neurologic outcome. The greatest decrease in these parameters was observed in patients with severe traumatic brain injury and fatal outcome. A direct correlation was found between the indices of dehydrogenases activity and A TP content in lymphocytes (r = 0.97, p = 0.005). The levels of NOx metabolites and 3-nitrotyrosine were significantly increased in children with severe TBI. CONCLUSION: obtained results suggest that mitochondrial dysfunction, impaired cerebral energy metabolism and oxidative stress contribute to cell death in the brain and thus represent therapeutic targets for the treatment of TBI.

Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis.

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1-2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 µM, glycine 10 µM, Mg2+-free) produced a rapid acidification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r(2) = 0.56) between the rate of [ATP]c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]c of as much as 15.9%. In the phase of high [Ca2+]i, the plateau of [ATP]c dropped to 10.4% compared to [ATP]c in resting neurons (100%). In the presence of the Na+/K+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]c in the phase of the high [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain (0.5 mM), led us to suggest that in addition to increase in proton conductivity and decline in [ATP]c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na+/Ca2+ exchange.

Effects of selective inhibitors of neuronal and inducible NO-synthase on ATP content and survival of cultured rat cerebellar neurons during hyperstimulation of glutamate receptors.

We studied the effects of selective inhibitors of neuronal and inducible NO-synthase (7-nitroindazole and aminoguanidine) and non-selective NO-synthase inhibitor L-NAME on ATP content and survival of cultured rat cerebellar neurons during hyperstimulation of glutamate receptors with toxic doses of glutamate. Application of 100 µM glutamate reduced ATP content in the primary culture of 7-8- and 14-15-day-old cerebellar granule cells by 66 and 49%, respectively, in comparison with the control. Inhibition of nitric oxide synthesis with 7-nitroindazole during glutamate exposure in the culture of 7-8-day-old neurons and with 7-nitroindazole and aminoguanidine in the culture of 14-15-day-old neurons ensured better protection of cells from ATP level decrease than non-specific inhibition with L-NAME. In addition, inhibition of neuronal and inducible NO-synthase during glutamate exposure decreased death of "young" neurons, whereas death of "old" neurons remained high under these conditions.

[Hereditary thrombophilia in children: current views of etiology and pathogenesis].

This review is focused on the role of genetic factors in etiology and pathogenesis of thrombophilia in adults and children. Their knowledge permits to elucidate the main causes of this pathology and choose adequate measures for its prevention.

[Autoantibodies to α7-subunit of neuronal acetylcholine receptor in children with traumatic brain injury].

An objective of the study was to search for new biologically significant markers of brain damage. Levels of blood serum autoantibodies (aAB) to different fragments of α7-subunit of acetylcholine receptor (ACR) were studied in children with traumatic brain injury of different severity. The more severe was trauma, the higher was the level of aAB to fragments of α7-subunit of ACR in the first week after trauma. The data obtained suggest that α7-subunits of ACR and aAB to them are involved in the pathogenesis of traumatic brain lesions and, probably, play a significant role in the course of post traumatic period.

Effect of enteropeptidase on survival of cultured hippocampal neurons under conditions of glutamate toxicity.

The effects of full-size bovine enteropeptidase (BEK) and of human recombinant light chain enteropeptidase (L-HEP) on survival of cultured hippocampal neurons were studied under conditions of glutamate excitotoxicity. Low concentrations of L-HEP or BEK (0.1-1 and 0.1-0.5 nM, respectively) protected hippocampal neurons against the death caused by 100 µM glutamate. Using the PAR1 (proteinase-activated receptor) antagonist SCH 79797, we revealed a PAR1-dependent mechanism of neuroprotective action of low concentrations of enteropeptidase. The protective effect of full-size enteropeptidase was not observed at the concentrations of 1 and 10 nM; moreover, 10 nM of BEK caused death of 88.9% of the neurons, which significantly exceeded the cell death caused by glutamate (31.9%). Under conditions of glutamate cytotoxicity the survival of neurons was 26.8% higher even in the presence of 10 nM of L-HEP than in the presence of 10 nM BEK. Pretreatment of cells with 10 nM of either form of enteropeptidase abolished the protective effect of 10 nM thrombin under glutamate cytotoxicity. High concentrations of BEK and L-HEP caused the death of neurons mainly through necrosis.

[S100B protein and autoantibodies to S100B protein in diagnostics of brain damage in craniocerebral trauma in children].

Levels of antibodies AB (AB) to S100B and S100B protein were studied in the blood serum of children with different severity and outcomes of traumatic brain injury (TBI) from the 1st to 15-75th days after TBI. Severity and outcomes were assessed using the Glasgow Coma Scale (GCS). Patients were stratified by outcomes into the following groups: complete recovery (group 1), moderate disability (group 2), high disability (group 3), vegetative state (group 4) and fatal outcome (group 5). In patients of groups 1-3, the changes of S100B in the blood serum didn't depend on the severity of brain's damage; the significant increase of S100B protein levels in the 1st day was accompanied by the decrease to the normal range in the following 2-3 days. On the contrary, the levels of nAB in these groups increased starting from 3-5 days corresponding to the severity of brain's damage. The development of vegetative state was accompanied by low S100B and high AB to S100B levels in the blood serum. The maximal level of S100B protein and increased levels of AB were observed in patients with fatal outcome. In most patients with combined TBI, the levels of both parameters were higher compared to those with separate TBI.

Neuroprotective effect of KB-R7943 against glutamate excitotoxicity is related to mild mitochondrial depolarization.

KB-R7943, an inhibitor of a reversed Na(+)/Ca(2+) exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Deltapsi(mit)), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca(2+)](i) and on Deltapsi(mit) changes in rat cultured cerebellar granule neurons. 15 micromol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca(2+)](i) overload, KB-R7943 considerably improved the [Ca(2+)](i) recovery and restoration of Deltapsi(mit) after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Deltapsi(mit). KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca(2+) uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca(2+) accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.

[EPR study of the influence of hypoxia on nitric oxide formation in the blood of Krushinskii-Molodkina rats].

The influence of hypoxia on nitric oxide formation in the blood of Krushinskii-Molodkina rats has been studied by electron paramagnetic resonance. It was found that nitric oxide synthesis in Krushinskii-Molodkina rats is increased compared with that in Wistar rats. A significant enhancement of the EPR signal of Hb-NO complexes in the animal blood was observed after hypoxia simulating the altitude of 5000 m above the sea level, in particular in the presence of sodium nitrite and the NO-synthase inhibitor L-nitroarginine. It was assumed that NO synthesis and nitro-/nitrite- reductase systems are activated under hypoxic conditions.

Autoantibodies to glutamate receptors and products of nitric oxide metabolism in serum in children in the acute phase of craniocerebral trauma.

Levels of serum autoantibodies (aAb) to glutamate receptors and products of nitric oxide (NO) metabolism, i.e., nitrates and nitrites, were assayed in children with recent craniocerebral trauma (CCT) of different levels of severity. All the children showed increases in serum aAb to both AMPA and NMDA receptor subtypes from day 1 to day 10 after trauma. The highest levels of serum aAb were to the NMDA subtype of glutamate receptor, which was characteristic of children with mild CCT (MCCT), with Glasgow Coma Scale (GCS) scores of 14-15 points. Levels of aAb to NMDA (NR2A) receptors in children with severe CCT (SCCT, GCS < 9 points) were lower than in children with MCCT, the lowest levels being seen in the group of children with lethal CCT (SCCT-2). Serum concentrations of NO metabolites increased by large factors in the group of children with SCCT, indicating marked brain hypoxia.

Activated protein C via PAR1 receptor regulates survival of neurons under conditions of glutamate excitotoxicity.

The effect of an anticoagulant and cytoprotector blood serine proteinase--activated protein C (APC)--on survival of cultured hippocampal and cortical neurons under conditions of glutamate-induced excitotoxicity has been studied. Low concentrations of APC (0.01-10 nM) did not cause neuron death, but in the narrow range of low concentrations APC twofold and stronger decreased cell death caused by glutamate toxicity. High concentrations of APC (>50 nM) induced the death of hippocampal neurons similarly to the toxic action of glutamate. The neuroprotective effect of APC on the neurons was mediated by type 1 proteinase-activated receptor (PAR1), because the inactivation of the enzyme with phenylmethylsulfonyl fluoride or PAR1 blockade by a PAR1 peptide antagonist ((Tyr1)-TRAP-7) prevented the protective effect of APC. Moreover, APC inhibited the proapoptotic effect of 10 nM thrombin on the neurons. Geldanamycin, a specific inhibitor of heat shock protein Hsp90, completely abolished the antiapoptotic effect of 0.1 nM APC on glutamate-induced cytotoxicity in the hippocampal neurons. Thus, APC at low concentrations, activating PAR1, prevents the death of hippocampal and cortical neurons under conditions of glutamate excitotoxicity.

[Autoantibodies to glutamate receptors and metabolic products of nitric oxide in blood serum of children in the acute period of brain trauma].

Autoantibodies (AB) to glutamate receptors of AMPA (Glur1) and NMDA (NR2A) types and nitric oxide metabolites, nitrates and nitrites (NOx), were studied in the blood serum of children with brain trauma of different severity. The level of both AB types increased from the 1st to the 10th day after trauma. The level of NMDA (NR2A) AB was higher comparing to AMPA (Glur1). Patients with mild brain trauma, scoring 14-15 on the Coma Glasgow Scale, had the highest AB concentration while patients with severe brain trauma (scores <9), had the lower level of NMDA (NR2A) AB. The lowest level of AB and the highest level of NOx in the blood serum were found in a group of children with the fatal outcome of severe brain trauma. The many-fold increase of NOx concentration in this group points to marked hypoxia after severe brain trauma.