Experimental Modeling of Cranial Injury and Defect Reconstruction Using Superconstructive Polymers (PEEK).

Experimental model of resection craniotomy with subsequent reconstruction of the defect with a polymer implant enables comprehensive assessment of functional and ultrastructural changes during replacement of the damaged tissue. Reconstruction of a skull defect was accompanied by transient motor disturbance in the acute period and did not cause functional disorders and neurological deficits in a delayed period. Histological examination of osteal and brain tissue revealed no pathological reactions that could be associated with the response to the chemical components of the implant.

Comparative Morphological and Molecular Genetic Characteristics of Cell and Tissue Strains of Experimental Rat Glioma 10-17-2 (Astrid-17).

In order to obtain models of gliomas of varying degrees of malignancy, we performed morphological and molecular genetic study of a tissue strain of glioma 10-17-2 (Astrid-17) obtained by intracranial passaging of tumor fragments of chemically induced rat brain tumor, and a cell strain isolated from it. More or less pronounced changes in the expression levels of Mki67, Trp53, Vegfa, and Gfap genes in the tissue and cell strain of glioma 10-17-2 (Astrid-17) compared with intact brain tissue were shown. The tissue model of glioma 10-17-2 (Astrid-17) according to the studied characteristics shows features of grade 3-4 astrocytoma and the cellular model - grade 2-3 astrocytoma.

The Effect of Therapy Regimen on Antitumor Efficacy of the Nanosomal Doxorubicin against Rat Glioblastoma 101.8.

One of the key problems of glioblastoma treatment is the low effectiveness of chemotherapeutic drugs. Incorporation of doxorubicin into PLGA nanoparticles allows increasing the antitumor effect of the cytostatics against experimental rat glioblastoma 101.8. Animal survival, tumor volume, and oncogene expression in tumor cells were compared after early (days 2, 5, and 8 after tumor implantation) and late (days 8, 11, and 14) start of the therapy. At late start, a significant increase in the expression of oncogenes Gdnf, Pdgfra, and Melk and genes determining the development of multidrug resistance Abcb1b and Mgmt was revealed. At early start of therapy, only the expression of oncogenes Gdnf, Pdgfra, and Melk was enhanced. Early start of treatment prolonged the survival time and increased tumor growth inhibition by 141.4 and 95.7%, respectively, in comparison with the untreated group; these differences were not observed in the group with late start of therapy. The results indicate that the time of initiation of therapy is a critical parameter affecting the antitumor efficacy of DOX-PLGA.

[Neuroprotective mechanisms of the ubiquinol action in experimental focal ischemia]. / Issledovanie neiroprotektornykh mekhanizmov deistviia ubikhinola pri éksperimental'noi fokal'noi ishemii.

Ischemic stroke is one of the most socially important diseases characterized by impaired cerebral circulation with focal damage of the brain tissue and decreased functionality. Despite the successes of modern pharmacology, possibilities of pharmacotherapy for stroke remain limited, and the research for new drugs with neuroprotective effects that can prevent brain cell death is still relevant. In this study we have investigated the neuroprotective activity of ubiquinol as a part of an innovative form on a rat model of irreversible 24 h-cerebral ischemia with evaluation of the mechanisms of its neuroprotective effect. Ubiquinol (30 mg/kg), administered intravenously in the acute period of irreversible 24 h focal cerebral ischemia, had a direct neuroprotective effect, characterized by a decrease in the volume of brain tissue necrosis. The protective effect of ubiquinol is due to its ability to inhibit the development of oxidative stress by the direct anti-radical action, preventing the increase in the lipid hydroperoxide content in the brain tissue adjacent to the focus of necrosis, lowering the lipid oxidation rate in plasma against under conditions of increased total antioxidant activity in the brain and blood of experimental animals. In vitro experiments have shown the ability of ubiquinol to prevent cell death in primary culture of cerebral neurons of rat brain under 4 h oxygen/glucose deprivation followed by 20 h reoxygenation.

Effect of Anesthetics on Efficiency of Remote Ischemic Preconditioning.

Remote ischemic preconditioning of hind limbs (RIPC) is an effective method for preventing brain injury resulting from ischemia. However, in numerous studies RIPC has been used on the background of administered anesthetics, which also could exhibit neuroprotective properties. Therefore, investigation of the signaling pathways triggered by RIPC and the effect of anesthetics is important. In this study, we explored the effect of anesthetics (chloral hydrate and Zoletil) on the ability of RIPC to protect the brain from injury caused by ischemia and reperfusion. We found that RIPC without anesthesia resulted in statistically significant decrease in neurological deficit 24 h after ischemia, but did not affect the volume of brain injury. Administration of chloral hydrate or Zoletil one day prior to brain ischemia produced a preconditioning effect by their own, decreasing the degree of neurological deficit and lowering the volume of infarct with the use of Zoletil. The protective effects observed after RIPC with chloral hydrate or Zoletil were similar to those observed when only the respective anesthetic was used. RIPC was accompanied by significant increase in the level of brain proteins associated with the induction of ischemic tolerance such as pGSK-3ß, BDNF, and HSP70. However, Zoletil did not affect the level of these proteins 24 h after injection, and chloral hydrate caused increase of only pGSK-3ß. We conclude that RIPC, chloral hydrate, and Zoletil produce a significant neuroprotective effect, but the simultaneous use of anesthetics with RIPC does not enhance the degree of neuroprotection.

The Influence of Proinflammatory Factors on the Neuroprotective Efficiency of Multipotent Mesenchymal Stromal Cells in Traumatic Brain Injury.

We studied the neuroprotective potential of multipotent mesenchymal stromal cells in traumatic brain injury and the effect of inflammatory preconditioning on neuroprotective properties of stem cells under in vitro conditions. To this end, the effects of cell incubation with LPS or their co-culturing with leukocytes on production of cytokines IL-1α, IL-6, TNFα, and MMP-2 and MMP-9 by these cells were evaluated. Culturing under conditions simulating inflammation increased the production of all these factors by multipotent mesenchymal stromal cells. However, acquisition of the inflammatory phenotype by stromal cells did not reduce their therapeutic effectiveness in traumatic brain injury. Moreover, in some variants of inflammatory preconditioning, multipotent mesenchymal stromal cells exhibited more pronounced neuroprotective properties reducing the volume of brain lesion and promoting recovery of neurological functions after traumatic brain injury.

The Use of Technetium-99m for Intravital Tracing of Transplanted Multipotent Stromal Cells.

We studied the possibility of in vivo tracing of multipotent mesenchymal stromal cells labeled with a radiophermaceutic preparation based on metastable isotope Technetium-99m and injected to rats with modeled traumatic brain injury. Accumulation of labeled cells occurred primarily in the liver and lungs. The cells distribution in internal organs greatly varied depending on the administration route. Cell injection into the carotid artery led to their significant accumulation in the damaged brain hemisphere, while intravenous injection was followed by diffuse cell distribution in all brain structures. Scintigraphy data were confirmed by magnetic resonance imaging and histological staining of cells. Visualization of stem cells labeled with Technetium-99m-based preparation by scintigraphy is an objective and highly informative method allowing real-time in vivo cell tracing in the body.

Relationship between Morphofunctional Changes in Open Traumatic Brain Injury and the Severity of Brain Damage in Rats.

A correlation between the severity of morphofunctional disturbances and the volume of brain tissue injury determined by MRT was demonstrated on the model of open traumatic brain injury in rats. A relationship between the studied parameters (limb placing and beam walking tests and histological changes) and impact force (the height of load fell onto exposed brain surface) was revealed.

Protection of Neurovascular Unit Cells with Lithium Chloride and Sodium Valproate Prevents Brain Damage in Neonatal Ischemia/Hypoxia.

Here we studied the cytoprotective effect of lithium chloride and sodium valproate in the in vivo model of neonatal cerebral ischemia/hypoxia and analyzed the influence of these substances on the death of the major neurovascular unit components in experimental ischemia in vitro. Lithium chloride and sodium valproate effectively prevented death of neurons, astrocytes, and endothelial cells in the oxygen-glucose deprivation. This treatment protected the brain of newborn rats from ischemia/hypoxia injury. The results suggest that lithium and sodium valproate can be used for the treatment of neurodegenerative pathologies associated with hypoxia and ischemia in newborns.

Structural alterations in the rat brain and behavioral impairment after status epilepticus: An MRI study.

Temporal lobe epilepsy (TLE) is one of the most common neurologic disorders often associated with behavioral impairments and cognitive deficit. Lithium-pilocarpine model of seizures in rodents reproduces many features of human convulsive status epilepticus (SE) and subsequent TLE. In this study, we have investigated changes in the rat brain after lithium-pilocarpine SE using a high-field MRI system for small animals in early and chronic periods after SE. We have studied the relationship between T2 relaxation time measured in these periods and the development of behavioral exploratory response to novelty and habituation in the open field test. A significant increase in T2 in the hippocampus and associated structures was found 2 days after SE and practically resolved by day seven, while an increase in T2 in the parietal and prefrontal cortex appeared 30 days after SE. High T2 values in the parietal cortex and thalamus on day two after SE were associated with an increased mortality risk. A substantial variability in T2 relaxation time was observed in the hippocampus and amygdala 30 days after SE. Rats survived after SE showed locomotor hyperactivity and disruption of long-term habituation in the open field test carried out 5 weeks after the seizures. Interestingly, T2 in the amygdala 30 days after SE had a strong correlation with hyperactivity in the novel open field. Therefore, the amygdala damage may be an important factor in the development of hyperactivity in the chronic period after SE.

CHANGES IN THE NUMBER OF NEURONS, ASTROCYTES AND MICROGLIA IN THE BRAIN AFTER ISCHEMIC STROKE ASSESSED BY IMMUNOHISTOCHEMISTRY AND IMMUNOBLOTTING.

It is known that the mechanisms of damage in the brain after stroke are regulated by combination of several types of cells, primarily of neurons, astrocytes, endothelium and microglia. Ischemic exposure disrupts the balance in the cellular composition of the brain; in the lesion, cells die by necrosis while in tissue surrounding ischemic zone the delayed induction of apoptosis occurs, and namely the ratio of death of different cells determines the clinical outcome of the disease. Thus, the assessment of death of various cell types of the neurovascular unit is an important part of fundamental studies of the mechanisms of brain damage and pre-clinical studies of potential neuroprotective drugs. In this line, we have conducted a comparative study of the two most often used methods: immunohistochemical staining of brain sections, allowing to determine the number and localization of specific cells in the tissue among other types of cells, and immunoblotting that detects specific proteins in the tissue homogenate. We have found that, depending on the type of cells, changes in their number and composition after stroke can be diffuse or localized, which imposes restrictions on the use of any method of estimation of the number of cells in brain tissue. In general, the most preferable is the use of immunohistochemistry, however, with certain limitations, immunoblotting can be used in estimating amounts of astroglia and microglia.

Neuroprotective and antiamnestic effects of mutant molecules of erythropoietin on model of photochemical thrombosis of rat brain prefrontal cortex.

Mutant EPO molecules, deprived of erythropoietic activity, but possessing cytoprotective action, were created by the method of genetic engineering. The assessment of the therapeutic effectiveness of the received mutant proteins was carried out by the retention of the conditioned reflex of passive avoidance (PA), developed before the ischemic injury of rat brain prefrontal cortex, and by the MRI-analysis of ischemic damage volume. Antiamnestic and neuroprotective action of mutant molecules - MERO-Fc and MEPO-TR is investigated on model of photothrombosis of rat brain prefrontal cortex at single intranasal introduction in 1 h after cortex ischemic damage. The neuroprotective (MRI) and antiamnestic (PA) effects of mutant molecules of erythropoietin derivatives are shown.

Effects of activated protein C on the size of modeled ischemic focus and morphometric parameters of neurons and neuroglia in its perifocal zone.

The effects of activated protein C (APC) on the quantitative parameters of neurons and neuroglia in the perifocal zone of infarction induced in the left hemispheric cortex were studied in two groups of rats. Group 1 animals served as control (control infarction). Group 2 rats were injected with APC (50 µg/kg) in the right lateral cerebral ventricle 3 h after infarction was induced, and after 72 h the infarction size was evaluated and the neurons and neuroglia in the perifocal zone were counted. APC reduced the infarction size 2.5 times in comparison with the control and reduced by 16% the neuronal death in the perifocal zone layer V, causing no appreciable changes in layer III, and did not change the size of neuronal bodies but increased (by 11%) the size of neuronal nuclei in layer III. The protein maintained the sharply increased count of gliocytes in the perifocal zone of infarction and promoted their growth. Hence, APC protected the neurons from death in the ischemic focus by increasing the gliocyte count and stimulating the compensatory reparative processes.

[Pathological changes after brain ischemia are similar to those observed in Alzheimer disease].

We studied the influence of ischemia/reperfusion of the middle cerebral artery in the rat's brain on the deferred violation of cognitive functions of the brain which are similar to main symptoms observed in the development of Alzheimer's disease. Using 8-hose radial labyrinth we demonstrated that 6 months after incidence of cerebral ischemia a significant impairment of working memory and a decrease in animals the ability to learn are developed. 7 months after focal cerebral ischemia we could observe the accumulation of a mature amyloid peptide and hyperphosphorylated form of the Tau pro- tein in ipsilateral cerebral hemisphere and of the the beta-amyloid peptide precursor in the contralateral hemisphere. Thus, after an experimental stroke in the brain pathological chanres occur as those typical of Alzheimer's disease.

Antitumor effects of monoclonal antibodies to connexin 43 extracellular fragment in induced low-differentiated glioma.

We studied the effect of intravenous administration of monoclonal antibodies to the second extracellular loop of connexin 43 (MAbE2Cx43) on the dynamics of glioma growth and survival of experimental animals. Morphometric analysis of magnetic resonance imaging data showed that weekly intravenous administration of MAbE2Cx43 in a dose of 5 mg/kg significantly reduced glioma volume starting from day 21 after tumor implantation. By day 29, the mean volume of glioma in the experimental group (therapy with specific antibodies) was 2-fold lower than in controls. Deceleration of glioma growth in rats receiving MAbE2Cx43 was accompanied by a significant prolongation of rat lifespan (according to Kaplan-Meier test) and even led to complete recovery without delayed relapses in 19.23% animals. The mechanism of tumor-suppressing effects of antibodies can be related to inhibition of specific functions of connexin 43 in glioma cells in the peritumoral zone.