Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

The Pathophysiological Aspects of Cerebral Diseases

Introduction. Cerebrovascular disorders are the main causes of heavy burden health worldwide, also, it is critical to understand the pathophysiological mechanism and then trying to prevent the neurological sequels. Objective. To discuss the inflammatory and oxidative stress aspects associated to the cerebrovascular diseases, focusing on biomarkers, also the role of omega oils, and the intracellular molecular network associated to the tissue burden on those conditions. Results. One of the most promising biomarkers it is Neuron-Specific Enolase (NSE). Serum NSE levels were elevated in stroke-patients compared to the non-stroke controls. Also, studies have demonstrated that in specific ratio omega oils 3, 6 and 9 can ameliorate the inflammatory and oxidative stress in nervous tissue and could be useful to the inflammatory and oxidative stress negative effects of cerebrovascular diseases. In addition, the study of the molecular mechanisms is essential to understand which molecules could be addressed in cascade of events preventing the permanent damage on the nervous tissue. Final considerations. The studies on cerebrovascular disorders must precisely identify the mechanisms and key molecules involved and improve the time of diagnostics and prognostics reducing the negative impacts of those conditions.

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals

The effect of a reduced deuterium (D) content in the incubation medium on the survival of cultured neurons in vitro and under glucose deprivation was studied. In addition, we studied the effect of a decrease in the deuterium content in the rat brain on oxidative processes in the nervous tissue, its antioxidant protection, and training of rats in the T-shaped maze test under hypoxic conditions. For experiments with cultures of neurons, 7–8-day cultures of cerebellar neurons were used. Determination of the rate of neuronal death in cultures was carried out using propidium iodide. Acute hypoxia with hypercapnia was simulated in rats by placing them in sealed vessels with a capacity of 1 L. The effect on oxidative processes in brain tissues was assessed by changes in the level of free radical oxidation and malondialdehyde. The effect on the antioxidant system of the brain was assessed by the activity of catalase. The study in the T-maze was carried out in accordance with the generally accepted methodology, the skill of alternating right-sided and left-sided loops on positive reinforcement was developed. This work has shown that a decrease in the deuterium content in the incubation medium to a level of −357‰ has a neuroprotective effect, increasing the survival rate of cultured neurons under glucose deprivation. When exposed to hypoxia, a preliminary decrease in the deuterium content in the rat brain to −261‰ prevents the development of oxidative stress in their nervous tissue and preserves the learning ability of animals in the T-shaped maze test at the level of the control group. A similar protective effect during the modification of the 2H/1H internal environment of the body by the consumption of DDW can potentially be used for the prevention of pathological conditions associated with the development of oxidative stress with damage to the central nervous system.

Benign ovarian teratoma in the dog with predominantly nervous tissue: A case report

Ovarian teratomas are rare neoplasms in female dogs, and they are characterised by the proliferation of tissues of embryonic origin. Most teratomas are benign, but a histological diagnosis is important for clinicians. The objective of this article is to describe a benign ovarian teratoma in a dog, which was found on the street and was appearing like pregnant. A veterinary inspection by palpation documented an enlarged abdomen with a mass of tough matter located on the right side in the abdominal-pelvic part. An ultrasound examination presumed neoplastic mass in region of ovary. A bilateral ovariohysterectomy was performed and the subsequent histological evaluation revealed a benign ovarian teratoma with a histochemically and immunohistochemically verified nervous tissue. After one year, no distant metastases were found and the dog was recognised as being clinically healthy without problems. On the basis of the ultrasonography diagnostics and histopathological analyses, we have demonstrated the occurrence of a benign ovarian teratoma in a dog.

Endoscopic confocal laser-microscopy for the intraoperative nerve recognition: is it feasible?

Surgeons lose most of their tactile tissue information during minimal invasive surgery and need an additional tool of intraoperative tissue recognition. Confocal laser microscopy (CLM) is a well-established method of tissue investigation. The objective of this study was to analyze the feasibility and diagnostic accuracy of CLM nervous tissue recognition. Images taken with an endoscopic CLM system of sympathetic ganglions, nerve fibers and pleural tissue were characterized in terms of specific signal-patterns ex-vivo. No fluorescent dye was used. Diagnostic accuracy of tissue classification was evaluated by newly trained observers (sensitivity, specificity, PPV, NPV and interobserver variability). Although CLM images showed low CLM image contrast, assessment of nerve tissue was feasible without any fluorescent dye. Sensitivity and specificity ranged between 0.73 and 0.9 and 0.55–1.0, respectively. PPVs were 0.71–1.0 and the NPV range was between 0.58 and 0.86. The overall interobserver variability was 0.36. The eCLM enables to evaluate nervous tissue and to distinguish between nerve fibers, ganglions and pleural tissue based on backscattered light. However, the low image contrast and the heterogeneity in correct tissue diagnosis and a fair interobserver variability indicate the limit of CLM imaging without any fluorescent dye.

Comparative Assessment of Silver Nanocomposites’ Biological Effects on the Natural and Synthetic Matrix

The aim of our investigation was to make a comparative assessment of the biological effects of silver nanoparticles encapsulated in a natural and synthetic polymer matrix. We carried out a comparative assessment of the biological effect of silver nanocomposites on natural (arabinogalactan) and synthetic (poly-1-vinyl-1,2,4-triazole) matrices. We used 144 three-month-old white outbred male rats, which were divided into six groups. Substances were administered orally for 9 days at a dose 500 μg/kg. Twelve rats from each group were withdrawn from the experiment immediately after nine days of exposure (early period), and the remaining 12 rats were withdrawn from the experiment 6 months after the end of the nine-day exposure (long-term period). We investigated the parietal–temporal area of the cerebral cortex using histological (morphological assessments of nervous tissue), electron microscopic (calculation of mitochondrial areas and assessment of the quality of the cell nucleus), and immunohistochemical methods (study of the expression of proteins regulating apoptosis bcl-2 and caspase 3). We found that the effect of the nanocomposite on the arabinogalactan matrix causes a disturbance in the nervous tissue structure, an increase in the area of mitochondria, a disturbance of the structure of nerve cells, and activation of the process of apoptosis.

A novel algorithm of the digital nervous tissue phantom creation based on 3D Voronoi diagram application

The essential part of mathematical modelling of nutrients convectional reaction-diffusion is creation of a digital phantom of considered biological object. This process becomes an especial problem which needs to be solved before numerical calculations of the concentration gradients will be done. There are two principal ways to get the solution in this case. The first approach is the reconstruction of a digital phantom on the base of the experimental data directly. The second one is the creation of a virtual object according to the experimental evidence and the known principals de novo. The main advantage of the created phantom is a high adaptability to modelling demands and a physical problem formulation. In the present study a new algorithm of a digital phantom creation has been established. The principles of the claimed procedures are demonstrated by the example of a nervous tissue. Initially, one needs to create N 3D objects according to Voronoi diagrams. Each object has 144 edges and 69 boundaries on average. Having chosen M rear objects, a long 3D structure mimicking neurons axons is created according to a loft procedure from the start boundaries to the end ones. Then, the set of Boolean operations has been applied to form continuous smooth objects. The remain (N-(M+s)) objects are combined into several whole bodies using the loft procedures between the closet neighbours. The final structure has a good conformity with a nervous tissue architecture. Furthermore, the obtained phantom is correct to the mesh application and further numerical calculations.

EMBRIOGENESIS OF NEURONAL ELENENTS (GLIOBLASTS AND GABAA RECEPTORS) IN THE HUMAN BRAIN NEUROIMMUNE SYSTEM UNDER PRENATAL ALCOHOL EXPOSURE

Exposure to alcohol causes imbalances in neuroimmune function and impaired brain development. Alcohol activates the innate immune signaling pathways in the brain. Neuroimmune molecules expressed and secreted by glial cells of the brain (microglia, oligodendroglia) alter the function of neurons and further stimulate the development of alcoholic behavior. Various signaling pathways and brain cells are involved in the transmission of neuroimmune signals. Glial cells are the main sources of immune mediators in the brain, which respond to and release immune signals in the central nervous system. The aim of this study was to study neuronal elements: morphometric parameters of glioblasts, synaptic structures and properties of synaptosomal GABAA-benzodiazepine receptors of the neuroimmune system in the embryogenesis of the human brain under perinatal exposure to alcohol. Changes in glioblasts in the brain tissue of human embryos and fetuses were revealed under conditions of chronic prenatal alcoholization with an increase in gestational age compared with control subgroups: a significant increase in the average number of glioblasts, the length of the perimeters of presynaptic terminal structures, postsynaptic density, presynaptic terminal regions were significantly less (p < 0.01) in the study group than in the control comparison group. Exposure to ethanol leads to a decrease in the affinity of GABAA-benzodiazepine receptors, which affects neuronal plasticity associated with the development and differentiation of progenitor cells (glioblasts and neuroblasts) during embryogenesis of the human brain and leads to suppression of GABAergic function in the brain. This causes a disruption in the interconnection of embryonic cells in the brain, leads to excessive apoptosis due to the activation of glial cells of the nervous tissue, disruption of neuroimmune function in the developing brain, changes in neuronal circuits, as well as a change in the balance of excitatory and inhibitory effects, which affects the functional activity in the central nervous system. Glial activation is a compensatory reaction caused by neuroplastic changes aimed at adapting the developing brain of the embryo and fetus under conditions of neurotoxicity and hypoxia under the influence of prenatal alcoholization of the maternal organism and the effect of ethanol on the fetus. The dynamics of changes in glial elements and receptor activity in the nervous tissue of human embryos and fetuses under conditions of prenatal exposure to alcohol indicates a more pronounced effect of alcohol on the earliest stages of human embryo development, which is of great practical importance in planning pregnancy and the inadmissibility of alcoholization of the mother in order to avoid negative consequences in offspring.

ROLE OF THE CELLULAR IMMUNITY IN THE PATHOGENESIS OF BRAIN CONTUSION

Traumatic brain injury (TBI) is one of the most common pathologies of the central nervous system in the world, and the use of structural neuroimaging methods – computed tomography (CT) and magnetic resonance imaging (MRI) – often doesn’t allow assessment of the severity of the brain injury that has occurred. This situation predetermines the need to search for new methods of differential diagnosis of the severity of TBI and predicting the risk of consequences.One of these promising areas is the study of the immune status, since traumatic brain injury is characterized by a high rate of complications.One of these promising areas is the study of the immune status in patients with TBI in the acute period. It is now known that in response to brain damage, a response from the immune system is triggered.The reactions from the immune system, which develop after brain injury and directed against its own antigens, in the early period of the disease are related to damage to the nervous tissue. However, according to the latest available data, they are subsequently able to stimulate the processes of repair and regeneration in the brain tissue. In the course of damage to the nervous tissue, in response to endogenous molecules formed during the destruction of cells and the extracellular matrix, the cells of the immune system are activated.Current evidence indicates that T-cells play a role in both the formation of secondary damage and repair mechanisms. They are able to protect neurons through the production of neurotrophic factors such as brain neurotrophic factor (BDNF), which stimulates the growth of neurons, the formation of synapses.Using multicolor cytometric analysis within the framework of this work, a study was carried out to determine the number of the main subpopulations of CD3+CD4+-lymphocytes. The relative number of Th17 (CXCR5- CXCR3- CCR6+CCR4- ) and Th17/Th22 (CXCR5- CXCR3- CCR6+CCR4+), Th1/Th17 (CXCR5- CXCR3+CCR6+CCR4- ) among total CD45RA-negative CD3+CD4+-cells population is significantly increased in comparison with the values in the control group, in turn, the Th1(CXCR5- CXCR3+CCR6- CCR4- ) subpopulations among total CD45RA-negative CD3+CD4+-cells are significantly decreased with the values in the control group. The results obtained so far make it possible to consider immune responses among the key links in the pathogenesis of brain contusion TBI. And, perhaps, a comprehensive immunological examination of the victims in the first day after the injury will determine the parameters that will help predict the nature of possible complications in patients with brain contusion.

PHYSIOLOGICAL ASPECTS OF ASTHIDHATU

Ayurveda is an ancient medical science which is related to health. Ayurveda described three basic physiological constitution of the body they are: Dosha, Dhatu and mala. Dhatu are structural unit of the body. Asthi is fifth dhatu among saptadhatu. In modern science asthidhatu relate with bones and cartilage. Bones store minerals such as calcium. Metabolic bone diseases are caused by minerals and vitamins deficiency. Asthidhatu is resultant of action of medoagni on medodhatu and is responsible for nourishing majjadhatu. It supports the basic structures, protect vital organs, nourishing the nervous tissue. Health refers to the moderate quantity of asthidhatu while dis- orders may develop when any vitiation occur. Just as asthi dhatu (Bones) contribute to health, diseases related to bones can disrupt the body. This paper attempts to understand concepts of Asthidhatu for maintain health and pre- vention from diseases related to the bone. Keywords: Health, Asthidhatu ksaya-Vruddhi, functions