Morphological Signs of Dystrophy, Regeneration and Hypertrophy of Neurons

Results: Dystrophic changes constitute an extensive group of neuronal disorders and are manifested at the morphological level by deformation of the perikarions and neuropil, wrinkling or swelling of the cell, and changes in the chromatophilia of the cytoplasm. At the electron microscopic level, disorganization of organelles is observed, reflecting gross violations of the vital processes of the neuron. There are several ways to regenerate neurons: intracellular regeneration, restoration of the neuropil, the formation of new neurons (in some parts of the nervous system - the hippocampus, the subventricular layer of the lateral ventricles and olfactory bulbs) and the formation of heterokaryons (fusion of a neuron with an oligodendrocyte). Hypertrophy of neurons may indicate both compensation and the development of a pathological process. To clarify the nature of this phenomenon, it is necessary to conduct an ultramicroscopic study of the organelles of the nerve cell.

Ultrastructural changes in the microvessels and perivascular space in cerebral infarction in the experiment

Background. Ischemic stroke is the second most common cause of death after coronary heart disease and the most common cause of disability worldwide. Much of the recent basic research on stroke is concerned with the mechanisms underlying the dysfunction and adaptation of the neurovascular block, which includes the blood-brain barrier structures, microglia, neurons, and the extracellular matrix of the basement membrane. Isolated studies of recent years have been devoted to the issues of morphology and in particular the ultrastructure of the brain in ischemic injury. Meanwhile, only morphological studies can reveal the peculiarities of the response of cellular structures to the influence of various adverse factors. Objective – to investigate ultrastructural changes in the vessels of the brain and perivascular space in experimental ischemic heart attack. Methods. Experimental cerebral infarction was reproduced on 15 white Wistar rats by injection of a suspension of barium sulfate in sterile saline in a ratio of 1: 3 in the amount of 0.1 -0.3 ml. Three animals formed a control group. The material was collected in terms of: up to 3, 9, 12 days and more than 12 days from the beginning of the experimental action, followed by standard processing of the material for electron microscopy. Results. In the early stages of ischemic brain damage perivascular edema, destructive changes of capillaries with destruction of basement membranes are registered. Some microvessels undergo irreversible changes with deformation of the vascular lumen, pyknosis and lysis of endothelial nuclei, destruction and vacuolation of cytoplasmic structures, microvacuolation and edema of mitochondria with partial destruction of cristae and enlightenment of the mitochondrial matrix. In the endothelium with signs of coagulation processes in the cytoplasm and nucleus, changes in cell contacts were observed. Structural changes of vessels are combined with changes of perivascular processes of astrocytes. On days 9 and 12, the structure of the endothelium, perivascular astrocytes, and intercellular contacts are restored. Hyperplasia of intracytoplasmic structures, increase in mitochondria and length of cytoplasmic network are noted. In the cells of the perivascular environment and in the cytoplasm of pericytes a significant number of phagolysosomes is detected, in the long term in the perifocal areas of irreversible ischemic changes around the vessels is reparative astrogliosis. Conclusion. Ultrastructural changes of the microcirculatory part in the perifocal areas of ischemic lesions within 3 days are characterized by perivascular edema and destructive changes in the endothelium of capillaries and pericytes, damage to basement membranes, changes in cell contacts. After 9-12 days in the endothelium, the processes of intracellular regeneration increase, the ultrastructure of intercellular contacts is restored. A significant number of phagolysosomes is registered in the cells of the perivascular environment and in the cytoplasm of pericytes, and reparative astrogliosis is detected in the perifocal areas of irreversible ischemic changes around the vessels.

Prevention of Postradiational Disorders in the Testes of Rats with the use of Magnetic Field

The action effects of the low-intensity low-frequency magnetic field (LMF) are mainly studied in its therapeutic use and to a much lesser extent in the mode of primary prevention. Aim. To identify adaptive metabolic and ultrastructural changes in the testes of rats under the preventive action of LMF under radiation conditions. Material and methods. The experiments were carried out on 28 mature non-linear male rats weighing 180-200 g. the animals were divided into 3 groups: in the experimental group, the animals received a course of LMF procedures followed by radiation exposure; in the control group, the animals were subjected only to radiation exposure; in the intact group, the animals were not exposed to any effects. The animals were slaughtered the day after the radiation exposure. the research methods were used: biochemical (to determine the content of RNA, DNA, antioxidant activity of the testes), light – optical (to count the number of convoluted seminiferous tubule – CST, exfoliated cells, spermatogonia, to determine the index of spermatogenesis); transmission electron microscopy; morphometric analysis of mitochondria (number, average and total area). Results. The preventive effect of LMF enhanced the adaptive capabilities of the body and increased the resistance of the testes to the effects of radiation. This was manifested in an increase in the power of the antioxidant system, activation oof the cellular and intracellular regeneration processes, and a decrease in the permeability of the structures of the CST’s own shell. Conclusion. The obtained data substantiate the possibility of using LMF as a means of protecting the organs of the reproductive system in the complex prevention of the body under the influence of radiation.

Enzymology of extracellular NAD metabolism

Extracellular NAD represents a key signaling molecule in different physiological and pathological conditions. It exerts such function both directly, through the activation of specific purinergic receptors, or indirectly, serving as substrate of ectoenzymes, such as CD73, nucleotide pyrophosphatase/phosphodiesterase 1, CD38 and its paralog CD157, and ecto ADP ribosyltransferases. By hydrolyzing NAD, these enzymes dictate extracellular NAD availability, thus regulating its direct signaling role. In addition, they can generate from NAD smaller signaling molecules, like the immunomodulator adenosine, or they can use NAD to ADP-ribosylate various extracellular proteins and membrane receptors, with significant impact on the control of immunity, inflammatory response, tumorigenesis, and other diseases. Besides, they release from NAD several pyridine metabolites that can be taken up by the cell for the intracellular regeneration of NAD itself. The extracellular environment also hosts nicotinamide phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase, which inside the cell catalyze key reactions in NAD salvaging pathways. The extracellular forms of these enzymes behave as cytokines, with pro-inflammatory functions. This review summarizes the current knowledge on the extracellular NAD metabolome and describes the major biochemical properties of the enzymes involved in extracellular NAD metabolism, focusing on the contribution of their catalytic activities to the biological function. By uncovering the controversies and gaps in their characterization, further research directions are suggested, also to better exploit the great potential of these enzymes as therapeutic targets in various human diseases.

Biogenic Ferrihydrite Nanoparticles: Synthesis, Properties In Vitro and In Vivo Testing and the Concentration Effect

Biogenic ferrihydrite nanoparticles were synthesized as a result of the cultivation of Klebsiella oxytoca microorganisms. The distribution of nanoparticles in the body of laboratory animals and the physical properties of the nanoparticles were studied. The synthesized ferrihydrite nanoparticles are superparamagnetic at room temperature, and the characteristic blocking temperature is 23–25 K. The uncompensated moment of ferrihydrite particles was determined to be approximately 200 Bohr magnetons. In vitro testing of different concentrations of ferrihydrite nanoparticles for the functional activity of neutrophilic granulocytes by the chemiluminescence method showed an increase in the release of primary oxygen radicals by blood phagocytes when exposed to a minimum concentration and a decrease in secondary radicals when exposed to a maximum concentration. In vivo testing of ferrihydrite nanoparticles on Wister rats showed that a suspension of ferrihydrite nanoparticles has chronic toxicity, since it causes morphological changes in organs, mainly in the spleen, which are characterized by the accumulation of hemosiderin nanoparticles (stained blue according to Perls). Ferrihydrite can also directly or indirectly stimulate the proliferation and intracellular regeneration of hepatocytes. The partial detection of Perls-positive cells in the liver and kidneys can be explained by the rapid elimination from organs and the high dispersion of the nanomaterial. Thus, it is necessary to carry out studies of these processes at the systemic level, since the introduction of nanoparticles into the body is characterized by adaptive-proliferative processes, accompanied by the development of cell dystrophy and tension of the phagocytic system.

Effectiveness of a combined agent with hemostatic and anti-adhesive activity in liver surgery in an experiment

The aim of this study was to develop a hemostatic agent with anti-adhesive properties and to study its effect on liver morphology, metabolic activity and hepatocyte regeneration in experimental liver injury. Methods. In 60 rats following experimental resection liver injury, the time of bleeding and the volume of blood loss were determined. Histological preparations were used to study the size of hepatocytes and their nuclei, the content of glycogen (PAS-reaction), the number of binucleated hepatocytes and the expression of Ki-67. Results. Compared with the control, an agent based on 6% sodium carboxymethylcellulose gel and 5% aminocaproic acid effectively and reliably reduces the bleeding time by 72% (217.91 s), the volume of blood loss by 74.7% (372.85 mg) (p ≤ 0.01) and the degree of blood filling of the sinusoid liver capillaries. In addition, the use of the novel gel prevents the adhesion formation. It stimulates mitotic activity of hepatocytes, accompanied by an increase in the number of binucleated hepatocytes and Ki-67 expression. By the 14th day, this activity significantly decreases. Hypertrophy of hepatocytes and their nuclei is observed by the 7th and 14th days of the experiment. This indicates both an increase in the metabolic activity of hepatocytes and intracellular regeneration. The use of the hemostatic gel does not alter the glycogen-storing function of hepatocytes, which indicates the lack of pronounced hypoxia due to effective control of bleeding. Conclusion. The local hemostatic gel based on 6% sodium carboxymethylcellulose gel and 5% aminocaproic acid can be recommended for local bleeding control in liver injuries and surgery.

Correction of the the liver morpho-functional state with stem cells in acute hepatitis

Цель работы - изучение влияния сочетанной трансплантации мультипотентных мезенхимальных стромальных и гемопоэтических стволовых клеток на регенерацию печени в физиологических условиях и в условиях токсического гепатита. Методика. Эксперименты выполнены на 84 белых лабораторных мышах-самцах, возраст 7-8 мес. Токсический гепатит вызывали внутрибрюшинным введением четыреххлористого углерода (CCl4 в дозе 50 мкг/кг. Животным опытной группы в хвостовую вену вводили мультипотентные мезенхимальные стромальные (ММСК, 4 млн клеток/кг) и гемопоэтические стволовые клетки (ГСК, 330 тыс. клеток/кг). Для инъекции полученные клетки суспендировали в 0,2 мл 0,9 % раствора NaCl. Животным контрольной группы внутривенно вводили 0,2 мл 0,9 % раствора NaCl. Внутривенные введения осуществляли через 1 ч после введения четыреххлористого углерода однократно. Для трансплантации использовали ММСК третьего пассажа, а ГСК не подвергались культивированию. Изучали влияние сочетанной трансплантации ММСК и ГСК на биохимические показатели периферической крови и морфометрические показатели печени в физиологических условиях и после введения CCl4 на 1-е, 3-и, 7-е сут. Результаты. Показано, что проведение сочетанной трансплантации мультипотентных мезенхимальных стромальных и гемопоэтических стволовых клеток при токсическом гепатите приводило к снижению активности ферментов цитолиза, активации белоксинтезирующей функции печени. Также отмечалось увеличение количества гепатоцитов, повышение митотической активности, что указывает на активацию внутриклеточной регенерации. Обнаруженное увеличение числа двуядерных гепатоцитов, размеров ядра, ядерно-цитоплазматического соотношения говорит об усилении процесса внутриклеточной регенерации в печени. The aim of the work was studying the effect of combined transplantation of multipotent mesenchymal stromal and hematopoietic stem cells on regeneration of the liver under the physiological conditions and in toxic hepatitis. Methods. Experiments were performed on 84 white laboratory male mice aged 7-8 months. Toxic hepatitis was induced by administration of carbon tetrachloride (CCl4) at a dose of 50 µg/kg, i.p. Mice were divided into experimental and control groups. The experimental group received caudal vein injections of multipotent mesenchymal stromal (MMSC) and hematopoietic stem cells (HSC) derived from the placenta chorion of female mice at respective doses of 4M cells/kg and 330K cells/kg suspended in 0.2 ml of 0.9% NaCl. Control animals were given 0.2 ml of 0.9 % NaCl, i.v. Intravenous injections were performed once 1 hour following the administration of carbon tetrachloride. MMSCs of the third passage were used for transplantation, while transplanted HSCs were not cultured. Effects of the combined MMSC and HSC transplantation on blood biochemistry and liver morphometry were studied under the physiological conditions and at 1, 3, and 7 days after administration of CCl4. Results. In toxic hepatitis, the combined transplantation of multipotent mesenchymal stromal and hematopoietic stem cells resulted in decreased activity of cytolytic enzymes, activation of hepatic protein synthesis, increased number of hepatocytes, and increased mitotic activity indicative of activation of intracellular regeneration. Increases in the number of binuclear hepatocytes, nucleus size, and the nuclear-cytoplasmic ratio suggested an enhancement of intracellular regeneration in the liver.

THE EFFECT OF MULTIPOTENT MESENCHYMAL STROMAL CELLS TRANSPLANTATION ON LIVER MORPHOMETRIC PARAMETERS OF MATURE AND OLD LABORATORY ANIMALS WITH TOXIC HEPATITIS

Цель - изучение влияния трансплантации мультипотентных мезенхимальных стромальных клеток (ММСК) на морфометрические показатели печени зрелых и старых лабораторных животных в условиях токсического гепатита. Материал и методы. Эксперименты выполнены на зрелых и старых мышах-самцах. Токсический гепатит вызывали путем внутрибрюшинного введения CClв дозе 50 мкг/кг. Трансплантация клеток осуществлялась в хвостовую вену через 1 ч после введения четыреххлористого углерода однократно. Исследовалось влияние ММСК на морфометрические показатели печени в физиологических условиях и условиях токсического гепатита на 1-, 3-, 7-е сутки после трансплантации клеток. Результаты. У зрелых лабораторных животных на 3-и сутки после введения ММСК на фоне токсического гепатита обнаружено увеличение митотической активности, повышение количества гепатоцитов, площади ядра гепатоцитов и ядерноцитоплазматического индекса. В то же время, у старых лабораторных животных выявлено лишь увеличение площади ядра гепатоцитов и ядерно-цитоплазматического индекса. На 7-е сутки после введения ММСК на фоне токсического гепатита в обеих возрастных группах выявлены активация митотической активности, повышение количества гепатоцитов, увеличение площади ядра гепатоцитов и ядерно-цитоплазматического индекса. Выводы. Изменение морфометрических показателей печени у зрелых и старых лабораторных животных реализуется через механизмы как клеточной, так и внутриклеточной регенерации. При этом у старых лабораторных животных на 3-и сутки после введения ММСК выявлена активация лишь внутриклеточной регенерации, в то время как у зрелых лабораторных животных имеет место повышение клеточной и внутриклеточной регенерации гепатоцитов. В более поздние сроки в обеих изучаемых возрастных группах изменение основных морфометрических показателей печени реализуется через активацию как клеточной, так и внутриклеточной регенерации. Objective - to study the influence of multipotent mesenchymal stromal cells (MMSC) transplantation on morphometric parameters of the liver of mature and old laboratory animals with toxic hepatitis. Material and methods. The experiments were performed on mature and old male mice. Toxic hepatitis was caused by intraperitoneal administration of CCl4 at a dose of 50 μg/kg. The cells were transplanted via the tail vein 1 hour after administration of a single dose of carbon tetrachloride. The effect of MMSC on liver morphometric parameters in physiological conditions and after toxic hepatitis development was studied on days 1, 3, 7 after cell transplantation. Results. An increase in mitotic activity, an increase in the number of hepatocytes, hepatocyte nucleus area, and nuclear cytoplasmic index were found in mature laboratory animals with toxic hepatitis on the 3 day after the introduction of MMSC. At the same time, only an increase in the area of hepatocyte nucleus and nuclear cytoplasmic index was revealed in old laboratory animals. On the 7 day after the introduction of MMSC to the animals with toxic hepatitis, both age groups demonstrated activation of mitotic activity, an increase in the number of hepatocytes, an increase in the area of hepatocyte nucleus and nuclear cytoplasmic index. Conclusions. Changes in liver morphometric parameters in mature and old laboratory animals are realized through mechanisms of both cellular and intracellular regeneration. In addition, the activation of only intracellular regeneration was found in old laboratory animals on the 3rd day after the introduction of MMSC, while in mature laboratory animals there was an increase in cellular and intracellular regeneration of hepatocytes. In later periods in both studied age groups, the change in the main liver morphometric parameters is realized through the activation of both cellular and intracellular regeneration.

Possibility of using combined transplantation of multipotent mesenchymal stromal cells and hepatic stellate cells to activate reparative liver regeneration in mature and old organism

The aim of the study was to examine the effect of combined transplantation of multipotent mesenchymal stromal (MMSC) and hepatic stellate (HSC) cells on liver regeneration after resection. Research has been carried out on laboratory animals of mature and old age. After the subtotal resection of the liver, MMSC and HSC were introduced in the tail vein in the amount of 4 million cl/kg of body weight and 9 million cl/kg of body weight respectively. Evaluation of reparative liver regeneration was performed on the 1st, 3rd, 7th days after subtotal liver resection. Features of reparative liver regeneration in mature and old organism were revealed. In mature organism against the background of combined cell transplantation, regeneration activation is achieved by increasing cellular and intracellular regeneration mechanisms. In this case, the old organism responds to cell transplantation by activating only intracellular mechanisms. In both age groups, decreased mutagenesis and inhibition of programmed cell death against the background of MMSC cotransplantation and HSC were observed.