Research areas:
1. Investigation of the molecular mechanisms of hibernation that ensure the physiological and biochemical stability of the body in hypothermia and hypoxia.
Hibernation is an adaptive strategy for animal survival in conditions of physiological stress: low temperatures, lack or absence of food and water. There are quite a large number of hibernating animals in the world, but the hibernation strategy of animals varies. All of them are united by the fact that during hibernation, as a rule, metabolism decreases. At the same time, body temperature drops, respiration and heart rate slow down, oxygen consumption decreases, cerebral circulation and organ perfusion decrease. During hibernation, all body tissues undergo significant changes, including the most numerous blood cells — erythrocytes. The changes occurring in them are associated with metabolic restructuring, the effects of oxidative stress, a large structural restructuring of membranes, and a change in the affinity of hemoglobin to oxygen. However, the animals have learned to cope with these difficulties. At this time, the body’s antioxidant defense system is activated, the rate of free radical processes is regulated, the metabolism of erythrocytes is rebuilt from glycolysis to the pentose phosphate pathway and vice versa, the lipid and protein composition changes, as well as the deformability of erythrocyte membranes. Therefore, a decrease in physiological functions does not lead to irreversible changes in the organs and tissues of the hibernators. After awakening from hibernation, the metabolism in the body quickly recovers and, despite prolonged immobility at low temperatures, as well as periodic awakening with activation of thermogenesis, the animal comes out of hibernation without any damage. We have shown that in the torpid state, the degree of generation of reactive oxygen and nitrogen species, the degree of oxidative modification of membrane lipids and erythrocyte proteins is at a low level compared to the summer control. At the same time, the activity of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes significantly decreases, but not the level of glutathione (GSH). In the torpid state, SOD is activated by exogenous GSH depending on the concentration, which indicates reversible inhibition of the enzyme. During the awakening of ground squirrels, when the body temperature reaches 25 ° C, red blood cells are subjected to oxidative stress. This is confirmed by a maximum increase in the level of uric acid in plasma, a marker of oxidative modification of lipids (TBARS) and proteins (carbonyl groups) in erythrocyte membranes, as well as a decrease in the level of GSH in erythrocytes relative to the torpid state and the activity of SOD and CAT. After full recovery of body temperature, the GSH level increases, the SOD/CAT ratio is restored, which significantly reduces the degree of oxidative damage to lipids and proteins of erythrocyte membranes. Thus, the oxidative stress detected at a temperature of 25 ° C was transient and physiologically regulated.
The low intensity of free radical processes in torpor may be associated not only with the presence of a highly effective antioxidant system, regulation of the affinity of hemoglobin to oxygen, but also with the adaptive restructuring of cell membranes. Membrane lipids (phospholipids, fatty acids and cholesterol) are involved in the temperature adaptation of ectotherms by changing the viscosity of membranes, which is determined by the degree of unsaturation of fatty acids, the ratio of cholesterol/phospholipids and phospholipid composition. Fatty acids and cholesterol can determine the parameters of hibernation and play an important role in the activity of lipid metabolism enzymes. The participation of phospholipids, cholesterol and polyunsaturated fatty acids in seasonal changes in the membrane structure has also been established. Fatty acids are considered the main energy substrate during the hibernation of mammals. The fatty acid composition of phospholipids in the adaptation of erythrocytes of the small ground squirrel (Spermophilus pygmaeus) at different stages of hibernation was studied. It was found that a significant restructuring concerns the fatty acid composition of all studied phospholipids: phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. In phosphatidylcholine, changes affect saturated, monoenic and polyenic acids, in phosphatidylethanolamine – saturated and polyunsaturated acids, and in phosphatidylserine – only polyunsaturated acids. We have shown for the first time that, despite large changes in the composition of fatty acids in erythrocyte membranes, the unsaturation index remains constant (Chebotareva MA, Shukolyukova EP, Nikitina EP).
2. Investigation of changes in the rheology of gopher blood in the non-hibernation period and during hibernation. The study of rheological disorders associated with various types of diseases (diabetes, metabolic syndrome, ischemia, oncology) in vitro and in vivo
2.1. The annual cycle of small ground squirrels (Spermophilus pygmaeus) consists of 2 periods: wakefulness (spring-autumn) and hibernation (winter). During the active period, ground squirrels reproduce (spring), accumulate fat reserves (summer) and prepare for hibernation (autumn). We assumed that the rheological properties of blood and the deformability of erythrocytes may change in different seasons of the year of the non-hibernation period in order to adequately provide tissues with oxygen. The purpose of our research was to identify possible adaptive changes in the deformability of erythrocytes and hematological parameters of ground squirrels during the active period. The deformability
of erythrocytes was studied by ectacytometry in an osmotic gradient. It was found that in the spring, after the awakening of ground squirrels, erythrocytes have the highest
deformability (El_max), hydration (O_hyper), water permeability (El_min) and osmotic stability (ΔO). In summer, compared with spring, the deformability of red blood cells decreases, and the average volume of red blood cells (MCV) decreases. In autumn,
before hibernation, the integral deformability of erythrocytes and their hydration increase, and the range of osmotic stability of erythrocytes relative to the summer period also increases. In summer and autumn, unlike in spring, the average
concentration of hemoglobin in the erythrocyte (MCHC) increases. At low
-shear voltage (1 Pa) in summer and autumn, the osmoscan has a pronounced polymodal
a shape indicating a change in the viscoelastic properties of the membrane of gopher erythrocytes. Thus, for the first time, we discovered seasonal variability in the deformability of gopher erythrocytes, which corresponds to the spring-summer activity of animals and preparation for hibernation.
Our further research was related to the study of the temperature dependence of the deformability of red blood cells of ground squirrels in the dynamics of hibernation. It was maximum in the torpid state (at the body temperature of the animal – Tb 5oC). This is probably due to the fact that at low body temperature, the blood flow rate decreases sharply, blood viscosity increases and in order for red blood cells to penetrate through narrow capillaries, they must have the ability to deform as much as possible. When the animal’s body was heated to 15 ° C, processes associated with oxidative stress began to develop at a high rate, causing lipid peroxidation and contributing to damage to membrane proteins and lipids, which cannot but affect the deformability of red blood cells. We have shown that at Tb 15 °C, the deformability of membranes is significantly reduced compared to torpor. Later, in the process of emerging from hibernation (at a body temperature of 25 ° C), deformability continued to decrease, and at 37 ° C (normothermal state), deformability was restored and returned to control values (Katyukhin L.N., Nikitina E.R., 2023). In general, the data obtained indicate adaptive changes occurring in the membranes of erythrocytes, allowing them to function under conditions of significant temperature changes and hypoxia. Further understanding of the mechanisms underlying such resistance of hibernators to oxidative stress may be very useful for developing a strategy to combat human diseases associated with the control of oxidative stress and protection from ischemic reperfusion injury.
2.2. The deformability of erythrocytes plays an important role in their transport through blood vessels and capillaries, the delivery of nutrients and oxygen to various organs and tissues. As is known, the main factors determining the deformability of erythrocytes are the shape of cells (the ratio of area to volume), as well as internal viscosity (determined by the concentration of cellular hemoglobin). The degree of compressibility of the lipid bilayer and its relationship with the protein structures of the cell membrane also affect its deformability. Since erythrocytes are the most glucose-consuming cells, with prolonged hyperglycemia in diabetes mellitus, the morphology and function of erythrocytes undergo significant changes, which further affects hemorheology and microcirculation. It has been shown that in streptozotocin-induced type 1 diabetes mellitus in rats, the deformation properties of erythrocytes decrease. The use of amitriptyline (a drug used for neuropathic pain in diabetes mellitus) increased the deformability of red blood cells to control values. Serious violations of blood rheology are a distinctive feature of the pathophysiology of many diseases, including: sickle cell anemia, various types of diabetes, ischemic injuries, cardiovascular diseases, oncology, as well as one of the key factors predisposing patients to pain crises, organ damage and early mortality. Therefore, the ultimate goal of any treatment should be to eliminate hemorheological disorders caused by diseases and normalize blood flow in order to prevent clinical complications.
