Hematological Characteristics and Erythrokinetic Indiсes in Little Ground Squirrels During Arousal from Hibernation

During hibernation, the small mammals pass through multiple cold (torpor) and warm (arousal) phases, as well as adaptive changes in red blood cells are vital for an adequate oxygen supply to tissues. Here, we have analyzed the structural and functional indices of red blood cells in little ground squirrels during arousal from hibernation. In a torpid state, a count of red blood cells, HGB and MCH levels in blood were established as not significantly changed, but MCV and HCT decreased, while MCHC and RDW increased as compared to the control. In hibernating animals, the reticulocyte count in blood decreased and the period of erythrocyte elimination in bloodstream was shortened, but the ability to produce the red blood cells remained at the control level. During arousal, the body temperature of 25-30°C was critical for rearrangement of structural and functional parameters of red blood cells. This was evidenced by RDW increase and the tendency to rise in HCT, MCV; high reticulocyte count in blood and extended time of their maturation; increased half-life of red blood cells. When the body temperature was fully normalized, the production of red blood cells was significantly increased due to the reduced reticulocyte maturation time.
 
Probl Cryobiol Cryomed 2020; 30(2): 132–147

Under low ambient temperatures and shortages of food resources, some mammals enter a torpid state, which is called hibernation. Hibernation is an example of evolutionary adaptation of mammals to extreme living conditions [17]. It is characterized by a controlled decrease in metabolic rate, body temperature and other physiological functions (heart rate, lung ventilation), as well as peripheral vasoconstriction [6,11,16]. Herewith, in hibernators, the function of organs and systems and biochemical processes are rearranged so that, even at a very low temperature of a body, it maintains a certain homeostasis [6,36].
The red blood cells (RBCs) play a key role in oxygen transport and supply to tissues. The RBCs are known to have the metabolic signaling cascades, promoting the oxygen delivery and tissue oxygenation as a result of systemic hypoxemia. The regulation of hemoglobin affi nity for oxygen can make an essential contribution into preventing oxidative stress during hibernation. The hemoglobin affi nity for oxygen in hibernating mammals was shown to increase, that reduced its release into tissues in accordance with their low metabolic activity [32]. It should be noted that the oxygen content in tissues will be determined not only by hemoglobin affi nity for it, but its content in RBCs as well, and the size of the latter, rheological properties of blood, and etc.
The fi ndings on the main hematological variables of diff erent animals during hibernation are controversial. For example, the study of seasonal changes in RBC count in blood of little ground squirrel (Spermophilus pygmaeus) showed its increase in winter months, when the animals were in a torpid state [28]. An increased counts of RBCs, hemoglobin and hematocrit were found out in the northern birch mouse (Sicista betulina) during hibernation [41]. In the hedgehogs (Erinaceus europaeus) within the initial months of hibernation [37] and in hibernating thirteen-lined ground squirrels (Spermophilus tridecemlineatus) [35] the hematological variables were reduced as compared with the summer control. At the same time S.T. Cooper et al. [8] have revealed no significant changes in RBC count, hemoglobin and hematocrit levels in S. tridecemlineatus in a torpid state and after interbout arousal as compared with the summer control.
Для моделювання гібернації та пробудження тварин випадковим чином розподілили на 7 груп (n = 6 у кожній): контроль у літній період; стан глибокої гібернації; різні етапи індукованого пробудження (тварини з температурою тіла 10, It is known that during hibernation, the cell division in hematopoietic tissue is signifi cantly reduced [4], and the lifespan of circulating RBCs is greatly increased [5]. Also, it has been found that in a torpid state, the average RBC volume in S. tridecemlineatus decreases, but in the period of interbout arousal it increases [8]. Due to the fact that during circulation in bloodstream the RBC size usually diminishes because of a loss of membrane part, so an increase in RBC size during interbout arousal is associated with the erythropoiesis activation and the entry in blood of larger reticulocytes than those in circulating RBCs. However, the results of a direct determination of reticulocyte count in blood of long-tailed ground squirrels (Spermophilus undulatus) in a torpid state showed a signifi cant decrease in their number as compared with summer control, and after stimulated arousal this index did not increase as compared with a torpid state of animals [13].
Thus, to date, a change in hematological parameters and physiological characteristics of erythrocytes has been investigated in animals in a torpid state and during interbout awakening, but the features of such changes during their arousal have remained unstudied.
The research aim was to investigate the hematological variables, RBC and erythrokinetic indices in ground squirrels during hibernation and within the induced arousal.

Materials and methods
The research was carried-out in little ground squirrels (S. pygmaeus Pall.), weighting 250-300g. The animals were caught in Buynaksk district of the Republic of Dagestan (42° 55'S, 47° 20'W; 320 m above sea level). The experiments were implemented in compliance with all the standards and requirements approved by the Directive 2010/ 63/EU of the European Parliament and of the Council 'On the Protection of Animals Used for Scientifi c Purposes'.
Визначення цитокінетичних показників еритроцитарного балансу крові. Для забарвлювання ретикулоцитів 0,3 мл крові змішували з 0,1 мл 1%-го розчину брильянтового крезилового синього, приготованого на 0,9%-му розчині NaCl («Арбис + », Росія) і витримували 40 хв при кім-a temperature of 2-5°C. After a few days, the animals went into hibernation with a decrease in temperature of body (Tb) down to (3.8 ± 0.4)°C. After two months of hibernation, the average duration of bout was (14 ± 0.5) days. For experiments, the animals were selected in a torpid state in the middle of the bout (7 days from the beginning of re-hibernation). To initiate the arousal, the ground squirrels were moved to a room with a temperature of 20°C. A complete arousal of animals and Tb rise up to 37°C occurred within (2.4 ± 0.2) hrs approximately. After reaching Tb of 10, 20, 25, 30 and 37°C, the animals were used in the experiment. Body temperature was measured with a digital thermometer MS 6501 (Mastech, Hong Kong), the sensor was inserted into the rectum to a 3-4 cm depth.
Study of hematological variables. Blood samples were collected from ground squirrels during arousal after Tb rising up to 20°C and higher, as well as from the control group animals after decapitation of anesthetized animals by intraperitoneal administration of sodium pentabarbital (85 mg / kg body weight). In the ground squirrels in a torpid state, and those with 10ºC Tb after arousal onset, blood was sampled without anesthesia. Blood samples were collected into special vacutainer blood collection tubes with anticoagulant K2-EDTA (Guangzhou Improve Medical Instruments, China). The count of red blood cells (RBCs), hemoglobin concentration (HGB), hematocrit (HCT), as well as the RBC indices such as: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) were measured with hematology analyzer Sysmex KX-21 (Sysmex Corporation, Japan).
Determination of cytokinetic indices of RBC balance of blood. To stain reticulocytes, 0.3 ml of blood was mixed with 0.1 ml of 1% diamond cresil blue prepared with 0.9% NaCl solution (Arbis + , Russia) and kept for 40 min at room temperature. A part of blood with anticoagulant was kept for 4 hrs in a thermostat at 37°C, then the samples were stained as described above. The smears were prepared from all the samples and then fi xed in methanol. They were air-dried and examined with immersion microscope Micromed-5 (×100 objective lens, ×10 ocular lens) (LOMO, Russia). Reticulocytes were counted per 1,000 red blood cells.
Величину добової продукції еритроцитів на 1 мкл крові визначали за рівнянням: Період напіввиведення еритроцитів оцінювали за формулою: and outfl ow of the same number of RBCs per time unit. We have assumed that an equilibrated balance of RBCs in blood of animals is maintained both in a torpid state and during arousal from hibernation as well. Cytokinetic indices of RBC balance of blood were counted by the formulas proposed by A.V. Ilyukhin et al. [15,21]. The half-life of reticulocytes (T 1/2r ) in a sample, the value of red blood cell production per day (P RBC / day ) and the half-life of red blood cells in blood circulation (T 1/2RBC ) were calculated by RBC and reticulocyte counts prior to and after 4 hrs of incubation. The reticulocytes' half-life, stipulated by their maturation was calculated by the formula: where t is incubation time; N r0 is reticulocyte number in the sample prior to incubation,%; N rt is reticulocyte number in the sample after incubation,%.
The value of daily RBC production per 1 μl of blood (PRBC / day) was found with the equation: where T 1/2RBC is bloodstream RBC half-life (days).
The RBC half-life was estimated by the formula: Our fi ndings were statistically processed using one-way analysis of variance (ANOVA) and Pearson's correlation coeffi cient using Statistica 8.0. software (StatSoft, USA). Signifi cance of diff erences was determined by Fisher's test at the level of p ≤ 0.05. The data in the Table and diagrams are given as the mean ± mean error.
It should be noted that the hemoglobin level in S. pygmaeus blood both in a torpid state and during warming, did not considerably change with respect to the control (Fig. 1B). After 2 months of hibernation in Daurian ground squirrels (Spermophilus dauricus), the hemoglobin level in blood remained unchanged as well [14]. At the same time, the hematocrit in S. pygmaeus signifi cantly decreased in a torpid state (Fig. 1C). A similar phenomenon was also found in bats and small marsupials [10]. Of interest is to note that in large hibernating mammals, the brown bear (Ursus Примітки: відмінності значущі порівняно з контролем (*) та сплячкою ( # ), р ≤ 0,05. Notes: diff erences are signifi cant if compared with the control (*) and hibernation ( # ), р ≤ 0.05.
It should be noted that an increase in MCV and HCT, revealed after reaching Tb of 25°C ( Fig.  1C and D), may have a biological meaning. It was found out that a moderate rise in blood viscosity promoted the tissue perfusion, the augmented functional capillary density in them and reduced peripheral vascular resistance [34], which in turn entailed an increased shear stress on vessel wall, endothelial activation with subsequent release of vasodilator molecules: nitric oxide (NO) [23]. In tissues of liver, lungs and heart of Asia Minor ground squirrels (Spermophilus xanthoprymnus) the level of NO stable metabolites after arousal was shown to be higher than in the animals during hibernation [3]. Electronimmunocytochemistry demonstrated that in endothelial cells of renal and mesenteric arteries of golden hamsters (Mesocricetus auratus) in a torpid state, the number of cells containing nitric oxide synthase (NO-synthase) was much lower than in the control, and at a late stage of animal arousal, when Tb increased from 9 up to 32°C within 2 hrs, the number of these cells did not virtually diff er from the control [33]. The expression of NO-synthase in endothelial cells during animal arousal, the authors also associated with an increased shear stress on the vessel wall.
What are the reasons for decrease in RBC volume during hibernation? S.T. Cooper et al. [8] showed a MCV decrease of in in hibernators at a torpid state and suggested it to be associated with the RBC loss of a part of damaged membrane due to a prolonged circulation in bloodstream. It is possible that during circulation in the hypobiosis-arousal cycle a part of RBC membrane undergoes changes due to oxidative modifi cation of lipids and proteins, band 3 protein clustering, externalization of phosphotidylserine to outer monolayer, binding to autologous antibodies because of aging or erythroptosis [30], with subsequent RBC elimination by cells of reticuloendothelial system. The removal of a membrane part by vesiculation allows preserving more than 80% of RBC membrane, preventing thereby their pre-mature elimination from bloodstream [40].
Показано, що у S. pygmaeus у торпідному стані RDW збільшується на 9,7% (р < 0,001) по in size. The initial stages of animal rewarming contribute to further RDW increase, i. e. at Tb of 25°C it reaches the maximum values (18.01 ± 1.10)%, that is 29.8% higher than in animals of the control group (p < 0.001), and by 18.3% higher than in those in a torpid state (p < 0.001). Of note is the fact, that after complete animal rewarming, this index is not normalized.
in RBCs decreases, reducing thereby the oxygen delivery to tissues. During arousal of hamsters, there was noted an increase in 2,3-DPG level in RBCs as compared with a torpid state [38]. So, during arousal from hibernation, an increase in Tb and 2,3-DPG concentration can reduce the hemoglobin affi nity for oxygen and promote rapid tissue oxygenation.
Thus, during the hibernation-rewarming cycle in blood of ground squirrels there is a signifi cant variation in the volume, hemoglobin saturation and heterogeneity of RBC population, while there is no strong alteration in their number. Altogether, this testifi es to the changes in erythron system of ground squirrels, being in a torpid state and at diff erent stages of arousal.
The equilibrium in the erythron system is stipulated by two factors, i. e. natural death of RBCs and their production by bone marrow. The technique of counting the bone marrow production of RBCs proposed by A.V. Ilyukhin et al. [15], consisting in examining the reticulocyte count prior to and after incubation of blood sample for a certain time period, allows to obtain some cytokinetic characteristics.
The RBC cytokinetic indices, presented in the Table demonstrate a decrease in an initial number of reticulocytes (N r0 ) in blood of ground squirrels in a torpid state by 33.6% (p < 0.05) versus the control. The rewarming contributed to an increase in reticulocyte count, which reached the maximum at Tb of 30°C, i. e. an increment was 34.9 and 103.3% versus the control and torpid state, respectively. After complete recovery of Tb of animals up to 37°C the reticulocyte count decreased and corresponded to that at the initial stages of arousal. A low reticulocyte count was also found in blood of dormant and artifi cially aroused S. undulatus [13]. Thus, the animal rewarming induces the reticulocyte release from bone marrow into the blood.
The calculation of reticulocyte half-life (T 1/2r ), determined by the time of their maturation, showed its reduction under torpid states by 30.4% as compared with the control. This may be due to a higher potential for rapid maturation in reticulocytes of hibernating animals than the control ones. This property is most likely associated to a change in gene expression level, responsible for this process. S.T. Cooper et al. showed [8] that during hibernation in heterotherms, the expression of most genes, associated with erythropoiesis was not signifi cantly changed. The exception was a threefold increase in SF3B1 gene expression. A high level of SF3B1 gene expression is suggested as проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 30, №/issue 2, 2020 ріння еритроцитів, внаслідок чого такі клітини прискорено виводяться з кровообігу [27].

Conclusions
1. Under torpid states, a count of RBCs, hemoglobin and MCH were not considerably changed, but MCV and HCT were signifi cantly reduced, and MCHC and RDW were signifi cantly increased as compared with the control. Erythrokinetic parameters testifi ed to the fact, that in animals in a torpid state, despite a decreased count of reticulocytes in blood and the reduced RBC bloodstream half-life, the ability to produce RBCs remained at the control level.
2. During arousal, a rise in body temperature up to 25°C entailed an increase in RDW relative to the torpid animals. Herewith, the tendency to MCV and HCT increase was revealed. With a rise in the body temperature, the reticulocyte count in blood and their maturation time augmented with the maximum at Tb of 30°C. At this body temperature, the RBC half-life in bloodstream was prolonged as well.
3. In blood of ground squirrels, after complete rewarming, the reticulocyte count reduced relative to both control and Tb of 25-30°С, but their maturation time was signifi cantly decreased, resulting in a strong increase in RBC production. Herewith, the RBC half-life reduced relative to the control and previous stage of rewarming.