Duration of Whole-Body Air-Cryo-Exposure ( – 120 ° C ) Affects Some Properties of Murine Erythrocytes

An influence of whole-body air-cryo-exposure (ACE) (–120°C) performed in cryochamber for experimental animals rendered on the populations, osmotic fragility and the hemolysis level in erythrocytes of peripheral blood (PB) of mice was studied. The analysis of our findings showed that the duration of one session of ACE (40, 60 and 90 sec) affected the qualitative and quantitative composition of erythrocytes, i. e. their total number in the PB was reduced, in particular on an account of discocytes, the part of reversibly modified forms (stomatocytes) was significantly increased. Whole-body ACE led to an increased osmotic fragility and elevated hemolysis of erythrocytes. An hour following the ACE we have not found any normalization in quantitative and population composition of erythrocytes of mice PB. Thus the observed effect, rendered by a whole-body cooling on the hemolysis in erythrocytes, enabled to state that the longer (60 and 90 sec) staying of the animals at –120°C had less impact on erythrocytes comparing to whole-body short-term extreme cooling of mice for 40 seconds.

Adaptive responses appearing in an organism may gain a damaging effect, if the action of the stimulus or duration of its exposure exceed the certain limits.Sharp fluctuations in ambient temperature can lead to the development of pathological processes in the body, in particular to affect significantly cell membranes [11,18].Preventing the above mentioned negative effects of the temperature changes should consider the duration of its impact on the body.To date the clinicians widely apply artificial hypothermic states and cryotherapy (-120°C) utilizing a certain frequency and duration of air-cryoexposure (ACE); usually these are used both for a general improvement of the functioning of main regulators of the organism's adaptive systems, or in the cycle of treatment of psychosomatic disorders [1,3,6,12].
The persistent interest within many decades to a therapeutic hypothermia is explained by the fact that in various fields of medicine the method of artificial lowering of body temperature has been widely applied.
The method is based on the feature of hypothermia to slow metabolic rate, and in such a way to increase an organism resistance to adverse conditions and, above all, to hypoxia, which is usually accompanied with various pathologies [1,3,6,11].
Extreme factors, in particular, low and ultra-low temperatures act on an organism resulting in the changes in homeostatic indices of the blood system.Blood is a self-regulating structure and therefore could make the body able to resist the extreme influences due to improved regulation of physiological functions, genetic conservatism of receptors and plasticity of executive apparatus [15].Furthermore, the blood circulatory system integrates the activity of all the functional links of an organism involved into homeostasis.Thus, blood characteristics can serve as sensitive indicators of stress and could be used when studying the processes of adaptation, resistance and constancy of inner environment of the body.
The largest population of blood cells (erythrocytes) is the first one involved into the body's response to various influences and factors.Numerous recent reports described the relationship between the changes in erythrocyte membrane properties and homeostasis characteristics of the cells of internal organs [8][9][10]15].
In this regard, the revealing of changes in structuralfunctional organization of erythrocytes under temperature stress conditions is important for investigation of the consequences appeared due to the extremely low temperatures exposure of an organism at the level of biological membranes, as well as for assessing the individual and species-specific resistance to air-cryotherapy.
The research aim was to study the effect of duration of whole-body extreme air-cryo-exposure (-120°C) on osmotic fragility, hemolysis level and the occurrence of mouse erythrocyte shapes estimated by sphericity index.The need of these studies consisted in the selection of the optimal for these animals time of staying in cryochamber (-120°C) to perform further investigations.

Materials and methods
The research was performed in autumn-winter period in 3-month-old CBA mice (n = 34), weighing 18-20 g.The animals were housed in the animal facility with natural light/dark cycle (at temperature of 22...24°C) and a standard diet.
The experiments were carried out in accordance with the General Principles of Experiments in Animals, approved by the 6 th National Congress in Bioethics (Kyiv, 2016) and consistent with the statements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
Air-cryo-exposure of mice was performed in cryochamber for extreme cooling of the experimental animals developed at the Department of Cryophysiology of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine [4,13].For the independence of the experimental conditions just one animal was placed into the cryochamber.Assuming that the average duration of a single recommended procedure for humans and rats at an exposure temperature of -120°C makes 120 seconds [3,6,12], in preliminary studies the mice were placed into the experimental well of cryochamber for 120 seconds.The mentioned time of staying for small rodents during 120 seconds at a temperature of -120°C caused a fibrillar twitching of their muscles and bluish coloring of ears, indicating a strong overcooling of the body, due to these facts we have not used this duration of exposure in further experiments.
Based on the differences between species as for the resistance of animals to many adverse factors including cold [14], we have decided to reduce the duration of the ACE session for mice from 120 down to 90, 60 and 40 seconds.
In all the groups of animals the rectal temperature was measured, the number of erythrocytes in peripheral blood (PB) was counted, and the transformation of the shapes of PB erythrocytes was studied.The animals were sacrificed by decapitation.
The dynamics of erythrocyte transformation was investigated by small-angle light scattering with the Cryocon device (Ukraine), developed at the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine [7].The light scattering intensity in erythrocyte suspension depending on the number of cells in the suspension was studied at an angle of 9° to the falling beam.Measuring well was filled with 3.0 ml NaCl solution of various concentrations (from 0.15 to 0.05 mol/L) and 3 µl of erythromass obtained after sedimentation of blood and plasma aspiration were introduced.All the studies were carried out at a temperature of 37°C.The number of survived erythrocytes (%) was counted.Distribution of erythrocytes by sphericity index (SI) was determined using the dependence of osmotic fragility and physical-mathematical model of hypotonic hemolysis of red blood cells in the solution of non-penetrating substance [7,19,20].The SI values are directly proportional to the surface-to-volume ratio (S/V), and characterize the shape of cells.The pre-dominant shapes of red blood cells corresponded to the following SI intervals: spherocytes (1-1.3),stomatocytes (1.3-1.7),normal (1.7-2.1) and flattened (2.1-3) discocytes.
The results were statistically processed by Student-Fisher test after checking the distribution normality using the Excel software (Microsoft, USA).Differences between the samples were considered as statistically significant at p < 0.05.

Results and discussion
The consequences of cold effect on a body depend on the duration of cooling [3,6,12].As a result of ACE in experimental animals their rectal temperature decreased insignificantly in all experimental groups immediately after a session of general extreme cooling at -120°C (Table 1).An hour after the ACE procedure this index restored in all experimental groups.
The blood system counts have long been used to assess the state of an organism.The study of quantitative and qualitative states of erythrocytes during hypothermia is of particular interest, since hypothermia and hypoxia increase an organism demand for oxygen [6,9,11].экстремального охлаждения при -120°C (табл.1).Через час после процедуры АКВ этот показатель восстанавливался у всех экспериментальных групп.
To investigate the possible causes of strong changes in the number of peripheral blood erythrocytes in mice we have carried out the studies of their osmotic resistance after the ACE, because these studies could reflect not only the qualitative composition of functioning blood cells and the structural-functional state of their membranes, but also to reveal the changes in other organs and tissues [8][9][10].
The level of erythrocyte osmotic fragility is assumed as numerical value of NaCl concentration in a solution where 50% hemolysis occurs.The obtained experimental curves of mouse erythrocyte osmotic fragility (Figs.1-3A) and the diagrams of density distribution of murine erythrocytes by the SI (Figs. 1-3B) showed that following the ACE the osmotic fragility of mouse erythrocytes was reduced, but the extent varied among the groups.In particular, 50% hemolysis of mouse erythrocytes in the control group was observed in 0.486% NaCl solution.After staying of a mouse in the cryochamber for 40 seconds the 50% hemolysis of erythrocyte suspension occurred in 0.526% NaCl solution (Fig. 1A), after 60 seconds of exposure the threshold was found at 0.543% concentration (Fig. 2A), and in case of 80 seconds duration this was at 0.508% NaCl (Fig. 3A).
Simultaneously, the number of normal and flattened discocytes significantly reduced, which might serve as an evidence of an increased osmotic fragility of erythrocytes in Group 2.
Можно предположить, что степень изменения осмотической резистентности эритроцитов зависит от длительности гипотермии.Нами показано, что динамика трансформации эритроцитов не находилась в прямой зависимости от продолжительности низкотемпературного воздействия.Так, зна-After 60 seconds of the animals' staying in the cryochamber at -120°C the erythrocyte population was more homogeneous (Fig. 2, B; Table 2), the cells of Group 3 mice were mainly stomatocytes ((86.68 ± 4.78)%) (possible reversible transformation of erythrocytes).The number of normal and flattened discocytes as well as spherocytes (terminal stage of development of red blood cells, i. e. of echinocytes, acanthocytes and stomatocytes in case of irreversible damage Пребывание мыши в криокамере (-120°C) в течение 90 с: -контроль; -после криокамеры.Fig. 3. Osmotic fragility (А), density of erythrocyte distribution by sphericity index (В).The mouse was in cryochamber (-120°C) for 90 seconds: -control; after cryochamber.appearance and natural aging) herewith was reduced (p < 0.05).During 90 seconds the ACE contributed to a statistically significant reduction of spherocytes and flattened discocytes amount, the level of stomatocytes and discocytes did not differ from the control (Fig. 3B; Table 2).One hour later the ACE the amounts of normal discocytes in subgroups 2A and 4A were not significantly changed in comparison with the data obtained immediately after cryo-exposure, whereas in subgroup 3A the number of normal discocytes increased (p < 0.05, compared with group 3).V.V. Lomako et al. [14] have shown that regardless of the depth (rectal temperature was lowered down to 32.5, 27.5 and 16.5°C) and the way of achieving the hypothermia (craniocerebral, general and total under hypoxiahypercapnia) in rats an increase of erythrocyte osmotic fragility was observed and their hemolysis strengthened.At the same time there was remained unclear how the duration of mild hypothermia affected an osmotic resistance of peripheral blood erythrocytes.
Note: * -statistically significant changes as compared with the control, p < 0.05.
of osmotic fragility allowed to determine the level of hemolysis of erythrocytes depending on the duration of mice staying in experimental cryochamber with the temperature of -120°C (Table 3).
It can be assumed that the depth of change of erythrocyte osmotic resistance depended on hypothermia duration.We have shown that the dynamics of erythrocyte transformation was not directly dependent on duration of exposure to low temperature.For example, a significant hemolysis in Group 2 was observed even in 0.6% NaCl solution and continuously increased with a decrease in the medium osmolarity (Table 3) that might be associated with a statistically significant increase in the percentage of spherocytes in the population of red blood cells immediately after one session of ACE (see Table 2).In Group 3, the reduction in NaCl concentration down to 0.5% led to the hemolysis of the majority of cells (81.75 ± 8.27), whereas in Group 4 we observed no significant changes of erythrocyte hemolysis level if compared with the control.
Similar results were reported by M.A.M. Al-Rabia et al. [2].Body temperature of rats was reduced within 30 min down to 30°C (short-term moderate hypothermia), in the following series of experiments this temperature was maintained for 90 and 180 minutes (prolonged moderate hypothermia).According to the study, moderate hypothermia for 90 minutes resulted in an increase of osmotic fragility of erythrocytes, and its prolongation up to 180 minutes allowed to reduce this value.
Cell osmotic resistance is a considerably constant value, nevertheless it decreases with the aging of cells, following its functional activation, as well as under pathology conditions or due to the extreme exposures [9].In our case, an altered osmotic fragility of erythrocytes of experimental animals could be related both to a rise in functional activity of the cells resulted from an increased oxygen demand of an organism during hypothermia [6], as well as other consequences of extreme cold stress.
It is known [16] that an osmotic fragility of erythrocytes mainly reflects the state of cytoskeleton of cells as well as cell shape and depends on the surface-tovolume ratio.Rise in osmotic fragility of red blood cells is probably caused by significant reduction in the content of discocytes and elevated number of erythrocytes with altered shapes (spherocytes).Presence of spherocytes  2. Экстремальное АКВ способствует повышению осмотической хрупкости и усилению гемолиза эритроцитов.
is indicative of the degree of hemolysis, as these cells are formed after erythrocytes losing the part of plasma membrane.In this connection, the cells become less osmotically resistant, as confirmed by our studies and other published reports [9,15].
It has been found that under extreme conditions, including hypothermia, the activity of antioxidant system decreases, lipid peroxidation is getting stronger and uncontrollable, that leads to a modification of lipids and proteins of blood plasma, membranes, erythrocytes and, ultimately, to hemolysis of the latter [5,17,18].M.A. Srour et al. [22] incubated rat's erythrocytes with oxidants (H 2 O 2 , ascorbate/Fe 2+ ) to increase their osmotic fragility.The data obtained allowed to suggest that the activation of oxidative damage in erythrocyte membrane could be a major cause of an increased osmotic fragility of erythrocytes after hypothermia.The authors believed that altered osmotic fragility of erythrocytes in dynamics of hypothermia was directly dependent on the state of thiol groups of proteins in erythrocyte membranes.This study also demonstrated that the prolongation of hypothermia contributed to a normalization of both osmotic resistance of erythrocytes, and the number of thiol groups in proteins of their membrane.This indicated that prolonged hypothermia in homeothermic organisms could trigger the mechanisms of adaptive rearrangements of metabolic processes at the level of individual cells [2,21].
Comparing our data on quantitative and qualitative compositions of erythrocyte population after one session of ACE, we have concluded that depending on duration of the procedure of extreme hypothermia one could achieve not only relative homogeneity of the population (see Table 2), but an increase in the number of erythrocytes in PB an hour later its performance too (see Table 1).A statistically significant increase in the erythrocyte count in PB of experimental mice after ACE was likely due to the elimination as a result of hemolysis of old and transformed shapes of erythrocytes and release of reticulocytes into the blood stream.
Thus, the study of structural-functional organization of erythrocytes enabled to determine the individual and species-specific resistance to the effect of extremely low temperatures and the duration of air-cryo-exposure being optimal for an organism.Using air-cryo-therapy as recreational or therapeutic procedures it is advisable to conduct studies to determine the duration of staying of an individual at a temperature of -120°C.
2. Extreme ACE promoted an increase in osmotic fragility and strengthening of erythrocyte hemolysis.
3. One hour later the ACE there was no normalization of quantitative and population compositions of PB erythrocytes in mice, regardless of the used duration of extreme cooling.
4. Short-time cooling during 40 seconds rendered less impact on erythrocytes than longer (60 and 90 seconds) staying at a temperature of -120°C.

Table 2 .
Effect of duration of one stay in cryochamber (-120°C) on the ratio of murine erythrocyte shapes estimated by sphericity index immediately after the ACE procedure and following one hour (n = 5, M ± SE)