key: cord-0072015-ntvabroj authors: Kondashevskaya, M. V.; Artemieva, K. A.; Aleksankina, V. V.; Tikhonova, N. B.; Boltovskaya, M. N. title: Indicators of Hypoxia Tolerance as Determined by Cellular Elements of Rat Blood date: 2021-12-20 journal: J Evol Biochem Physiol DOI: 10.1134/s002209302106003x sha: 704d3d71cc0325f3fa01df06bec4911041002982 doc_id: 72015 cord_uid: ntvabroj Although hypoxia tolerance is mainly determined genetically, it is important to study individual variability of animal organisms in order to identify the factors that underlie their tolerance to hypoxic exposure. We investigated blood cell counts and coagulograms in Wistar rats as predictors allowing the animal population to be split into hypoxia-tolerant and hypoxia-intolerant individuals. The validity of the specific predictors’ choice was proved by a coincidence between the population split in accordance with the detected individual parameters and the results of testing animals in a decompression chamber at a rarefaction corresponding to the “rise to an altitude” of 11500 m above sea level. Circulating blood cells were quantitatively assessed by eighteen indicators before and after hypoxic exposure. The differences between animals low-tolerant (LT), high-tolerant (HT), and medium-tolerant (MT) to hypoxia were determined by five indicators: white blood cell count (WBC), granulocyte count (Gran#), red blood cell count (RBC), reticulocyte count/percent (RTC), and mean corpuscular hemoglobin (MCH). The RBC, RTC, and MCH values in HT rats were significantly higher than in LT animals (by 1.4, 1.9, and 1.1 times, respectively). The WBC and Gran# values in HT rats were lower than in LT individuals. The hypoxia tolerance indices (HTI) were calculated using the original formula. It was established that in LT rats, the HTI ≤ 0.203, in HT rats ≥ 0.335, and in MT rats < 0.335 but > 0.203. After testing in a decompression chamber, the activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) decreased, but the fibrinogen level increased. LT rats were characterized by the lowest APTT, TT, and PT values and the highest values of the fibrinogen level. Our results indicate that one of the most important mechanisms underlying a high hypoxia tolerance in rats consists in sustaining reciprocal relationships between the complex of RBC indicators, which tend to increase under hypoxia, and Gran# indicators, which tend to decrease after hypoxic exposure. It has long been known that virtually all dis eases, no matter infectious or noninfectious, as well as in extreme stressful conditions, cause pathological endogenous hypoxia (low oxygen content) [1, 2] . Specifically, hypoxia is one of the pathogenetic mechanisms, as well as a factor, that determines the resistance to standard methods of cancer therapy [3] . With a decrease, for whatever reason, in the efficiency of tissue oxygen supply, there are microvascular dysregulation, extravasa tion and activation of white blood cells (WBCs) in the zone of hypoxic microenvironment. There are many types of immune and pro inflammatory resident cells, infiltrating this zone in a state of activation, which produce significant amounts of reactive oxygen species leading to oxidative stress, oxidative damage and mitochondrial dysfunction in most of the surrounding cells. The majority of the cells infiltrating the hypoxic zone is repre sented by activated neutrophils that release pro coagulant bioactive substances into the extracellular space [4] . Generally, all processes caused by hypoxia, oxidative stress and mitochon drial dysfunction provoke the secretion of signal ing molecules that additionally stimulate the inflammatory response [5, 6] . This situation is often accompanied by a damage to the endothe lial glycocalyx, which performs the anticoagulant function. This results in a release of such procoag ulant endothelial products as endothelin 1, superoxide anions, and thromboxane A2, as well as a decrease in the bioavailability of nitric oxide (NO) [6, 7] . Consolidation of the prothrombotic effects of neutrophils and endothelium leads to the formation of a "vicious circle" of the pro cesses that enhance dysfunction of the anticoagu lant system, causing the dominance of the blood coagulation system and the possibility of transi tion of a local inflammatory reaction to a systemic level [4] . The individual threshold of sensitivity to oxy gen deficiency, directly related to genetic factors, largely determines the duration and severity of diseases [8, 9] . The study of the relationship between the tolerance to hypoxia and the pecu liarities of biochemical, cellular, organ and phe notypic properties of the animal organism, aimed at extrapolating the obtained information to humans, is important for solving the senescence problems, as well as increasing the tolerance to hypoxia in various diseases and the working capacity of people engaged in professions associ ated with large psychophysiological and environ mental loads. This is the most common cause of anthropogenic impacts associated with air pollu tion, one of the effects of which is a reduction in the oxygen concentration. At present, the priority trend in experimental biology and medicine is searching for biomarkers of animal tolerance and receptivity to hypoxia in order to determine therapeutic targets for the modeling of various human diseases and imple mentation of an individual approach to treatment. In most cases, it becomes necessary to split the population of laboratory animals into low toler ant (LT), medium tolerant (MT), and high tol erant (HT) to hypoxia before starting the experiment. Most often, researchers work with groups of animals that differ sharply in all param eters, i.e. with LT and HT. Currently, it is known that LT individuals are characterized by uneco nomical consumption of oxygen-they consume a lot more oxygen per unit mass of tissue per unit time than HT individuals, whereas HT animals have developed the mechanisms for more effec tive adaptation to hypoxia, enabling them to with stand prolonged exposure to oxygen deficiency [10] . To divide the population of laboratory ani mals, they are tested individually in a decompres sion chamber, where conditions of acute hypoxia are created. Several types of hypoxia are repro duced in biomedical research, with the imitation of exogenous hypobaric hypoxia being the most commonly used [11] . This situation is artificially reproduced by a controlled pumping air out of a decompression chamber, thus a decrease in the environmental oxygen content leads to a decrease in the oxygen tension (pO 2 ) in the alveoli and arterial blood of the animal. Secondary tissue hypoxia develops in animals as a result of a decrease in the oxygen tension in blood and tis sues to levels below critical, at which the rate of oxygen utilization (consumption) in tissues begins to go down. If the strength and/or duration of hypoxic exposure exceed the adaptive capabilities of the body, then irreversible changes will appear in organs and tissues and the animal will die. Due to the fact that testing in a decompression chamber can damage the central nervous system and even cause death of animals, it is necessary a longer (at least 10 month) recovery period for the survived individuals to be involved in further experiments. The topical task is to reveal the rela tionship between certain physiological indicators in the pre hypoxic period and the level of hypoxia tolerance [9, 12] . Any hypoxic situation induces a complex of responses, which involves all the func tional systems of the organism. The main and most well known factor mediating this response is the HIF 1 transcription complex (HIF 1α and HIF 1β subunits) produced by most cells in response to oxygen deficiency. HIF 1β is a con stitutively expressed subunit, whereas HIF 1α is an oxygen regulated subunit [13] . It has been shown that the level of HIF 1α expression in human and animal WBCs varies, which indicates phenotypic differences in its regulation [14, 15] . However, HIF 1α is difficult to use as a predictor of hypoxia tolerance due to significant changes in its level depending on many factors. Since the contribution of HIF 1α to the pathogenesis of any diseases, as a rule, constantly changes, the issue of targeted pharmacological impacts on HIF 1α for the addressed regulation of the processes of urgent and long term adaptation to hypoxia in animals and humans is quite ambiguous [16] . The same is true for many neuroimmunoendocrine indicators. Undoubtedly, blood appears the most attractive object of studies aimed at identifying predictors of hypoxia, since it is the main conduit for transport ing oxygen from the lungs to tissues and carrying carbon dioxide in the opposite direction. Under any environmental challenges, including hypoxia, erythroid cells continue to perform their specific functions, while changing their numbers, size, oxygen content, etc. Under hypoxic conditions, WBCs, in small laboratory rodents mainly repre sented by neutrophils, are activated. It was deter mined, that one of the activators is HIF 1α, which plays a crucial role in the regulation of cel lular responses to hypoxia. Activated WBCs can affect coagulation directly by producing procoag ulant and anticoagulant molecules, and/or indi rectly by affecting platelets and endothelial cells. The appearance of a large number of activated WBCs can slow down the movement of red blood cells (RBCs), become a direct cause of microves sel occlusion, and reduce the efficacy of oxygen transportation by blood, thus provoking hypoxia in the microcirculation [17, 18] . We assumed that the analysis of blood cell parameters in the pre hypoxic period would allow the identification of predictors of the tolerance to acute hypoxic hypoxia. Therefore, the objective of this work was to study the cellular composition and coagulation system of the peripheral blood in Wistar rats before and after testing in a decom pression chamber, and then to distinguish a num ber of indicators as predictors allowing the rat population to be split on the basis of hypoxia tol erance. The study was carried out on 40 sexually mature male Wistar rats obtained from the "Stolbovaya" The personalized hypoxia tolerance of animals was determined by modeling acute hypoxic hypoxia in a decompression chamber. To achieve the similarity of recording conditions, testing was carried out in the morning (9-11 a.m.), consider ing the phase of infradian biorhythms, i.e. multi day (by to our data, 4 day), periodically recurring fluctuations in the intensity of many parameters in animals and humans [9, 19] . Namely, testing was performed between acrophase and bathy phase. i.e. the highest and lowest values of corti costerone levels, locomotor activity, etc. Hypoxia tolerance was determined by measur ing the time taken for the onset of gasping (gasp ing time). Rats were exposed, one at a time, to simulated hypobaric hypoxia caused by a rarefac tion equivalent to the "rise to an altitude" of 11500 m above sea level, in a decompression chamber coupled to a mercury barometer (equiv alent to 180 mm Hg). All the decompression and recompression instances were achieved gradually at a rate of 80 m/s to prevent any tissue injury due to a sudden fall or rise in the ambient pressure. Rats that had an impaired postural reflex "at an altitude" for less than 3 min were considered low tolerant (LT), more than 9 minutes-high toler ant (HT), more than 3 minutes, but less than 9 minutes -medium tolerant (MT ) [20] . Peripheral blood was collected from the caudal vein under zoletil anesthesia (5 mg/100 g, Virbac Santй Animale, France) into test tubes with EDTA as an anticoagulant, a day before the simu lation of acute hypoxic hypoxia and 5-10 min thereafter. The blood count was carried out for to 18 parameters, using a Mindray BC 2800 Vet Automatic Hematology Analyzer (China) with Rat software (WBC, Lymph#, Mon#, Gran#, Lymph%, Mon%, Gran%, RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDV, RТC). Parameters of hemostasis were determined by using a KC4 Delta Semi Automated Hemosta sis Analyzer (Tcoag, Ireland). The serum corti costerone concentration was determined by Enzyme Linked Immunosorbent Assay (ELISA, IBL, Germany). Statistical data analysis was carried out using Statistica 8.0. Normal distribution was checked by the Shapiro-Wilk test. It was found that the empirical distribution of our data is different from the normal. For statistical processing, nonpara metric methods for paired samples was used, the Mann-Whitney U test and the Wilcoxon-Mann-Whitney t test. The results were expressed as the median and interquartile range Me (25-75%). The differences were considered significant at p < 0.05. The analysis of all blood parameters with cate gorization of blood cells into drement in blood indicators vs. the mean in a given group, bacmean background value of blood indicators in a given group, RBC-absolute number of red blood cells, MCH-mean corpuscular hemoglobin level in the red blood cell, RTC-percent of reticulo cytes vs. the total number of red blood cells, WBC-absolute number of white blood cells, Gran#-absolute number of granulocytes. It was found that normal hypoxia tolerance indices are characterized by the values ≤ 0.203 (LT rats), ≥ 0.335 (HT rats), and < 0.335 but > 0.203 (MT rats). The major evidence that such indicators as the absolute number of WBCs, granulocytes and RBCs, as well as the percent of reticulocytes vs. the total number of red blood cells, and mean cor puscular hemoglobin in red blood cells can be predictors of hypoxia tolerance in rats was obtained when testing animals in a decompression chamber. It was found that "at an altitude" LT rats had impaired postural reflex for less than 3 min. In almost all cases, LT animals began rush ing about the decompression chamber, not reach ing the maximum "altitude" of 11500 m; some of them showed signs of seizures; 3 rats died after testing. MT animals endured a maximum "alti tude" for no longer than 9 min. The behavior of these rats was characterized by anxiety and vigor ous running at the onset of reaching a maximum "altitude", calming down after 4-5 minutes. HT individuals were distinguished by a pro nounced calmness and the ability to stay at an "altitude" of 11500 m for more than 9 min. Of the population of 40 rats, 30% turned out to be HT, 40% LT, and 30% MT. It should be emphasized that the individual values of the hypoxia tolerance index (HTI) in HT, MT, and LT rats, determined during testing in a decompression chamber, coin cided in their HTI values obtained before testing. Such cellular components of blood as WBCs, specifically granulocytes, make a significant con tribution to hemostasis, which has a strong influ ence on oxygen transport and utilization in tissues and, accordingly, on hypoxia tolerance [22] [23] [24] . In this regard, we studied hemostasis in rats before and after testing in a decompression chamber. It was found that values of hemostasis' indica tors of naive HT, LT and MT rats did not have statistically significant differences, whereas after testifferent subpopulations allowed us to reveal statistically significant differences between rats low tolerant (LT), high tolerant (HT) and medium tolerant (MT) to hypoxia only in five parameters: white blood cell counts (WBC), gran ulocyte counts (Gran#), red blood cell counts (RBC), % reticulocyte (RTC) and mean corpus cular hemoglobin (MCH). The RBC, RTC and MCH values were significantly higher in HT rats than in LT animals (by 1.4, 1.9 and 1.1 times, respectively) both before and after testing in a decompression chamber, as can be seen in Table 1 . The Gran# and WBC indicators in HT individuals were much lower than in LT rats under the same conditions (Table 1) . For instance, while in HT individuals the normal WBC was 2.2 times and the Gran# 1.3 times lower, after testing in a decompression chamber, the differences were 2.8 and 1.5 times, respec tively (Table 1) . Overall, immediately after testing in a decompression chamber, all blood cell parameters were found to increase in all animals ( Table 1) . The values of the same indicators were higher in MT compared to LT rats, but lower than the HT individuals. Since many hematological parameters of MT rats were statistically indistin guishable from those in LT and HT animals, their values are not shown Tables 1, 2. Since the amplitudes and units of measurement for blood parameters showed a great dispersion [21] , we developed a formula to calculate the hypoxia tolerance index (HTI) based on the for mula for the integrative assessment of functional reserves of an organism: where Δ-incing in a decompression chamber, the APTT, TT and PT values decreased, and the lev els of fibrinogen increased (Table 2 ). It was shown that LT animals were characterized by the lowest values of APTT, TT and PT and the highest levels of fibrinogen (Table 2) . Serum corticosterone levels did not differ sig nificantly between animals with different hypoxia tolerance before and after testing in a decompres sion chamber ( Table 2 ). The corticosterone con tent increased significantly after the hypoxia, as in any case of acute stress (Table 2 ) [25] . In this study, it was found that such indicators of peripheral blood of Wistar rats as the white blood cell count (WBC), granulocyte count (Gran#), red blood cell count (RBC), reticulo cyte count/percent (RTC) and mean corpuscular hemoglobin (MCH) can be used as predictors of hypoxia tolerance. The priority information obtained in the present study is that one of the most important mechanisms that underlie high hypoxia tolerance consists in sustaining reciprocal relationships between the specified set of indica tors of the erythroid hemopoietic branch, which tend to increase, and those of the granulocytic branch, which tend to decrease under hypoxia. At the same time, compared to LT, HT animals are characterized by significantly higher values of the RBC, RTC and MCH, as well as by lower values of WBCs and Gran# in naive rats. The same ratio of all these cellular elements remains after testing rats in a decompression chamber. Our data echo the results by L.A. Gridin, who found that hypoxia, as a specific stimulator of erythropoiesis, activates the mechanisms that lead to a compen satory adaptation, i.e. a decrease in the reproduc tion of white blood cells in the bone marrow [26] . In this study, we recorded, possibly genetically fixed, an increased mobilization readiness of the organism of HT rats to respond to hypoxia or other stressful exposures in the form of a mecha nism that suppresses the reproduction of granulo cytic cells and boosts the proliferation of erythroid cells. Perhaps this is due to the fact that, in the case of a significant predominance of WBC ele ments, counter to their protective function, a disaster may result, because granulocytes, which account for the majority of WBCs in animals and humans, instantly respond to an emergency situa tion by a release of large amounts of toxic sub stances that also have a negative impact on the host body. In this case, the vascular endothelium can be damaged, causing the activation of the coagulation system. These negative effects of neu trophils, representing a significant portion of the granulocytic elements of the WBC population, are now well documented when studying their action in COVID 19 [27, 28] . Increased blood coagulation plays a significant role in decreasing hypoxia tolerance [29, 30] . In the present work, we established that after being in conditions of acute hypoxic hypoxia, LT rats developed a state of hypercoagulation associated with the intrinsic (shortening of the APTT) and extrinsic (shortening of the PT) pathways of acti vation of blood coagulation, which was aggra vated by an increase in fibrinogen levels. This can be explained by a damage to the vascular wall, inflicted by reactive oxygen species, and an increased (vs. HT rats) number of hypoxia acti vated granulocytes that release procoagulant fac tors [17, 24, 31] . Our data are consistent with those obtained by other authors [32] [33] [34] . An increase in the blood fibrinogen level in response to stress induced corticosterone elevation may have been causative for the shortening of the TT indicator, which reflects a fibrinogen to fibrin conversion and depends on the blood fibrinogen level [35] . Testing of rats in a decompression chamber proved the fact that individual hypoxia tolerance in rats can be determined by HTI values. Besides hypoxic stimuli, other stress factors also act on liv ing organisms in a decompression chamber. In physiology, at all stages of evolution, it is customary to distinguish two qualitatively different strategies of survival: the active, resident strategy characterized by an active overcoming of stress factors, and pas sive, tolerance strategy implying a quiet perception of an external stimulus' intervention (a freezing response). These adaptation strategies are provided by different neuroimmunoendocrine mechanisms [36, 37] . As follows from our data, in a decom pression chamber, LT rats demonstrated a distinct resident strategy, due to which oxygen reserves were overspent, and the animals quickly lost the ability to sustain postural reflex. While HT rats held to a tolerance strategy that promoted long term saving in oxygen consumption, MT rats demonstrated an alteration of strategies, which determined the intermediate values retention time of postural reflex in HT vs. LT rats. The manifes tation of behavioral strategies, likewise hypoxia tolerance, is believed to be genetically fixed [37, 38] . As shown in our previous studies, moderate physical loads (swimming) allowed tolerant behavior conditioning in most of Wistar rats in the population. At the same time, it was established that animals adopted to tolerant strategy, became HT, while unlearned rats turned out to be LT [39] . Given that tolerance to hypoxia is geneti cally fixed and MT rats are probably the most tol erant to hypoxia, it can be assumed that MT rats, which learned the strategy of tolerance as the main principle of their behavior, became HT ani mals. Our studies give a reason to pose the prob lem of including behavioral mechanisms in the integrative response of animals and humans to acute hypoxia. Thus, it has been demonstrated for the first time that HT rats are characterized by an increased mobilization readiness (probably fixed geneti cally) to respond to hypoxia in the form of sup pressing the reproduction of WBCs and boosting erythropoiesis. We also identified the predictors of hypoxia tolerance, namely WBCs, granulocytes, RBCs, RTC, and MCH. We developed an origi nal formula for calculating the hypoxia tolerance index (HTI), which allows splitting the rat popu lation into HT, LT and MT individuals without testing them in a decompression chamber. It was established that in LT rats, the HTI values ≤ 0.203, in HT rats > 0.335, and in MT rats < 0.335 but > 0.203. Prediction of individual tolerance to hypoxia in naive animals is of great interest from both theoretical and practical points of view, since acute hypoxic hypoxia damages the central ner vous system, can cause death of animals, and pro longs the recovery period of surviving individuals (up to 1 month) for the involvement in further experiments. The identification of the relation ship between certain physiological indicators in naive animals or humans with the level of hypoxia tolerance makes it possible to improve the accu racy of predicting the outcome of acute hypoxic exposure, which is important for the professionals whose activities are associated with a high risk of hypoxia. Moreover, it is important to search for therapeutic targets when modeling various human diseases in order to develop a personalized approach to treatment. All applicable international, national and/or institutional principles for the care and use of ani mals have been observed. This article did not contain the results of any research involving people as research objects. 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