Impact of Temperature on Morphological Characteristics of Erythrocytes and Heart Weight: Experimental Study on Wistar Rats

Abstract

The aim was to find what happens to heart weight and forms of erythrocytes antemortemly and postmortemly as a result of exposure to high water temperature. Total of 40 adult Wistar rats is divided into three groups, depending on water temperature exposure of 37°C (KG, n = 8), 41°C (G41, n = 16), and 44°C (G44, n = 16). Depending on the length of time of exposure to water, temperatures of 41 and 44°C are further divided into G41-AM, G41-PM, G44-AM, and G44-PM. The anesthetized rats were exposed to preheated water using the water bath. May-Grünwald-Giemsa coloring technique was applied to blood samples. Light microscopy was performed to detect poikilocytes. Heart weight was measured after dissection with a scale. A statistically significant difference in heart weight was found in the experimental groups (p = 0.024). The lowest value was observed in KG37 and was 0.99 ± 0.11 g, and the highest values were found in rats of the G41-PM group, with a mean value of 1.26 ± 0.26 g. There is a statistically significant difference between the experimental groups in forms of poikilocytes.

1. Introduction

1.1 Body temperature – values, fluctuations, and regulation

The physiological range of human body temperature is 36.8 ± 0.3°C [1]. During physical activity, body temperature can rise from 38 to 40°C, and exposure to extremely low ambient temperatures can lead to a decrease in body temperature to 35°C [2]. In clinical thermometry, the mean physiological oral temperature of 36.8 ± 0.9°C correlates with the end product of the energy of all enzymatic reactions. Metabolism, through the sum of all the body’s cellular reactions, is usually measured as the amount of oxygen consumed. The standardized estimate of metabolism is the basal rate of metabolism, which depends on the activity of these physiological processes to maintain euthermia [3]. The physiological body temperature of the human body core is about 37°C and is controlled in a narrow range (33.2–38.2°C), and is further narrowed if oral measurements are neglected in favor of rectal, tympanic, or axillary measurements [4]. Abnormal deviations of the core temperature of even a few degrees will trigger the body’s thermoregulatory mechanisms, and changes in temperature outside the physiological range can prove fatal. Measured body temperature above 42°C leads to cytotoxicity with protein denaturation and impaired deoxyribonucleic acid (DNA) synthesis [5], resulting in organ failure and neuronal damage. If body temperature falls below 27°C (hypothermia), associated neuromuscular, cardiovascular, hematological, and respiratory changes may prove equally fatal [6]. The core temperature is maintained in the range of +/ 6°C in the environment from 10–55°C, while the skin temperature varies depending on the environment. The temperature measured orally is from 36.5 to 37°C, while the rectal temperature is 0.5°C higher [7]. In humans, body temperature varies by about 1°C during the day, with a gradual increase during wakefulness and a decrease during sleep [8]. Daily fluctuations in body temperature are a strong effect of circadian rhythms [9] associated with a number of physiological functions, such as metabolism and sleep [10, 11]. Evidence in humans and rats shows that circadian temperature rhythm is controlled separately from locomotor activity rhythms [12]. The amount of core temperature formation depends on the intensity of metabolism, and it depends on basal metabolism, muscle activity, thyroxine, adrenaline, noradrenaline, sympathetic nervous system activity, cell temperature, and digestive system activity. Heat release depends on the rate of conduction to the skin surface and the rate of heat transfer from the skin to the environment. The skin and subcutaneous tissue participate in the thermal insulation of the body. Blood vessels can regulate heat transfer by constriction and dilatation [13]. Body temperature varies depending on where it is measured. In thermoregulatory research, it is common for the body to be divided into two sections—the outer core, which includes the skin and which mainly varies in temperature with the environment, and the inner core, which includes the central and peripheral nervous system and has a relatively stable temperature [13, 14]. The preoptic area of the anterior hypothalamus plays a major role in the regulation of body temperature [15]. Most nerves are more sensitive to heat than to cold. Heating these areas of the brain increases the body’s sweating, and cooling interrupts any mechanism of heat loss. There are many more receptors on the periphery to register cold than heat and all act on the hypothalamus [16]. Heat receptors also exist in deep tissues and are exposed to body core temperatures. On both sides of the posterior hypothalamus at the level of the mammary corpuscles is the posterior hypothalamic region that integrates central and peripheral thermal sensations. The role in the regulation of body temperature is mediated by sweat glands that have cholinergic innervation (acetylcholine), and to some extent, they can be stimulated by adrenaline and noradrenaline, secrete primary secretion, which is a product of epithelial cells, depending on the intensity of sweating [17]. With poor sweating, the secretion takes more time to pass through the canals, and consequently, more sodium and chlorine ions are reabsorbed, and potassium, urea, and lactic acid ions are concentrated. The process of acclimatization is associated with the reduction of sodium and chlorine ions in sweat, which improves the preservation of body electrolytes [18]. The nervous system acts as a biological thermostat for heating and cooling inside the animal’s body. Because animals use resources, such as energy, water, and oxygen, for thermoregulation, the nervous system monitors the abundance of these resources and adjusts thermoregulatory mechanisms accordingly. Hunger, dehydration, or hypoxemia alter the activity of temperature-sensitive neurons in the preoptic region of the hypothalamus. Other regions of the brain work together with the hypothalamus on the adaptability of thermoregulation. For example, the amygdala is likely to inhibit neurons in the preoptic area, overriding thermoregulation when there is a risk of hypothermia or overheating. Moreover, the hippocampus allows the animal to remember microcells that allow safe and efficient thermoregulation [19].

1.2 The body’s response to hyperthermia

Hyperthermia is a condition of elevated body temperature, above the upper physiological limit [19, 20]. When the body is exposed to high temperatures, the secretion of interleukins 1 and 6 (IL-1 and IL-6) and tumor necrosis factor (TNF) alpha from excited immune cells, which act on thermoregulatory centers and consequently lead to setting the center to a higher temperature [20]. In the body’s response to hyperthermia, it is important to distinguish between endogenous and exogenous hyperthermia. Exogenous hyperthermia occurs when the influx of heat from the external environment increases significantly, such as in tropical areas, in small enclosed spaces that do not have adequate insulation and airflow with artificial increase in air temperature, in the bathroom during bathing, in saunas, and in Turkish baths. The fastest exogenous hyperthermia develops when there is a combination of increased heat influx from the outside with difficulty in heat transfer. Under these conditions, heat transfer mechanisms, despite maximum activation, do not remove heat from the outside, and body temperature begins to rise. Thermoregulation is actively aimed at raising the temperature by the process of overheating, all with the aim of faster heat transfer. In the 1990s, science showed that hyperthermia was teratogenic to both humans and animals. The state of hyperthermia can be the result of two processes. One is impaired production and release of heat, conditionally speaking the relationship between body temperature and ambient temperature, and the other is the setting of the thermoregulatory center to a higher level [21]. When there is an increased ambient temperature, the body temperature level rises slightly to the newly set temperature and hyperthermia occurs. Temperature rise occurs due to reduced temperature release and increased thermogenesis. High-energy consumption is required to raise the temperature, so a feeling of exhaustion may be present. When the body temperature equalizes that of the thermoregulatory center, thermogenesis ceases (if pyrogen secretion has ceased). After that, the set temperature of the thermoregulatory center returns to a lower value and there is a gradual decrease in body temperature due to reduced thermogenesis and increased heat release. Infectious diseases, exposure to elevated ambient temperature, hypothalamic damage, malignancies, tissue necrosis, and any other stimulus that could stimulate immune cells to secrete endogenous pyrogens can lead to hyperthermia [22, 23]. Hyperthermia occurs in combination with increased hypothalamic activity with values above the physiological range and occurs when the body’s thermoregulatory mechanisms are no longer able to efficiently emit heat (evaporate) [24]. Exogenous environmental stressors, such as high temperature; growth factors and ligands for surface receptors; and many drugs or chemical agents can cause apoptosis. However, cells that have undergone apoptosis show similar morphology, suggesting that these divergent apoptotic stimuli converge to induce a common cell-death pathway. Possible signaling molecules that ultimately lead to apoptosis are interleukin-1-enzyme (ICE)-like1 protease or caspase and other ceramide messengers [25]. If the body temperature of the nucleus does not decrease, a fatal outcome occurs in 30–80% of patients [26]. Heatstroke can cause severe damage to myocardial cells in rats, followed by an increase in apoptotic cells. Heatstroke causes oxidative damage to cellular proteins and DNA [27, 28]. Exposure to heatstroke for 1 hour seriously injures chicken myocardial cells, as evidenced by decreased cell vitality and the onset of apoptosis.

With an increase in body temperature, cardiac output and blood pressure drop drastically and are associated with myocardial oxygen consumption. Hypoxia causes numerous injuries to the heart muscle, from subendocardial hemorrhage, myocardial necrosis, and rupture among fibrin fibers. An increase in internal temperature in rats from 37–42°C also causes tachycardia and increases mean blood flow and vascular resistance by 13% [29]. In the state of heatstroke, large amounts of calcium are released from the sarcoplasmic reticulum of the heart muscle, causing a hypermetabolic state. Continuous increase in calcium allows excessive stimulation of aerobic and anaerobic glycolytic metabolism, leading to respiratory and metabolic acidosis, increased membrane permeability, and the occurrence of hyperkalemia. Rhabdomyolysis leads to an increase in potassium and myoglobin levels in the heart and edema occurs. Disseminated intravascular coagulation occurs as a consequence of thromboplastin release in tissues [30].

1.3 Hematological parameters

Monitoring of hematological parameters enables fast detection of changes in the physiological state because changes in hematological parameters manifest themselves very quickly and precede possible damage. Each species has its own characteristics of individual hematological parameters. It is evident that there are unfavorable endogenous and exogenous factors that can, in certain circumstances, change the original biconcave form of mammalian erythrocytes and thus partially or completely disable its physiological role in gas exchange.

1.4 Erythrocytes: shape and size

Erythrocytes or red blood cells make up the majority of blood cells. Although they are called cells, mature erythrocytes do not have a nucleus, mitochondria, or other organelles. Normal erythrocytes are actually biconcave plates with an average diameter of about 7.8 μm. In the thickest place, their thickness is about 2.5 μm, and in the center 1 μm or less. Their average volume is 90 to 95 μm³. Their membrane is too large in relation to the cell content, so the deformation will not cause the membrane to stretch, but neither will it burst, which would happen to many other cells. The cytoplasm of erythrocytes contains large amounts of the protein hemoglobin, which is able to temporarily bind gases to itself. It is because of this protein that erythrocytes have the ability to carry oxygen and carbon dioxide.

The total number of erythrocytes in the bloodstream is maintained within relatively narrow limits. The body strives to ensure that the number of erythrocytes is always sufficient to carry oxygen from the lungs to the tissues in appropriate quantities, without impeding blood flow through the blood vessels. Tissue oxygenation is the most important regulator of erythrocyte formation. Any condition in the body that reduces the amount of oxygen in the tissue increases the production of erythrocytes. If a person becomes anemic, due to bleeding or any other reason, the bone marrow immediately begins to produce a large number of erythrocytes. Erythropoietin is a circulating hormone that stimulates the production of erythrocytes, and its production increases in response to hypoxia. Under normal conditions, 90% of erythropoietin is produced in the kidneys and the rest is mostly in the liver. The production of erythropoietin is especially stimulated by adrenaline and noradrenaline, and some prostaglandins. Erythropoietic cells are among the fastest growing and proliferating cells in the human body. Therefore, their maturation and rate of formation are greatly influenced by a person’s general nutrition [31].

Erythrocytes, the main carriers of oxygen in the blood, are thought to play a key role in controlling local blood flow to the tissue. According to the hypothesis proposed by Ellsworth et al. (1995), when erythrocytes encounter an area where metabolic requirements are increased, a signaling mechanism associated with oxygen release is triggered, resulting in the release of ATP from erythrocytes into the vascular lumen. ATP acts on endothelial P2y receptors, triggering the release of nitric oxide, prostaglandins, and/or hyperpolarizing factors derived from the endothelium, which in turn act on surrounding smooth muscle cells causing vasodilation [32].

1.5 Poikilocytosis

Poikilocytosis is a term used for abnormally shaped red blood cells (RBCs) in the blood [33]. Poikilocytosis generally refers to an increase in the abnormal shape of red blood cells that make up 10% or more of total red blood cells. Poikilocytes may be flat, elongated, teardrop-shaped, crescent-shaped, may have pointed or thorny protrusions, or may have any other abnormal feature. Examination of the blood smear reveals various forms of erythrocytes. Spherocytes are small round cells that do not have a flat, brightly colored center of regular erythrocytes [34]. The central part of the stomatocyte is incised or elliptical, which differs from the regular round shape of erythrocytes. Dental cells have the shape of a mouth. Podocytes are also known as target cells because they resemble a bull’s eye. Sickle cells, also known as drepanocytes, are crescent-shaped and elongated erythrocytes [35]. Elliptocytes, also known as ovalocytes, are oval or cigar-shaped cells with blunt ends. Droplet cells or dacryocytes are abnormal erythrocytes that have one round and one pointed end. Acanthocytes are erythrocytes that have abnormal spike-like protrusions present on the cell membrane. Echinocytes similar to acanthocytes also have protrusions (spicules) on the cell membrane similar to acanthocytes, but the projections in echinocytes are evenly distributed and more frequently present. Schistocytes are fragmented erythrocytes [36, 37, 38, 39, 40].

Red blood cells usually carry oxygen and many nutrients to tissues and organs. In poikilocytosis, erythrocytes are irregular in shape and may be unable to carry enough oxygen. Poikilocytosis is caused by other medical conditions, such as anemia; red blood cell membrane defects, such as hereditary spherocytosis; many genetic causes, such as sickle cell disease and thalassemia; eating disorders, such as iron deficiency anemia and megaloblastic anemia; and other causes, such as kidney disease and liver [40].

1.6 Animal model of inducing hyperthermia

The physiological body temperature of rats is from 35.9 to 37.5°C [41]. The body temperature of 40.9°C is the upper limit before the compensating mechanisms are activated [42]. The development of techniques for the induction of hyperthermia in laboratory animals represents a significant contribution to experimental research. According to the available literature, hyperthermia in an animal model can be induced with dry (high temperature) and moist heat (immersion in heated water). Induction of hyperthermia and temperature measurement are important components in heatstroke studies to determine the degree of progression or regression of heatstroke. The electric thermometric method is more suitable and precise for continuous or consecutive measurements in comparison with a classical mercury thermometer. Common temperature measurement sites are the skin, oral cavity, axilla, rectum, and eardrum [43]. The superiority of tympanic measurement over rectal thermometry has not been demonstrated in animal studies.

Until the 21st century, rectal thermometry was the most appropriate technique for measuring temperature in heatstroke studies. At the beginning of the 21st century, the best indicator of the average core temperature of the body is considered to be the temperature of the blood in the pulmonary artery [44]. Due to the poor accessibility of the pulmonary artery, other anatomical locations (esophagus, rectum, and oral cavity) are most often used in the routine measurement of core temperature today [45]. Rats, dogs, monkeys, baboons, cows, rabbits, and sheep were used in experimental studies that allow manipulation of exposure conditions and experimental methodology. Among these species, rats, rabbits, and sheep are the most suitable models because of their resemblance to humans as a reaction to high temperature and given their availability, price, and ease of handling. Such models can be used to simultaneously study different pharmacological and laboratory parameters and functions.

Rats are used for routine experiments, while sheep are reserved only for large experiments in which several parameters and functions of the organism are examined at the same time. Several studies related to heatstroke in rats have been performed as experimental models [46, 47, 48]. The models were based on the exposure of rats to high temperatures, dry air, or water, until the core temperature reached a predetermined temperature (40.5°C).

A body temperature value of 40.5°C on exposure for 15 minutes was accepted as a reference for the diagnosis of heatstroke. No direct conditional-consequential relationship between hyperthermia and mortality (less than 10% death) was found in rats exposed to lower temperatures during the experiment [49]. Sharm et al. [50] in their study showed that the animal model for the induction of rat hyperthermia is comparable to the clinical situation. The model has proven useful for studying the effects of diseases associated with exposure to high ambient temperatures on changes in various organs and systems, including the central nervous system. Because hyperthermia is often associated with severe brain dysfunction, additional methods have been described to examine some key parameters of brain injury and the development of brain edema [50]. The research was mostly done for the purpose of proving hypo and hyperthermic therapeutic effects in malignant diseases. Several studies are known to go in the direction of the association between hyperthermia and survival time [47].

The first model of hyperthermia was developed on a dog in 1973 and on a rat in 1976 [51]. Hubbard et al. [47] induced rat hyperthermia by heating the cage at a high temperature and measuring rectal temperature [47]. A study by Weshler et al. [52] investigated the development of thermotolerance in the development of hyperthermia in rats in the aquatic environment. Following the historical sequence, more models of hyperthermia have been developed but most of them cause heatstroke by high-temperature dry air. In the animal model of hyperthermia, a study by Suzuki et al. [1] indicates hyperthermia as a cause of death during bathing and the association between high water temperature and survival time.

In the 19th century, an animal model of piglets was developed to investigate disorders caused by hyperthermia. This experimental study was a pioneer in later studies that demonstrated the role that hyperthermia can play in diseases, such as hemorrhagic shock and encephalopathy syndrome, and, in some cases, sudden infant death syndrome [53, 54, 55, 56].

1.7 Cardiovascular response to hyperthermia

When exposed to high temperatures, the circulating flow from the environment is redirected to the skeletal muscles and skin, to give off heat. Acute cardiogenic shock can also occur, leading to intracranial hypertension, cerebral hypoperfusion, cerebral ischemia, and neuronal injury. Prolonged exposure to elevated ambient temperatures can result in convulsions, exhaustion, and heatstroke. Thermoregulatory mechanisms relax, sweating stops, and body temperature rises. A condition accompanied by arrhythmias occurs, and disseminated intravascular coagulation, skeletal muscle, and myocardial necrosis may occur [57]. Rhabdomyolysis, which occurs in such heatstroke conditions, is characterized by rupture and necrosis of striated muscle cells, which can be caused by trauma under conditions of hyperthermia. If rhabdomyolysis is extensive, circulating myoglobin may produce acute renal failure [58]. The mortality rate for such patients exceeds 50%. Death caused by hyperthermia is diagnosed in a hospital or by autopsy mainly using serological and pathohistological methods. Postmortem diagnosis of death caused by hyperthermia and heatstroke presents certain difficulties [59].

Hyperthermia occurs and the result of thermoregulatory mechanisms is felt in many organs, including the heart, which is the first response in the chain. Cardiac dysfunction and degeneration occur secondarily in relation to the massive increase in catecholamine secretion, as well as hyperkalemia, acidosis, and hypoxia [60]. Thanks to the research that has been done, nonspecific abnormalities are noticeable on the electrocardiogram [61], angiograms [62], and pathohistological analyzes of the myocardium [63]. An increase in heart mass due to the hyperthermic effect is also observed [64].

2. Material and methods of research

The study was conducted as a prospective, randomized, controlled, experimental study done on an animal model of causing rat hyperthermia. This study was approved by the Ethical Committee of the Medical Faculty University of Sarajevo under registration number 02–3-4-1253/20, Bosnia and Herzegovina.

2.1 Experimental animals

The experiment used 40 adult albino Wistar rats, both sexes, weighing 250 to 300 g. All animals were kept under the same laboratory conditions, and 7 days before the experiment for acclimatization and adaptation were kept in a vivarium with a 12-hour light regime day-night and at room temperature (20°C ± 2°C). During the experiment, the animals received commercial feed for laboratory animals and running water ad libitum. The care and care of animals, as well as the implementation of all experimental procedures, were carried out in compliance with the International Guidelines for Biomedical Research on Animals-CIOMS (The Council for International Organizations of Medical Sciences) and ICLAS (The International Council for Laboratory Animal Science) [65, 66].

Hyperthermia model was used on 40 adult Wistar rats that were methodologically divided into three experimental groups, depending on water temperature exposure of 37°C (KG, n = 8), 41°C (G41, n = 16), and 44°C (G44, n = 16). Each of the trial groups exposed to 41°C and 44°C water temperature was further classified according to the time of analysis, as the antemortem group (G41-AM; G44-AM) with exposure time of 20 min and the postmortem group (G41- PM; G44-PM) with exposure until time of death.

2.2 Induction of hyperthermia in a rat model

The water bath was filled with water and heated to the target water temperature. The water temperature was continuously monitored on the display with additional measurements with a probe immersed in water and readings on a thermometer. A pre-anesthetized rat with a head above water level, fixed on a wooden board, was immersed in preheated water at the target temperature. Survival times were recorded, which included the time from the immersion of the rats in the water of the set temperature (41°C and 44°C) to the time of death. We defined hyperthermia as an increase in internal temperature by 0.5°C, and heatstroke as an increase in internal temperature above 40.5°C [67] (Figure 1).

Figure 1.

Experimental procedures in the laboratory of the Faculty of Veterinary Medicine, University of Sarajevo.

2.3 Measurement of heart mass

To measure heart mass, we used a 0.001 mg sensitivity scale (model GT410V, USA) after dissection and before immersion in formalin.

2.4 Microscopic examination and cell counting

Blood samples for analysis were taken from the abdominal aorta. At least two blood smears were made using standard laboratory blood staining techniques (May-Grünwald-Giemsa). Stained blood smears were analyzed by two independent researchers, with counting performed on representative single-layer visual fields where blood cells did not overlap or only touched their edges. Two thousand erythrocytes were analyzed on each stained blood smear using a Motic Type 102 M light microscope and a magnification of 1000 times to examine the presence of poikilocyte red blood cells. The average value of two independent measurements was taken for analysis and the percentages of the number and type of poikilocytes were presented. The most representative microscopic images were stored in electronic form using the software Motic Images Plis 2.0 [68, 69].

3. Results

The body weight of rats in the groups formed according to the length of exposure to elevated temperature ranged from 280.14 g in KG37 to 325.50 g in G44-AM, but there was no statistically significant difference in body weight between groups (p = 0.081) (Table 1).

Body mass (g)
Groups	X	±SD	95% CI
			LL	UP
KG37	280.14	43.71	239.71	320.57
G41-AM	315.86	32.29	285.99	345.73
G41-PM	286.75	29.77	261.85	311.65
G44-AM	325.50	47.27	285.98	365.02
G44-PM	281.25	37.90	249.56	312.94

Table 1.

Mean values of body weight of rats in the experimental groups according to the length of exposure to elevated temperature.

X - mean value; ± SD standard deviation; CI-confidence interval, LL-lower limit; UL-upper limit; KG37-control group of rats exposed to water temperatures of 37°C; G41-AM-antemortem group exposed to water temperature 41°C (exposure length 20 minutes); G41-PM-postmortem group exposed to water temperature 41°C (length of exposure to death); G44-AM-antemortem group of rats exposed to water temperatures of 44°C (exposure length 20 minutes); G44-PM-postmortem group of rats exposed to water temperatures of 44°C (length of exposure to death).

The lowest mean heart weight of rats was 0.99 ± 0.11 g in KG37, and the highest value was found in G41 and was 1.15 ± 0.23 g. No statistically significant difference in rat heart weight was found between the three groups, p > 0.05 (Table 2).

Group	X(g)	±SD	95% CI
			LL	UL	P
KG37	0.99	0.11	0.88	1.10
G41-AM	1.01	0.07	0.94	1.08
G41-PM	1.26	0.26	1.04	1.48	0.024
G44-AM	1.06	0.08	0.99	1.13
G44-PM	1.15	0.21	0.98	1.33

Table 2.

Mean values of rat heart mass in the experimental groups.

X - mean value; ±SD standard deviation; CI-confidence interval, LL-lower limit; UL-upper limit; p-probability; KG37-control group of rats exposed to water temperatures of 37°C; G41-AM-antemortem group exposed to water temperature 41°C (exposure length 20 minutes); G41-PM-postmortem group exposed to water temperature 41°C (length of exposure to death); G44-AM-antemortem group of rats exposed to water temperatures of 44°C (exposure length 20 minutes); G44-PM-postmortem group of rats exposed to water temperatures of 44°C (length of exposure to death).

A statistically significant difference in rat heart weight was found in the experimental groups (p = 0.024). The lowest value was observed in KG37 and was 0.99 ± 0.11 g, and the highest values were found in rats of the G41-PM group, with a mean value of 1.26 ± 0.26 g (Table 2).

The mean values of rat heart weight in the experimental groups differed in the KG37 and G41-PM groups, p = 0.04, and the 41-AM and PM groups, p = 0.08 (Table 3).

Group	Group	p	95% CI
			LL	UL
KG37	G41-AM	1.00	−0.29	0.25
	G41-PM	0.04	−0.53	−0.00
	G44-AM	0.33	−0.15	0.61
	G44-PM	1.00	−0.34	0.19
	G44-PM	0.73	−0.43	0.10
G41-AM	G41-PM	0.08	−0.51	0.01
	G44-AM	1.0	−0.31	0.21
	G44-PM	1.0	−0.40	0.12
G41-PM	G44-AM	0.27	−0.05	0.45
	G44-PM	1.00	−0.15	0.36
G44-AM	G44-PM	1.000	−0.34	0.16

Table 3.

Multiple comparisons of mean rat heart weight values in the experimental groups.

CI-confidence interval; LL-lower limit; UL-upper limit; p-probability; KG37-control group of rats exposed to water temperatures of 37°C; G41-AM-antemortem group exposed to water temperature 41°C (exposure length 20 minutes); G41-PM-postmortem group exposed to water temperature 41°C (length of exposure to death); G44-AM-antemortem group of rats exposed to water temperatures of 44°C (exposure length 20 minutes); G44-PM-postmortem group of rats exposed to water temperatures of 44°C (length of exposure to death)

Table 4 shows the differences in poikilocytotic forms between the antemortem groups (41°C and 44°C) and the control group (37°C).

	A: Temperature 37 C			B: Temperature 41 C			C: Temperature 44 C
	Med	Per 25	Per 75	Med	Per 25	Per 75	Med	Per 25	Per 75	p ^A v B v C	p ^A v B	p ^A v C	p ^B v C
Ovalocytes	1.0	0.0	2.0	3.50	1.00	6.00	3.00	2.00	3.00	0.011	0.005	0.038	0.155
Dacryocytes	1.0	0.0	2.0	8.50	1.00	12.0	5.00	2.00	9.0	0.003	0.003	0	0.793
Annulocytes	1.0	0.0	3.0	39.50	31.0	55.0	47.0	25.0	74.0	0.003	0.008	0.001	0.141
Echinocytes	0.0	0.0	1.0	2.50	0.0	38	0.00	0.00	15.0	0.029	0.011	0.079	0.28
Stomatocytes	1.0	0.0	2.0	10.00	4.0	22.0	17.0	6.00	35.0	0.001	0.003	0	0.402
Drepanocytes	0	0.0	0.0	0.00	0.0	0.00	0.00	0.00	0.00
Schistocytes	0.0	0.0	2.0	1.00	1.0	6.00	1.00	1.00	2.00	0.097	0.056	0.079	0.756
Leptocytes	0.0	0.0	0.00	0.00	0.0	0.00	0.00	0.00	0.00
Acanthocytes	0.0	0.0	0.0	0.00	0.0	1.00	0.00	0.00	0.00	0.687	0.536	0.636	0.867
Spherocytes	1.0	0.0	2.0	1.00	1.0	8.00	2.00	1.00	15.0	0.023	0.019	0.007	0.981
Reticulocytes	1.0	1.0	1.0	1.50	0.0	2.00	1.00	1.00	4.0	0.027	0.019	0.02	0.685
Target cells	1.0	0.0	1.0	24.50	20	34.0	12.0	3.0	24.0	0.013	0.005	0.02	0.375

Table 4.

Differences in poikilocytotic forms between antemortem group and control groups.

Variables are represented as median values with an interquartile range. P ^{A v B v C} was tested with the Kruskal-Wallis H test, and differences between two groups were tested with the Mann-Whitney U test. P – probability with p < 0.05 deemed as significant.

There is a statistically significant difference between the antemortem group and the control group in ovalocytes, dacryocytes, annulocytes, echinocytes, stomatocytes, spherocytes, reticulocytes, and target cells. Statisticaly significant difference was found between control and antemortem group exposed to 41°C in ovalocytes, spherocytes, reticulocytes, dacryocytes, annulocytes, echinocytes, stomatocytes, and target cells, while the difference between the control group and antemortem at 44°C exposure is in ovalocytes, annulocytes, spherocytes, reticulocytes and target cell. There was no difference between antemortem at 41°C and 44°C (Tables 4 and 5).

	Temperature 41°C			Temperature 44°C
	Median	Per 25	Per 75	Median	Per 25	Per 75	P
Ovalocytes	9	4	13	3	1	10	0.094
Dacryocytes	7	5	26	16	8	19	0.481
Annulocytes	50	3	55	100	28	123	0.110
Echinocytes	8	4	59	7	1	13	0.405
Stomatocytes	10	2	51	15	8	26	0.698
Drepanocytes	0	0	0	0	0	0	1.000
Schistocytes	1	1	2	1	1	2	1.000
Leptocytes	0	0	0	0	0	0	1.000
Acanthocytes	0	0	1	0	0	1	0.591
Spherocytes	47	40	84	46	25	54	0.481
Reticulocytes	8	3	11	4	1	10	0.304
Target cells	2	1	4	1	1	2	0.584

Table 5.

Differences in poikilocytotic forms between postmortem groups at 41°C and 44°C.

Differences in values are tested with Mann-Whitney U test, p - probability with p < 0.05 deemed as significant.

When comparing rats’ antemortem and postmortem groups exposed to a water temperature of 41°C, there are significant differences in the presence of spherocytes, reticulocytes, and target cells (Table 6).

	Antemortem			Postmortem
	Temperature 41°C			Temperature 41°C
	Median	Per 25	Per 75	Median	Per 25	Per 75	P
Ovalocytes	3.5	1	6	9	4	13	,051
Dacryocytes	8.5	1	12	7	5	26	,295
Annulocytes	39.5	31	55	50	3	55	,731
Echinocytes	2.5	0	38	8	4	59	,445
Stomatocytes	10	4	22	10	2	51	,731
Drepanocytes	0	0	0	0	0	0	1000
Schistocytes	1	1	6	1	1	2	,945
Leptocytes	0	0	0	0	0	0	1000
Acanthocytes	0	0	1	0	0	1	,836
Spherocytes	1	1	8	47	40	84	,001^*
Reticulocytes	1.5	0	2	8	3	11	0,041
Target cells	24.5	20	34	2	1	4	,001^*

Table 6.

Differences in poikilocytotic forms between antemortem and postmortem groups at 41°C.

*

Represents a significant difference between groups.

When comparing rats’ antemortem and postmortem exposed to a water temperature of 44°C, a significant difference in dacryocytes and spherocytes was observed (Table 7).

	Antemortem			Postmortem
	Temperature 44°C			Temperature 44°C
	Median	Per 25	Per 75	Median	Per 25	Per 75	P
Ovalocytes	3	2	3	3	1	10	,902
Dacryocytes	5	2	9	16	8	19	,002^*
Annulocytes	47	25	74	100	28	123	,165
Echinocytes	0	0	15	7	1	13	,318
Stomatocytes	17	6	35	15	8	26	1000
Drepanocytes	0	0	0	0	0	0	1000
Schistocytes	1	1	2	1	1	2	,535
Leptocytes	0	0	0	0	0	0	1000
Acanthocytes	0	0	0	0	0	1	,383
Spherocytes	2	1	15	46	25	54	0,017^*
Reticulocytes	1	1	4	4	1	10	,383
Target cells	12	3	24	1	1	2	,053

Table 7.

Differences in poikilocytotic forms between antemortem and postmortem groups at 44°C.

*

Represents a significant difference between groups.

4. Discussion

The aim of the study was to develop and use an animal model of rat hyperthermia and to examine the effect of hyperthermia on erythrocyte shape and heart mass.

The rats included in the study were distributed in groups according to the water temperature to which they were exposed. The bodyweight of rats in groups formed according to the length of exposure to elevated temperature ranged from 280.14 to 325.50 grams (g). Analysis of heart weight by groups did not show a significant difference in the division into three groups according to water temperature, but by division into groups according to water temperature and length of exposure showed that the hearts of postmortem groups had significantly higher mass. The difference between cardiac weight in antemortem and postmortem measurements is due to edema, congestion, and accumulation of blood in the heart cavities as antemortem characteristics and redistribution of blood caused by thoracic dissection during the autopsy, as a postmortem response in cardiac weight [70]. In a study by Michiue et al. [71] in situ cardiac blood volume in cardiac cavities and dilatation index were higher in sudden deaths and lower in cases of bleeding, suffocation, and hyperthermia. In most cases, systolic and/or diastolic function may be reduced in heart failure. Minute volume is also reduced as well as oxygen delivery with vasoconstriction and redistribution of circulating blood. At the same time, due to reduced beating heart volume, renal perfusion is reduced, antidiuretic hormone release is increased, and water and salt retention occur. The result of increased venous pressure is the transudation of fluid into the intercellular space and the appearance of edema. With the gradual development of heart failure, compensatory mechanisms are developed that facilitate the work of the heart and improve the supply of oxygen to the tissues. As a consequence of a long-term compensatory mechanism, the myocardium hypertrophies. This is also a response to the increase in heart weight in groups that have been exposed to hyperthermia for the longest time, and later to heatstroke and experienced death due to exhaustion of compensatory mechanisms. With an increase in body temperature, cardiac output and blood pressure drop drastically and are associated with myocardial oxygen consumption. Hypoxia causes numerous injuries to the heart muscle, from subendocardial hemorrhage, myocardial necrosis, and rupture among fibrin fibers. The effect of hyperthermia on heart weight and erythrocyte shape was studied in rat embryos. An increase in the internal temperature in rats from 37–42°C also causes tachycardia and increases mean blood flow and vascular resistance by 13% [29].

In the state of heatstroke, large amounts of calcium are released from the sarcoplasmic reticulum of the heart muscle, causing a hypermetabolic state. Abnormal forms of red blood cells depending on exposure and length of exposure to higher temperatures have been demonstrated. There is a statistically significant difference between the experimental groups and the control group in ovalocytes, dacryocytes, annulocytes, echinocytes, stomatocytes, spherocytes, reticulocytes, and target cells.

In the antemortem groups (41°C and 44°C) and the control group (37°C), there is a statistically significant difference in almost all poikilocytotic forms, which indicates a direct effect of temperature on erythrocyte shape in 20-minute exposure length in antemortem groups.

Hyperthermia affected changes in the percentage of certain forms of poikilocytes, especially in groups that had longer exposure to high ambient temperatures (aquatic environments). In any case, the thermal process of overheating gives the same effect as a stress reaction that can be caused in different ways and make it a nonspecific reaction.

The lowest temperature at which red blood cells undergo thermal fragmentation is 45°C [72].

In our study, the most pronounced poikilocytotic forms occurred in the postmortem groups at 41°C and 44°C by echinocyte and spherocyte type. In the antemortem group of 41°C, there is a pronounced poikilocytosis for the target cell, which is 100%, while in the antemortem group of 44°C, there is 100% anulocytosis. After statistical analysis between all groups, it is noticed that the number of expressed poikilocytes increased in postmortem groups, that is, with prolonged exposure to high temperatures. In the antemortem groups (41°C and 44°C) and the control group (37°C), there is a statistically significant difference in almost all poikilocytotic forms, which indicates a direct effect of temperature on erythrocyte shape in 20-minute exposure length in antemortem groups.

When comparing antemortem and postmortem rats exposed to a water temperature of 41° C, there are significant differences in some forms of erythrocytes (spherocytes, reticulocytes, and target cells), which suggests that poikilocytosis is more pronounced and associated with the length of exposure to high temperature than temperature between the antemortem and postmortem groups at 41°C. It has been noticed that erythrocytes in organisms that are exposed to heat for a long time are more sensitive and hemolyze very quickly. Their osmotic and mechanical resistance are significantly reduced. The assumption is that the result is damage to the erythrocyte membrane, which becomes permeable, and spherocytes with significantly reduced resistance appear in the blood. Due to erythrocyte damage, hemoglobinemia and hemoglobinuria occur and, consequently, hemolytic anemia. However, unlike erythroptosis, significant hemolysis is activated only at high temperatures with a sharp increase in hemolysis at 41°C and above [73].

When comparing rats exposed to antemortem and postmortem to a water temperature of 44°C, there are significant differences in individual erythrocyte forms (dacryocytes and spherocytes) that agrees with the results of Lucijanović et al. [74]. The higher presence of spherocytes in the blood smear is most commonly associated with anemia and the immune type of hereditary spherocytosis [75]. Mortality can occur at body temperatures of 41°C and above where erythrocytes undergo hemolysis in vivo. Metabolic processes within erythrocytes contribute to cell shape change when experiencing suicidal cell death and consequently, nonspecific poikilocytotic forms of erythrocytes occur as a result of hyperosmolarity, oxidative stress, and xenobiotic exposure [76].

Optimal erythrocyte functionality is closely related to ambient temperature. Using digital holography in the microscopic configuration, changes in erythrocyte membrane profile, mean corpuscular hemoglobin (MCH), and cell membrane fluctuations (CMF) of healthy erythrocytes under different temperatures were analyzed. Erythrocytes were exposed to an increase in temperature from 17–41°C for a period of less than 1 hour, after which holograms were recorded. Reconstruction of the obtained holograms showed that there are changes in the 3D profiles of erythrocytes. The amplitude of cell membrane fluctuation was correlated with the curvature curve of erythrocytes, and the changes observed in the indentation of erythrocytes were greater at higher temperatures. Regardless of shape changes, no changes in mean corpuscular hemoglobin concentration were observed with temperature variations [77]. In examining the effect of temperature on syringomycin E pores of lipid bilayer erythrocyte membranes, it was found that different temperatures and pore formation were only slightly affected, while inactivation was strongly influenced by elevated temperature [78]. The movement of erythrocytes through blood vessels at elevated temperature is an interesting and useful task in separating blood cells from the buffer in which they are suspended based on their size or density, and for further analysis. It has been found that increasing the temperature increases the cell-free area near the blood vessel wall due to the inertia of the cell flow after the narrowing of the blood vessel [79]. The movement of erythrocytes through the blood vessel at elevated temperature in this way (increased area without cells near the blood vessel wall), enabled the production of a hybrid microfluidic device that uses hydrodynamic forces to separate human plasma from blood cells. The blood separation device includes an inlet that is reduced by approximately 20 times to a small constrictor canal, which then opens toward a larger outlet canal with a small lateral plasma collection canal. When tested, the device separated plasma from whole blood using a wide range of flow rates, between 50 and 200 microl/min, at higher flow rates injected manually and at temperatures ranging from 23 to 50°C, resulting in an increase in the cell-free layer to 250%. It was also tested continuously using between 5% and 40% of erythrocytes in plasma and whole blood without channel blockage or cell hemolysis. The mean percentage of plasma collected after separation was 3.47% from a 1 ml sample. The change in temperature also affected the number of cells removed from the plasma, which was between 93.5 +/− 0.65% and 97.01 +/− 0.3% at 26.9–37°C, respectively, using the flow rate from 100 microl/min. Due to its ability to work in a wide range of conditions, it is envisaged that this device can be used in in vitro “lab on a chip” applications, as well as a hand-held care device (POC) [80].

During cardiopulmonary bypass surgery, perfusion at low temperatures (33–35°C) is recommended to avoid high-temperature cerebral hyperthermia during and after surgery. Also, high body temperatures (40–41°C) affect proteins in both blood plasma and those involved in building red blood cells. The ideal temperature for uncomplicated cardiac surgery is still an unresolved issue. Precisely because of this, the goal of scientific studies was to establish the effect of both low and high temperatures on blood flow and viscosity through blood vessels.

In a study examining the effects of low temperature on blood viscosity, the aim was to determine the effects of temperature, shear rate, hematocrit, and various volume expanders on blood viscosity in conditions that mimic deep hypothermia in cardiac surgery. Dilutions were prepared to 35%, 30%, 22.5%, and 15% hematocrit using plasma, 0.9% NaCl, 5% human albumin, and 6% hydroxyethyl starch. Viscosity was measured in the range of shear rates (4.5–450 s (−1)) and temperature (0–37°C). A parametric expression for predicting blood viscosity based on the studied variables was developed and its agreement with the measured values was examined. Viscosity was higher at low-shear rates and low temperatures, especially at temperatures below 15°C. Reducing hematocrit, especially to less than 22.5%, reduces viscosity. The theoretical model for blood viscosity predicts independent effects of temperature, shear rate, and hemodilution on viscosity over a wide range of physiological conditions, including thermal extremes of deep hypothermia in an experimental setting. Moderate hemodilution to hematocrit of 22% reduced blood viscosity by 30%–50% at a blood temperature of 15°C, indicating the potential to improve microcirculatory perfusion during deep hypothermia [81]. In a study investigating the effects of elevated temperature, it was investigated at which temperature the breakdown of blood plasma proteins occurs after 2 hours of heat exposure. As a result, blood plasma proteins were exposed to heat in the range of 37–50°C for 2 hours. Protein degradation was first established between 43 and 45°C exposure to heat [82]. The importance of the influence of temperature on the cellular elements of blood, its proteins, and thus on its viscosity, has conditioned a large number of scientific researches that have dealt with this problem. Blood viscosity measurements are widely used to monitor patients during and after surgery, which requires the development of a high-precision viscometer that uses a minimum amount of blood. The devices were also used to construct blood viscosity models based on temperature, shear rate, and anticoagulant concentration.

The model has an R-square value of 0.950. Finally, the protein content of the blood can be altered to simulate disease states. Simulated disease states were clearly detected by comparing the estimated viscosity values using the model and the measured values using the device, which demonstrated the applicability of the setting in anomaly detection and disease diagnosis [83]. Taking into account the influence of temperature on erythrocyte shape, blood plasma proteins, and blood viscosity, the optimal temperature for human life activity was determined, assuming that this parameter corresponds to the most intensive oxygen transport in arteries and the most intensive chemical reactions in cells. It was found that oxygen transport mainly depends on blood oxygen saturation and blood plasma viscosity, with both parameters depending on blood temperature and acid-base balance. Additional parameters that affect the volume of erythrocytes and, accordingly, the temperature of the most intensive oxygen transport are taken into account. It is assumed that erythrocytes affect the shear viscosity of the blood in the same way because the impurity particles change the viscosity of the suspension. It has been shown that the optimum temperature is 36.6°C under normal ambient conditions [84].

5. Conclusion

In this study, in antemortem groups, water temperature directly affected morphological forms of erythrocytes, while in postmortem groups, the length of body exposure to high temperature was more important than the direct temperature on the morphological characteristics of red blood cells. Hyperthermia affected the changes in the percentage of certain forms of poikilocytes, especially in the groups that had a longer exposure to high temperatures of the aquatic environment. Heart mass varied with the length of exposure and the duration of debilitating compensatory mechanisms.