**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

*Impact of Temperature on Morphological Characteristics of Erythrocytes and Heart Weight… DOI: http://dx.doi.org/10.5772/intechopen.105101*

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

*Impact of Temperature on Morphological Characteristics of Erythrocytes and Heart Weight… DOI: http://dx.doi.org/10.5772/intechopen.105101*

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 [36–40].

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 [46–48]. 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 *Impact of Temperature on Morphological Characteristics of Erythrocytes and Heart Weight… DOI: http://dx.doi.org/10.5772/intechopen.105101*

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 [53–56].

#### **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.
