**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**).

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

#### **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**).

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**).

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**).


*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).*

#### **Table 1.**

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

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**).

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

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


*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).*

#### **Table 2.**

*Mean values of rat heart mass 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 3.**

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


**Table 4.**

*Differences in poikilocytotic forms between antemortem group and control groups.*

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

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**).

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**).

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**).


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

#### **Table 5.**

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


#### **Table 6.**

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

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


#### **Table 7.**

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

### **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

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

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.

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