a b s t r a c t

Background: Heatstroke is one of the most common clinical emergencies. Heatstroke that occurred in a dry- heat environment such as desert is usually more seriously effective and often leads to death. However, the report of the pathophysiologic mechanisms about heatstroke in dry-heat environment of desert has not been seen.

Objectives: Our objectives are to establish a rat model of heatstroke of dry-heat environment of desert, to assess the different degrees of damage of organ, and to preliminarily discuss the mechanism of heatstroke in dry-heat environment of desert.

Methods: The first step, we have established a rat heatstroke model of dry heat environment of desert. The second step, we have accessed changes in morphology and blood indicators of heatstroke rats in dry-heat environment of desert.

Results: The heatstroke rats have expressed the changing characteristics of mean arterial pressure, core temperature, and heart rate. The organ damage changed from mild to serious level, specifically in the morphology and blood enzymology parameters such as alanine aminotransferase, aspartate aminotransfer- ase, creatinine, urea, Uric acid, creatine kinase-MB, creatine kinase, and blood gas parameters such as base excess extracellular fluid and bicarbonate ions (HCO₃-).

Conclusions: We have successfully established the rat heatstroke model of dry-heat environment of desert. We have identified heatstroke rats that presented changing characteristics on physiological indicators and varying degrees of organ damage, which are aggravated by the evolution of heatstroke in dry-heat environment of desert. We have preliminarily discussed the mechanism of heatstroke in dry-heat environment of desert.

(C) 2014

Introduction

Heatstroke is a life-threatening disease characterized by high body temperature and symptoms of central nervous system [1]. Manifested as delirium, convulsions, coma, and high temperature, heatstroke is often fatal [2]; even if not fatal, it often leaves permanent nerve damage. So far, we still lack satisfactory prevention and treatment of heatstroke.

People who are exposed to the dry-heat environment of desert [3], such as workers, soldiers, travelers, and explorers in the desert, risk a higher incidence of heatstroke [4]. Dry-heat environment of desert has a series features such as extremely hot and dry climate, much temperature difference, strong ultraviolet radiation, less

? Funding: This study was funded by “The Clinical Medicine Major Projects of New and High Technology Research of the PLA (2010gxjs016).”

* Corresponding author. Department of Hepatobiliary Surgery, Urumqi General Hospital of Lanzhou Military Region of PLA, Urumqi 830000, PR China. Tel.: +86 0991 4991830.

E-mail address: [email protected] (J.W. Liu).

rainfall and water resources, poor water quality, and sparse vegeta- tion and population.

Currently, the world desert area is approximately 48000,000 km², accounting for approximately 10% of the total area of the world, and these data do not include any area of the large human settlements and special workplaces, which also have the same features as the dry-heat environment.

Discovering the mechanisms of heatstroke in the dry-heat environment becomes the most urgent task.

The objectives of this study are the following: first, to establish a rat model of heatstroke in dry-heat environment of desert for the study of the pathogenesis of heatstroke in dry-heat environment, and second, to further study the organ injury during the evaluation of heatstroke, such as liver, kidney, heart, and lung. In this study, we used a simulated climate cabin to establish the dry-heat environment of desert; we monitored the changes in mean arterial pressure (MAP), heart rate (HR), and core temperature (Tc) of rats to diagnose heatstroke; and we divided the process of heatstroke into 3 phases. Then, we collected the blood and isolated organs of rats at each phase to have a further study about changing Organ function and organ morphology.

http://dx.doi.org/10.1016/j.ajem.2014.02.017

0735-6757/(C) 2014

Materials and methods

Animals

Adult male Sprague-Dawley rats (weighing 338-372 g) were obtained from the Animal Resource Center of Xinjiang Medical university hospital of China. The animals were housed individually at an ambient temperature of 22?C +- 1?C with a 12-hour light-dark cycle. Pelleted rat chow and tap water were administered ad libitum. The experimental protocol was approved by the Animal Committee of the Xinjiang Medical University Hospital of China. Animal care and experiments were conducted according to the National Science Council guidelines.

Anesthetization and intubation

Adequate anesthetization was maintained to abolish the corneal reflex and pain reflex induced by tail pinching throughout all experiments by an intraperitoneal dose of sodium pentobarbital (50 mg/kg of body weight). The rat was anaesthetized and posed in a supine position, its right femoral artery was separated, and then a venous indwelling needle (24 G) was intubated in the right femoral artery. The process of intubation was performed carefully and must be sure to keep the injury to a minimum while in the process of intubation. All rats were killed by taking off the cervical spine.

Establishment of heatstroke model

Experiments were carried out in 2 steps: step 1, 16 anaesthetized rats were randomly divided into the following groups. Each group had 8 rats. (a) A control group was placed in a normothermic environment (25?C, 35% humidity) continuously; (b) a heatstroke group was placed in a cabin (The Simulated Climate Cabin for Special Environment of Northwest of China, Urumqi of China), which simulated the dry-heat environment of desert (41?C, 10% humidity), continuously until death. Each rat was anesthetized and then fixed in a supine position. Its right femoral artery was separated by intubating a venous indwelling needle (24 G), 3 electrodes were attached to its limbs, and a precise mercury-in-glass thermometer was inserted into its anus and pushed to the area of rectum. After that, rats were allowed 20 minutes to calm down in a normothermic environment (25?C, 35% humidity). Then the rats in the heatstroke group were moved into the cabin of simulation of dry-heat environment of desert (41?C 10% humidity), and the rats in the control group were still placed in the normothermic environment (25?C, 35% humidity). At the same time, the rats in the 2 groups were connected to the multichannel polygraph ( BL420F; Taimeng, Chengdu, China) by the pressure sensor (which was linked to the venous indwelling needle) and electrocardiogram sensor (which was linked to the electrode). Thus, the dynamic changes in MAP and HR were monitored by the multichannel polygraph, and the dynamic changes in Tc were monitored by the thermometer. Based on these results, we divided the heatstroke process into 3 phases of mild heatstroke, moderate heatstroke, and serious heatstroke and calcu- lated the median time point of each phase.

Changes in morphology and blood indicators

Step 2, 48 anesthetized rats were randomized into the following groups: (c) mild heatstroke group and (d) its control group, (e) moderate heatstroke group and (f) its control group, (g) serious heatstroke group and (h) its control group. Each group had 8 rats. All the rats were treated the same as the rats in step 1. Then, the rats of the 3 heatstroke groups were connected to multichannel polygraph by the pressure sensors and electrocardiogram sensors and moved into the cabin of dry-heat environment of desert, and the rats of the

3 control groups were still placed in the normothermic environ- ment. Continuously monitoring each heatstroke group in the cabin until each heatstroke group presented signs of each heatstroke phase, we immediately collected the blood of the abdominal aorta and isolated organs in each heatstroke group and its control group at the median time point of each heatstroke phase. The blood was used for detecting blood indicators, and the organs were used for a further morphology study.

Outcome measurements

Blood enzymology parameters such as alanine aminotransferase, aspartate aminotransferase, creatinine, urea, uric acid, creatine kinase- MB, and creatine kinase were examined by an automatic biochemical analyzer (Abbott Aeroset, Abbott Park, IL). Blood gas parameters such as base excess extracellular fluid (BEecf), bicarbonate ions (HCO₃-) were examined by a Blood gas analyzer (ABL-800; Radiometer, Copenhagen, Denmark).

The tissues (liver, kidney, heart, and lung) were isolated from the animals by killing them, and the tissues were fixed in Bouin’s solu- tion and embedded in paraffin blocks. Sections of 5 mm were ob- tained, deparaffinized, and stained with hematoxylin and eosin. The tissue was examined, evaluated, and photographed in random order under blindfold conditions with standard light microscope (Optiphot 2; Nikon, Tokyo, Japan).

For electron microscopic observation, specimens in 1 x 2 mm size were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, and after primary fixations, tissues were washed in 0.1 M phosphate buffer overnight. The tissues were postfixed with 1% osmium tetroxide in phosphate buffer for 1 hour at 4?C. Then, the postfixed tissues were washed in 0.1 M phosphate buffer and dehydrated by graded ethyl alcohol and finally with propyleneoxide. Then the dehydrated tissues were processed for making araldite blocks. Ultrathin section were obtained by ultramicrotome (RMC-MTX Ultramicrotome, Boeckler Instruments, Tuscon, Arizona) and collected on copper grids for double staining (uranyl acetate and Reynold’s lead citrate). Stained sections were finally observed under an electron microscope (JEM-1230; JEOL, Tokyo, Japan).

Statistical analysis

Data are expressed as mean +- SD. Statistical analysis was per- formed using the program SPSS 15.0 (SPSS, Chicago, IL). The 1-sample Shapiro-Wilk test was used to examine whether the values in the 6 individual groups follow a normal distribution. The test indicated that values were normally distributed. The 1-way analysis of variance was used to test the difference between multiple means of the inde- pendent groups. The Duncan new multiple range method was used for multiple comparisons of the independent groups. P b .05 was considered statistically significant.

Result

Core temperature

As shown in Fig. 1a, after being placed in the cabin of dry-heat environment, at the first 20 minutes, Tc almost remained unchanged and remained at 35.41?C +- 0.54?C. Then Tc rose continuously until the highest point of 42.63?C +- 1.00?C at the 170th minute was reached. Rats died immediately while Tc reached the highest point. Then Tc stopped rising and drop slightly to 42.38?C +- 1.14?C at the 175th minute.

Heart rate

As shown in Fig. 1b, at the first 30 minutes, HR almost remained unchanged and remained at 401.75 +- 45.03 beats per minute. From the 30th to 85th minute, HR fluctuated in a tight range and declined to

Fig. 1. Compared with control rats of normothermic environment, the dry-heat environment-exposed rats presented significantly different changes in Tc, HR, and MAP. The dry-heat exposure was commenced at 0 minute for 175 minutes. Core temperature, HR, and MAP were monitored at a 5-minute interval for 175 minutes. Data are expressed as mean +- SD. +P b .01 compared with control group; *P b .05 compared with control group.

353.13 +- 32.11 beats per minute. From the 85th minute, HR rose continuously until the highest point of 543.25 +- 32.31 beats per minute at the 155th minute was reached. Then HR fell sharply to

213.25 +- 43.68 beats per minute, and at the same time, the rats died. We saw an interesting phenomenon that, even when the rats already died, HR still remained 20.63 +- 4.17 minutes at a level of

213.25 +- 43.68 beats per minute until it could be detected. From being placed into the cabin to death, the survival time of heatstroke rats lasted 171.38 +- 10.50 minutes.

Mean arterial pressure

At the first 40 minutes, MAP almost remained unchanged and remained at 118.38 +- 6.61 mm Hg. From the 40th to 100th minute, MAP fluctuated in a tight range and declined to 103.25 +-

22.69 mm Hg. From the 100th to 145th minute, MAP rose and reached at the highest point of 134.00 +- 23.10 mm Hg. Then MAP declined rapidly to 0 mmHg. Rats died immediately while MAP fell below 50 mm Hg.

Heatstroke phase

Generally speaking, Tc almost rose from start to finish, HR and MAP fluctuated after remaining steady for a short period, then rose up to a high level for a short period, and declined suddenly and

separately. According to these changes, we divided the process of heatstroke in dry-heat environment into mild, moderate, and serious; the 3 phases mainly depend on the change in Tc. During the first 30 minutes, Tc, HR, and MAP almost maintained a relatively steady state. From the 30th to 85th minute, the Tc continuously rose from 35.89?C +- 0.80?C to 39.10?C +- 1.22?C, the HR fluctuated in a tight range, and MAP dropped down slightly. We classified this period as mild heatstroke phase. (To facilitate differentiating, we designated the interval during which Tc rose from 36?C-39?C as the mild heatstroke phase.) From 85th to 125th minute, Tc continuously rose from 39.10?C +- 1.22?C to 40.95?C +- 1.04?C, HR rose slightly, and MAP still remained at a slight decline and then rose slightly to reach the level of control group. We classified this period as mode- rate heatstroke phase. (To facilitate differentiating, we designated the interval during which Tc rose from 39?C-41?C as the moderate heatstroke phase.) From the 125th minute to the end, Tc continu- ously rose from 40.95?C +- 1.04?C to the highest point of 42.38?C +- 1.14?C, HR rose to a significantly high level and then declined rapidly, and MAP continuously rose slightly to a relatively high level and then declined rapidly until it could be detected. So we classified this period as serious heatstroke phase. (To facilitate differentiating, we designated the interval during which Tc rose beyond 41?C as the serious heatstroke phase. The instant in which the HR rose to a value of 100 beats per minute compared with basic level was also taken as the start of serious heatstroke phase.)

We have calculated the median time point of 37.5?C (37.5?C is

the average Tc value of mild phase.), 40?C (40?C is the average Tc value of moderate phase), and 41.8?C (41.8?C is the average Tc value of serious phase.). The 3 median time points of each phase are the

52.5th minute, the 100th minute, and the 145th minute.

Organ damage

At the second experiment, compared with the rats of each control group, rats of 3 heatstroke phases expressed the changing charac- teristics on morphology of liver, kidney, heart, and lung and also blood indicators of each organ such as alanine aminotransferase, aspartate aminotransferase, creatinine, urea, uric acid, creatine kinase-MB, creatine kinase, BEecf, and HCO₃-. Combining the changes in enzymology, blood gas parameters, and morphology, it indicated that they were basically consistent. It also showed that the damage to organs was aggravated with the time prolonging.

Morphology of normothermic control groups

Each organ of the 3 normothermic control groups was observed without any obvious change under the light microscope and the electron microscope, and each organ was observed without obvious difference between the 3 normothermic control groups.

Changes in morphology under light microscope

Liver. In the mild group, significant Pathologic changes ap- peared; part of hepatic sinusoids, part of central veins, and part of portal area vessels presented dilatation and congestion. The structure of hepatic lobules was still normal. In the moderate group, further changes appeared. Part of the hepatocytes presented acidophilic de- generation, and particle-like bleeding could be seen in part of hepatic mesenchyme occasionally. In the serious group, dilatation and con- gestion of hepatic sinusoids, central veins, and portal area vessels became progressively aggravated. The number of swelling hepato- cytes and acidophilic degeneration increased. The structure of normal liver became blurred.

Kidney. In the mild group, obvious pathologic changes could be seen in the kidney tissue. Bowman capsule became narrow. Epithelial

cells of proximal convoluted tubules presented edema, and the lumens of tubules became narrow. Part of interstitial vessels presented angiectasis and hyperemia, and some of them presented a high degree of angiectasis and hyperemia. The mesenchyme of distal convoluted tubules presented obvious hyperemia. In the moderate group, except the previous changes of the mild group, there appeared some further changes in this group. Part of capillaries of renal paren- chyma presented extensive dilatation and congestion. The degree of narrowing of Bowman capsule became aggravated. In the serious group, angiectasis and hyperemia of renal mesenchyme became progressively aggravated. Angiectasis and hyperemia of glomerulus became progressively aggravated. The degree of narrowing of Bowman capsule became further aggravated, and the protein casts could be seen in lumens of proximal convoluted tubules occasionally, and also some thrombosis could be seen in part of lumens of tubules.

• Heart. In the mild group, part of myocardium presented mild hyperemia and edema, and punctate hemorrhages could be observed in the myocardium occasionally. Myocardial fibers still arranged regularly. In the moderate group, the dilatation and congestion of myocardial vessels became further aggravated, tiny capillaries of myocardium presented mild congestion, and punctate hemorrhage increased. Myocardial mesenchyme presented edema. In the serious group, the dilatation and congestion of myocardium vessels became progressively aggravated; a large number of punctate hemorrhage existed. Interstitial edema of myocardium became progressively aggravated, myocardial mesenchyme thickened, and interstitial vessels of myocardium presented dilatation. Part of myocardial fibers fractured.

• Lung. In the mild group, a minority of pulmonary alveoli presented emphysema, a minority of pulmonary alveolar walls mildly thickened, and capillary congestion and punctate hemorrhage appeared in the thickened alveolar walls. The structure of the majority of pulmonary alveoli was basically normal. In the moderate group, part of pulmonary alveolar walls thickened, and vascular congestion appeared in the alveolar walls; the extent was more serious than the mild group. A small number of inflammatory cells infiltrations appeared. Some thrombosis could be seen occasionally in part of blood vessels. In the serious group, most of pulmonary alveolar walls thickened, and there was obvious vascular hyperemia in it. Patches of inflammatory cells infiltrations appeared in part of lung tissue.

  Changes in morphology under electron microscope

  Liver. In the mild group, hepatocytes presented edema, and the gap between hepatocytes widened. The number of glycogen granules was decreased. A proliferation of mitochondria appeared. In the moderate group, the number of glycogen granules was further reduced. The smooth endoplasmic reticulum and the capillary bile duct proliferated. The number of marrow shaped structures increased. The number of dual-core and multinucleated cells increased. In the serious group, there is almost no glycogen granule to be seen. The rough endoplasmic reticulum presented hyperplasia. The part of hepatic sinusoids presented atresia and edema. The part of hepatic sinusoids and part of surface villi of hepatocytes membrane presented unclear structures. The Endothelial cells presented edema, and physiologic changes“>neutrophils could be seen in part of hepatic sinusoids.

• Kidney. In the mild group, endothelial cells presented slight edema, and arch-like changes appeared. Podocytes presented slight edema, and foot processes presented microvilli change. In the mode- rate group, there appeared some further changes except the previous changes of the mild heatstroke group. The edema of endothelial cells became progressively aggravated. Deposition of erythrocytes was

  observed in blood vessels, and some other sediments were also could be observed. The gap between tubular membranes widened. In the serious group, epithelial cells of tubules presented edema, and some of them presented ballooning degeneration, and the density of matrix reduced. Mitochondria of tubules presented proliferation. Lysosomes could be observed. Mitochondria presented edema, and vacuolar degeneration and the density of matrix reduced. Exfoliated cells appeared in the lumens of tubules. Endothelial cells of capillaries presented serious edema, and capillaries presented a state of adverse opening. Sediments appeared in lumens of vessels. Mesangial area widened, and mesangial cells presented proliferation. Bleedings and broken cells appeared in the mesenchyme between tubules.

  • Heart. In the mild group, the accumulations formed by a large number of mitochondria appeared and presented like protuberances under the sarcolemma. More mitochondria could be seen near the nuclei. Individual muscle fiber presented edema. Individual muscle bundle dissolved. Papillary proliferation of endothelial cells appeared. In the moderate group, intravascular blood deposition appeared, mitochondria increased, glycogen particles reduced, and swelling endothelial cells appeared. Nuclei of myocardial cells concentrated at the edge; heterochromatin increased. Macrophages appeared and the mitochondria of macrophages presented vacuolar degeneration. In the serious group, the sarcolemma presented incomplete shape and many organelles and some erythrocytes could be seen between muscle cells. Z line disappeared and the myofilament arranged in an extreme disorder. Part of muscle fibers dissolved. Lymphocytes ap- peared in capillaries between myocardial cells. Mitochondria pre- sented slight edema and its matrix density decreased. Capillary endothelium presented papillary hyperplasia.

  • Lung. In the mild group, some sediments of erythrocytes appeared in capillary lumens. A slight expansion of endoplasmic reticulum appeared in plasma cells. The structure of pneumocytes type II was still normal. In the moderate group, erythrocytes could be seen in alveolar spaces. Increased macrophages could be seen in alveolar spaces. Alveolar spaces were basically clear. The number of pneumocytes type II and lamellar bodies increased. In the serious group, surface microvilli of pneumocytes type II decreased. Lamellar bodies reduced and vacuole-like structure appeared in pneumocytes type II. Alveolar spaces presented some deciduous myelinfigure. Alveolar spaces contained some contents. Alveolar spaces were relatively vague.

    Blood indicators

    Fig. 2 showed the changes in blood enzymology parameters and blood gas parameters of control rats and heatstroke rats (x +- s, n = 8). Each indicator changed significantly during the 3 phases (P b .05). Each indicator changed significantly compared with its control group at each phase (P b .05), except the difference of BEecf between the mild and moderate heatstroke phases (P N .05), as shown in Fig. 2h.

    Discussion

    Past studies of heatstroke mainly concentrated on the aspect of the moist-heat environment. This study firstly discussed heatstroke in dry-heat environment of desert.

    Physiologic changes

    Generally speaking, compared with some studies about heat- stroke model of moist-heat environment from physiologic indicators, this study presented its own characteristics. The same point was that, after being exposed to the dry-heat environment, Tc, HR, and MAP all rose to a high level. Then MAP and HR declined immediately

    

    Fig. 2. Changes in the blood indicators of normothermic control rats and dry-heat environment-exposed rats. From left side to right side in each histogram, the columns are mild heatstroke group and its control group, moderate heatstroke group and its control group, and serious heatstroke group and its control group, respectively; n = 8. Data are shown as mean +- SD. In 3 heatstroke groups, means with a different lowercase letter are significantly different (P b .05). *Compared with its control group, the heatstroke group is significantly different (P b .05).

    when they reached the highest point separately, and this point also could be observed in some heatstroke models of moist-heat envi- ronment [5]. Because rats almost died immediately when MAP and HR declined rapidly, as shown in Fig. 1b and c, it was also indicated by us that it was very important to rescue the animal or human with heatstroke in dry-heat environment before the serious heatstroke

    phase occurred. The different point was Tc, HR, and MAP presented many specific changes in this study. As shown in Fig. 1b and c, after being exposed to the dry-heat environment, MAP and HR firstly remained steady for a period, then fluctuated in a tight range, and began to rise sharply. The Tc also firstly remained steady for a short period and then began to rise as shown in Fig. 1a. Description of

    MAP, HR, and Tc in studies of heatstroke in moist-heat environment was that all of the 3 rose immediately after being exposed to the moist-heat environment [6]. It can be explained in this way that the body had a certain ability to adapt the dry-heat stress, which can keep its HR, Tc, and MAP steady while being placed in the dry-heat environment. When the body can no longer adapt to the dry-heat environment, HR, Tc, and MAP begin to rise. Heatstroke in moist-heat environment usually lasts a relatively short time, approximately 68 or some more minutes before reaching serious heatstroke state or death [7]. This experiment lasted a relatively long time, approxi- mately 175 minutes, so the presence of HR, MAP, and Tc was more specific and detailed. In the first step of this study, when MAP could not be detected, HR still lasted 20.63 +- 4.17 minutes at a level of

    213.25 +- 43.68 beats per minute. We speculated that this was an electromechanical dissociation [8].

    Some researches about heatstroke in moist-heat environment show that the tissue damage can occur at Tc greater than 42?C in rats and humans [9,10]. This was very different from our study. The reasons may be as follows: First, because of the relatively short process of some heatstroke in moist-heat environment, organ damage could not occur at the first dozens of minutes. Second, because of the relatively short process of some heatstroke in moist-heat environ- ment, researchers may seldom study organ damage, during the period that was before the onset of heatstroke.

    Why divide the process mainly by Tc? Fig. 1a showed that Tc presented the most significant changes and the most stable trend compared with HR and MAP. So dividing the process by Tc was more feasible and scientific. In some rat heatstroke models in moist-heat environment, the occurrence of both hyperthermia (Tc N 42.0?C) and hypotension (MAP b 50 mm Hg) was taken as the time point for heatstroke onset [11]. In addition, in some other rat heatstroke models, an instance in which the MAP dropped a value of 25 mm Hg from the peak was arbitrarily taken as the onset of heatstroke [5]. Why divide the heatstroke process of dry-heat environment into 3 phases and not 2 phases like some previous heatstroke models of moist-heat environment? Fig. 1 showed that the rats survived almost 175 minutes in the dry-heat environment, which was much longer than some rat heatstroke model of moist-heat environment [12,13], and in the experiments, we found that tissue damage that occurred in mild and moderate heatstroke phases also occurred in serious heatstroke phase; this was very different from some researches about heatstroke in moist-heat environment, which showed that tissue damage can occur at Tc greater than 42?C in rats and humans. So it was more appropriate to divide the process into 3 intervals. Although in some researches about the rat heatstroke model of moist-heat environment, the 42?C of Tc seemed to be the onset of heatstroke [14], why designate the interval during which Tc rose beyond 41?C as the serious heatstroke phase? Fig. 1a showed that when Tc reached 42.29?C +- 0.97?C at the 155th minute, MAP was

    121.63 +- 25.52 mm Hg and HR was 543.25 +- 32.31 beats per

    minute. Then all of them presented large variation in the next 15 minutes, including Tc that soon rose to 42.38?C +- 1.14?C, MAP that soon declined to 13.75 +- 6.30 mm Hg, and HR that soon declined to

    213.25 +- 43.68 beats per minute. So it could be observed that rat’s health was in urgent situation when Tc reached 42?C and soon it would deteriorate further. By the way, the objective of division is for rescuing critical patients and animals before the coming of dangerous phase of the disease; if designated a phase as serious phase after the dangerous phase already took place, it lost the meaning of division. So it is more appropriate to designate 41?C as the beginning of serious heatstroke phase. Why did we use HR and MAP as another basis for identifying serious heatstroke phase? Fig. 1b and c showed that during the last interval, HR and MAP presented significant difference compared with the control groups, so both of them could be used as another basis to identify the serious heatstroke phase, and it was also feasible and scientific. In short, this

    method of dividing facilitated the follow-up studies and also provided references for clinical practice.

    From the significant change of blood functional indicators, it suggested that it is useful and necessary to monitor the organ’s functional indicators while treating patients who suffer from heatstroke in dry-heat environment. From the rapidly aggravated trend of this disease, it also strongly suggested that monitoring the organ’s functional indicators should be carried on as early as possible; thus, we could get enough time to make a plan for rescuing the patient considering the outcomes.

    The performance of damaged organs by light microscope and electron microscope can visually be observed. Light microscope can observe organizational changes from a larger visual field. Electron microscope can observe them from a more detailed and accurate aspect. Both of them play important roles in this study.

    Morphological changes

    It presented that organs were damaged more and more seriously dependent on the progression of heatstroke.

    Electron microscope changes

    Liver, kidney, and heart presented proliferation of mitochondria in the mild and moderate groups. It showed that while rats were exposed to the dry-heat environment, its energy consumption increased and mitochondria presented compensatory hyperplasia [15]. Glycogen granules of liver and heart decreased gradually in the mild and moderate groups. It also showed the increased energy consumption in the dry-heat environment. Liver of moderate heatstroke group presented an increase of dual-core and multicore cells, which indicated the exuberant function of Protein Synthesis and could not be observed in the serious group. Lung of moderate group presented an increase of pneumocytes type II and lamellar bodies and then lamellar bodies decreased and vacuole-like structures appeared in pneumocytes type II in the serious group. Vacuole-like structures were deemed as substances that were transformed from the vanished lamellar bodies after the lamellar bodies were secreted to the alveolar spaces [12]. These changes of liver and lung indicated that organ functions evolved from compensatory phase to discompensatory phase while being exposed to the dry-heat environment continuous- ly. Obviously, rescuing patient at the compensatory phase is easier and more effective than at the discompensatory phase. So rescue patient as soon as possible, especially at the mild heatstroke phase while heatstroke took place in dry-heat environment in desert.

    All organs (liver, kidney, heart, and lung) presented dilation and congestion of blood vessels in all heatstroke groups (mild, moderate, and serious).

    These changes reflected that blood deposition could occur in various organs while heatstroke takes place in the dry-heat environ- ment. The blood deposited in vital organs and voluminal blood vessels dilated. Finally, sharp drop in effective Circulating blood volume led to hypovolemic shock. So use of antishock therapies such as giving plasma expander and supplying the blood volume is necessary while heatstroke takes place in dry-heat environment in desert.

    Liver, heart, and lung presented infiltrations of inflammatory cells (mainly in the serious group).

    Liver

    Electron microscope showed neutrophils in hepatic sinusoids in the serious group.

    Heart

    Electron microscope showed macrophages in the moderate group and lymphocytes in capillaries between myocardial cells in the serious group.

    4.3.3. Lung

    Light microscope showed a small amount of infiltrations of inflammatory cells in the moderate group and patches of infiltra- tions of inflammatory cells in the serious group. Electron micro- scope showed increased macrophages in alveolar spaces in the moderate group.

    It indicated that, when heatstroke took place in dry-heat envi- ronment of desert, systemic inflammatory response syndrome caused by the dry-heat environment damage appeared [16]. Systemic in- flammatory response syndrome is critical, hard to be corrected, and could lead to necrosis of tissue, disseminated intravascular coagu- lation syndrome, and multiple-organ failure, so treatments of anti- inflammatory should be applied as soon as possible while heatstroke takes place in dry-heat environment in desert, for example, suppres- sing the activity of various protease and lipid hydrolases; stabilizing structures of lysosomal membranes and regulating production of interleukin, tumor necrosis factor, and other inflammatory cytokines effectively; bettering microcirculation and tissue perfusion; and reducing damage factors [17,18].

    According to the results of this study, it is also indicated that multiple-organ dysfunction syndrome occurred during the process of heatstroke in dry-heat environment in desert.

    From this study, we observe that the function of vital organs of rat such as liver, kidney, heart, and lung was seriously damaged while heatstroke takes places in dry-heat environment in desert. We infer that the mechanism may be as follow as: tissue damage caused by dry-heat environment damage activated various Inflammatory mediators in vivo [19], which made a cascade of reactions of cyto- kines and an excessive activation of white blood cells [20]. Activated white blood cells released a large number of oxygen free radicals and lysosomal enzymes [21], which directly damaged the mem- branes of cells and capillary endothelium. Thus, all of them aggra- vated the organs damage, caused a series of complications, and even led to failures of vital organs in severe cases.

    Enzymatic detection has been used as an important basis for evaluating organ damage [22]. Because organs were rich in enzymes, once they were damaged, it may result in the changing of serum enzyme due to the increased cell permeability, cell necrosis, and so on. From Fig. 2a to g, it showed that the cardiac function, renal function, and Hepatic function were significantly damaged at the 3 heatstroke phases, especially in the serious phase. Blood gas param- eters could reflect organ function, especially the function of kidney and lung. In Fig. 2h and i, it shows that the acidosis occurred at the 3 heatstroke phases and especially in the serious phase. So if possible, take a blood test for enzymology analysis and blood gas analysis; it could easily discover the health condition of patient while heatstroke takes place in dry-heat environment. Actually, in recent years, some portable blood gas analyzer and biochemistry analyzer are applied in clinics. When a patient is affected suddenly by heatstroke in dry- heat environment in desert and then large equipment cannot be carried to the spot in time, the portable devices are the most useful tools for diagnosis and assessment of health.

    All organs (liver, kidney, heart, and lung) presented tissue damage in all heatstroke groups (mild, moderate, and serious). All these changes reflected that the damage to vital organs took place during the process of heatstroke in dry-heat environment.

    Combining the changes in enzymology, blood gas parameters, and morphology, it indicated that they were basically consistent. In sum- mary, the rat heatstroke model in dry-heat environment presented its unique features of morphology, physiology, zymology, and blood gas parameters. Because the anesthetization was used in the expe- riments, the impact of the anesthetic on the results of this study still needs to be discussed, and this is a limitation of the present study.

    This experiment firstly discussed the mechanisms of heatstroke in dry-heat environment of desert. It provided a possibility for a further study of heatstroke in dry-heat environment. It also pro- vided a theoretical basis for a further study of multiple-organ dys- function syndrome induced by heatstroke in dry-heat environment. This experiment firstly established a rat heatstroke model of dry- heat environment and divided it into mild, moderate, and serious phases. It created the conditions for a further implementation of drug interventions.

    Acknowledgments

    The authors wish to thank Jiang Yin Song and Zhou Lan for the helpful advice on hematoxylin and eosin staining and also the tech- nical assistance in intubation and blood collection.

    References

    1. Abookasis D, Zafrir E, Nesher E, et al. Diffuse near-infrared reflectance spec- troscopy during heatstroke in a mouse model: pilot study. J Biomed Opt 2012;17 (10):105009 [PubMed: 23085983].
    2. Yan YE, Zhao YQ, Wang H, et al. Pathophysiological factors underlying heatstroke. Med Hypotheses 2006;67(3):609-17 [PubMed:16631316].
    3. Knochel JP. Environmental heat illness: an eclectic reviews. Arch Intern Med 1974;133(5):841-64 [PubMed:4821779].
    4. Montain SJ, Sawka MN, Cadarette BS, et al. Physiological tolerance to uncompen- sable heat stress: effects of exercise intensity, protective clothing, and climate. J Appl Physiol 1994;77(1):216-22 [PubMed:4821779].
    5. Yang HH, Chang CP, Cheng JT, et al. Inhibition of acute lung inflammation and injury is a target of brain cooling after heatstroke injury. J Trauma 2010;69

       (4):805-12 [PubMed:4821779].

       Yang HH, Hou CC, Lin MT, et al. Attenuating heat-induced acute lung inflam- mation and injury by dextromethorphan in rats. Am J Respir Cell Mol Biol 2012;46

       (3):407-13 [PubMed: 22033269].

       Lin CY, Hsu CC, Lin MT, et al. Flutamide, an androgen receptor antagonist, im- proves heatstroke outcomes in mice. Eur J Pharmacol 2012;688(1-3):62-7 [PubMed: 22609231].

    6. Yanagihara AA, Shohet RV. Cubozoan venom-induced Cardiovascular collapse is caused by hyperkalemia and prevented by zinc gluconate in mice. PLoS One 2012;7(12):e51368 [PubMed: 23251508].
    7. Bynum GD, Pandolf KB, Schuette WH, et al. Induced hyperthermia in sedated humans and the concept of critical thermal maximum. Am J Physiol Regul Integr Comp Physiol 1978;235(5):R228-36 [PubMed: 727284].
    8. Hubbard RW, Matthew WT, Criss REL, et al. Role of physical effort in the etiology of rat heatstroke injury and mortality. J Appl Physiol Respir Environ Exerc Physiol 1978;45(3):463-8 [PubMed: 727284].
    9. Lin XJ, Li YJ, Li ZL, et al. Pre-existing lipopolysaccharide may increase the risk of heatstroke in rats. Am J Med Sci 2009;337(4):265-70 [PubMed: 19365172].
    10. Wu WS, Chou MT, Chao CM, et al. Melatonin reduces acute lung inflammation, edema, and hemorrhage in heatstroke rats. Acta Pharmacol Sin 2012;33

        (6):775-82 [PubMed: 22609835].

        Yang TH, Ho WY, Shih MF, et al. Effects of combination treatment with dexa- methasone and mannitol on neuronal damage and survival in experimental heat stroke. Biol Pharm Bull 2010;33(9):1522-8 [PubMed: 20823568].

    11. Hsu SF, Niu KC, Lin CL, et al. Brain cooling causes attenuation of cerebral oxidative stress, systemic inflammation, activated coagulation, and tissue ischemia/injury during heatstroke. Shock 2006;26(2):210-20 [PubMed: 16878031].
    12. Liu CT, Brooks GA. Mild heat stress induces mitochondrial biogenesis in C2C12 myotubes. J ApplPhysiol 2012;112(3):354-61 [PubMed: 22052865].
    13. Chen Y, Tong H, Zhang X, et al. Xuebijing injection alleviates liver injury by inhibiting secretory function of Kupffer cells in heatstroke rats. J Tradit Chin Med 2013;33(2):243-9 [PubMed: 23789225].
    14. Li L, Pan J, Yu Y. Development of sorbent therapy for multiple organ dysfunction syndrome (MODS). Biomed Mater 2007;2(2):R12-6 [PubMed: 18458435].
    15. Chang CK, Chang CP, Chiu WT, et al. Prevention and repair of circulatory shock and cerebral ischemia/injury by various agents in experimental heatstroke. Curr Med Chem 2006;13(26):3145-54 [PubMed: 17168703].
    16. Heled Y, Fleischmann C, Epstein Y. Cytokines and their role in hyperthermia and Heat stroke. J Basic Clin Physiol Pharmacol 2013;24(2):85-96 [PubMed: 23509213].
    17. Leon LR, Helwig BG. Heat stroke: role of the systemic inflammatory response. J Appl Physiol 2010;109(6):1980-8 [PubMed: 20522730].
    18. Lambert GP, Gisolfi CV, Berg DJ, et al. Selected contribution: hyperthermia- induced intestinal permeability and the role of oxidative and nitrosative stress. J Appl Physiol 2002;92(4):1750-61 [PubMed: 11896046].
    19. Alzeer AH, el-Hazmi MA, Warsy AS, et al. Serum enzymes in heat stroke: prog- nostic implication. Clin Chem 1997;43(7):1182-7 [PubMed: 9216454].