Heat stroke (HS) is an acute heat-induced disorder of central nervous system and/or cardiovascular system characterized by disturbances in heat balance and/or salt and water metabolism under high temperature and high humidity. Patients with HS manifest symptoms of hyperthermia (core temperatures [Tc] >40°C), disorders of central nervous system (delirium, convulsion, or coma), and hyperpyrexia with systemic inflammation responses, which induce multiple organ dysfunction syndromes (MODS). Classic HS primarily occurs in individuals with immune dysfunction during increasing heat waves every year (1–4). The average mortality rate of HS is 10% to 15% (5), which may increase to over 40% in severe cases with MODS (6). Cytokines such as interleukin (IL)-1β (7, 8), IL-1 (8, 9), tumor necrosis factor-α (7, 8), and IL-12p40 (9) produced by heat stress disturb the thermoregulatory mechanisms of sweating and blood vascular system, and alter the vascular permeability, and organ microcirculation, which induces hypotension, hyperthermia, inflammatory response, MODS, and even multiple organ failure.
Microcirculation mainly ensures adequate oxygen supply to the tissues (10). Microcirculation disorder usually presents decreased microvascular blood flow; damage to endothelial cells and high vascular permeability; lower microvascular reactivity; and leukocyte adhesion, incarceration, and migration. Studies suggested that microcirculatory disorders were closely correlated with MODS and directly affected the prognosis of hemorrhagic or septic shock (11, 12). Oxygen free radicals produced in the interaction between endothelium and leukocytes contributed to microcirculatory disorders in an ischemia/reperfusion model, similar to hemorrhagic shock (13). Recent study also found that not only pretreatment but also posttreatment of antioxidant could attenuate microcirculation disorder (14, 15).
In severe HS, microcirculation disorders are inextricably associated with pathophysiological processes, including circulatory failure, tissue edema, and bacterial translocations. As reported in a previous study by our group, heat stress caused explosive increase of ROS, which in turn led to apoptosis of endothelialcells and probably altered endothelial permeability (16). However, microcirculatory changes in heat stress and the role of oxidative stress in microcirculatory disorder remain unknown.
By reproducing the classic severe HS model of rats, this study observed the alteration of microcirculation during heat stress, and examined the relation between microcirculatory disorder and systemic hemodynamic disorder. We also investigated the role of oxidative stress in HS by observing the effect of antioxidant, superoxide dismutase (SOD), during heat stress.
MATERIALS AND METHODS
One hundred and forty-seven specific pathogen free male Wistar rats, weighing between 160 and 180 g and receiving standard diet and water, were fasted for 12 h before experiment but had free access to water. Rats were divided into three groups, i.e., HS group (HS group), group pretreated with SOD (Sigma-Aldrich, St Louis, MO, Product No.: S5395SOD) (SOD+HS group), and group pretreated with normal saline (NS+HS group). In SOD+HS group, rats were injected with 5 mg/kg SOD in 0.2 mL NS, and in NS+HS group, rats were injected with 0.2 mL NS (14). All solutions were injected through rat tail vein. Rats designated for heat stress were subjected to continuously heat stress in infant incubator (humidity: 65%, temperature: 40°C). The rectal temperatures were recorded every 10 min during heat stress, and the body weight was measured, respectively, before and after heat stress (Table 1).
All surgical interventions were performed under deep anesthesia with sodium pentobarbital (60 mg/kg/body weight) using a standardized protocol established in our laboratory. All experiments were conducted in the Key Laboratory of Shock and Microcirculation (Southern Medical University, Guangzhou, P.R. China), in accordance with the Chinese National Guidelines for the Use and Care of Experimental Animals, and approved by the Institutional Experimental Animal Ethics Committee of the Southern Medical University, Guangzhou (P.R. China). The Chinese animal guidelines adhere to the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines. All efforts were made to reduce the number of animals used and to minimize animal discomfort.
Preparation of spinotrapezius muscle and intravital microscopy
The spinotrapezius muscles located in the mid-dorsal region, which inserted onto the spine of the scapula. The preparation of spinotrapezius muscle was performed as previously described by Gray (17). Briefly, the exteriorization of spinotrapezius muscle was performed with marginal damage to the fascia, only. No evidence of local trauma, which may impact regional blood flow in this model, has been reported. Throughout the surgical preparation and experimental course, the exposed tissue was continuously superfused with a heated Krebs-Henseleit bicarbonate-buffered solution to maintain a constant 37°C temperature, humidity, pH, and ionic strength of the sample. The solution was heated to prevent loss of heat via perfusion. The exposed spinotrapezius muscle was fixed at six equidistant sites around the caudal periphery to ensure consistent shape and length of the selected vessels. Unbranched, third-order arterioles and venules (diameter range 20–60 μm) were selected at random and videotaped at 2,000 frames/s using a CR5000X2 high-speed camera (Optronis, Puchheim, Germany). The vessel diameters and erythrocyte velocities (VRBC) were assessed at a rate of 25 frames/s (18). Assuming cylindrical geometry, the blood flow rate was calculated using the formula as previously described: π × Vmean × (D/2)2, when Vmean = VRBC/1.6 and D = diameter of the vessel (18).
The prepared spinotrapezius was covered with thin gauze which was continuously superfused with a heated Krebs-Henseleit bicarbonate-buffered solution, and right femoral artery catheterization was operated. Mean arterial pressure (MAP) was observed continuously for at least 20 min until MAP was stable. SOD and NS were injected accordingly as mentioned above. One hour later, rats were subjected into infant incubator for heat stress, and microcirculation was observed by intravital microscopy. The arteriolar/venular red blood cell (RBC) velocity, vascular diameter, and threshold of norepinephrine (NE) were detected at the time before heat stress, heat stress to rats’ rectal temperature of 38°C (Tc = 38°C), and heat stress to HS at rats’ rectal temperature of 41°C (Tc = 41°C, representing the group with severe HS). The survival time and the changes of rats’ body weight were calculated after Tc = 41°C. Because the rats have to move out of the infant incubator at each time point, the microcirculation observation was conducted as fast as possible to avoid dropping of rats’ Tc (Fig. 1A).
Different concentrations of NE solutions (Table 2) were prepared, and spinotrapezius was prepared as described above. Under the intravital microscopy, appropriate vision spot was selected, and the vessel location was recorded by drawing. Different concentrations of NE solutions were added from low to high concentration in droplets (200 μL each time). The concentration was used as the arteriolar threshold to NE when the arterioles contracted and blood flow stopped for 20 s. The observation spot was repeatedly superfused with Zenker's fluid at 37°C until the vessel condition and blood flow recovered to its original state every time before addition of next concentration of NE solutions. The threshold to NE solution was evaluated, respectively, before exposure to heat stress at Tc = 38°C and Tc = 41°C.
SOD and NS were injected accordingly as mentioned above, and 3% Evans Blue (EB) solution (MP Biomedicals, Santa Ana, Calif, Product No.: 5G) at 1.5 mg/kg was injected to all rats in the three groups. One hour later, rats were subjected to infant incubators for heat stress. At the time before heat stress, heat stress to rats’ Tc of 38°C, and heat stress to rats’ Tc of 41°C, representing the rats with severe HS, 10 rats in each group at each observing time point were killed for detecting wet-to-dry weight ratio, the concentration of EB, and histopathology of lung. All rats were killed by exsanguination under anesthesia (Fig. 1B).
Measurement of water content of lung
The right lung was harvested; the lung tissues were rinsed with NS before filter papers were used to absorb the water on the surface. Lung tissues were weighted to obtain the lung wet weight. The lung was maintained in a baking oven at 60°C for 48 h and dried to a constant weight, which represented the lung dry weight. The wet-to-dry weight ratio = (lung wet weight − lung dry wet)/lung wet weight.
Measurement of EB concentration in lung tissue
EB (MP Biomedicals, LLC, Product No.: 5G) standard curve was drawn using the deionizedformamide as the blank control. The tail vein was used for intravenous injection of 3% EB at a concentration of 1.5 mL/kg, and the subjects were left alone for 1 h before anesthesia. Then the chest cavity was opened, and saline was perfused via the left ventricle, whereas the right atrium was cannulated for flushing out all the blood. The left lung was then harvested and separately weighed before transferring into 3 mL of deionized formamide solution. The solution was maintained in the water bath set at a constant water temperature of 50°C for 48 h to enable the EB contained in lung tissues to fully integrate into the deionized formamide solution before centrifuging lung tissues at 4,000 rpm for 20 min. The supernatant was used to assess the OD of lung tissues and the concentration of EB. The EB of every gram of lung tissues was calculated as EB concentration (μg/mL) × 3 mL.
The right lungs of the rats were immersed in 10% neutral formalin overnight and processed the next day. To assess lung injury, four sections were cut from the lung base toward the apex at 3-μm intervals and stained with hematoxylin & eosin (H&E). Ten random fields per animal were read under 200× magnification by a pathologist (ICL) who was blinded to the grouping of the rats. Histologic variables of acute lung injury were examined, each graded from 0 to 3, by intraalveolar infiltration of neutrophils, interstitial infiltration of neutrophils, perivenous infiltration of neutrophils, pulmonary congestion, and alveolar hemorrhage (19).
Weight alteration and the survival time after exposure to Tc of 41°c
Rats’ body weight was measured before heat stress and at Tc = 38°C and Tc = 41°C, respectively. The weight alteration was calculated as follows: (weight before heat stress − weight when Tc = 38°C/41°C)/weight before heat stress × 100%. The survival time counting was started immediately when the rectal temperature reached 41°C.
All data are expressed as means ± SD. To compare the differences between groups at different time points, the analysis of variance (ANOVA) for repeated measurements was used. Pairwise comparisons were conducted using the t test. The level of statistical significance was set at P < 0.05. All data were analyzed using IBM SPSS software (IBM SPSS 19, Chicago, IL).
Arteriolar RBC velocity decreased with the rise of temperature, and the changes in the three groups were all significant. Arteriolar RBC velocity in SOD+HS group was higher than that in the other groups at Tc = 38°C and Tc = 41°C, the disparities were significant. However, no significant differences were observed between NS+HS and HS groups, as shown in Figure 2A.
Venular RBC velocity of NS+HS and HS groups significantly decreased with the rise of temperature and, meanwhile, that of SOD+HS group increased insignificantly. Venular RBC velocity of SOD+HS was significantly higher than that in the two other groups after severe HS (Tc = 41°C), as shown in Figure 2B.
With rise of temperature, no remarkable changes were seen in the arteriolar diameter of HS and NS+HS groups between Tc = 38°C and Tc = 41°C. However, the arteriolar diameter in SOD+HS group was significantly reduced as the temperature increased from Tc = 38°C to Tc = 41°C (P = 0.004). At Tc = 38°C, arteriolar diameter of rats in SOD+HS was starkly larger than that of the two other groups, and the disparities were meaningful but the divergence among the three groups became insignificant as the temperature increased to Tc = 41°C, as shown in Figure 2C.
No venular diameter changes or disparities were observed among the three groups with the rise of temperature, as shown in Figure 2D.
Arteriolar flow rate of HS and NS+HS groups appeared to show a statistical decrease when the temperature increased to Tc = 38°C. SOD+HS group showed no obvious reduction at this temperature, but the decrease became significant when the temperature reached Tc = 41°C. When Tc = 38°C, the arteriolar flow rate of SOD+HS was apparently higher than that of the two other groups, whereas there was no significant divergence between the HS and the NS+HS groups. At Tc = 41°C, the disparities became insignificant among the three groups, as shown in Figure 2E.
Venular flow rate of the three groups started declining at Tc = 38°C, which was statistically significant, whereas there was no obvious divergence among the three groups. When the temperature increased to Tc = 41°C, venular flow rate of both HS and NS+HS groups exhibited a stark reduction, excluding that of the SOD+HS group, which was significantly higher than that in the other two groups (Fig. 2F).
Altered microvascular threshold to NE after heat stress
The microvascular threshold of the three groups to NE ranged from 0.5 to 1.0 mg/L before heat stress, with no significant differences among groups. The threshold of SOD+HS group was slightly increased as Tc increased to 38°C, but there is no statistical significance. At Tc = 41°C, the threshold of HS and NS+HS was reduced starkly (P < 0.05 in HS group, P < 0.01 in NS+HS group), which represented the excessive reaction toward NE, whereas there was no such change in SOD+HS group. In SOD+HS group, the microvascular threshold to NE was obviously higher than that in the other two groups, with significant disparities, as shown in Figure 3.
The alteration of pulmonary permeability
The lung water content of SOD+HS and NS+HS groups was higher than that of HS group before heat stress or when Tc = 38°C. The lung water content of HS group increased with the rise of temperature and was obviously higher than the level before heat stress, whereas that of the two other groups displayed no significant changes (Fig. 4A).
In HS group, the EB concentration of lung tissues increased significantly with the rise of Tc. In contrast, there was no apparent increase in EB concentration of lung tissues with the rise of the Tc in subjects of SOD+HS and NS+HS groups. The EB concentration of lung tissues in the SOD+HS group was obviously lower than that in the HS group, whereas the difference between NS+HS and HS was not statistically significant (Fig. 4B).
Changes in histopathological injury
H&E staining of lung tissue in different groups: Histopathological injury of lung tissue became more serious with increase of Tc in all three groups, but in SOD+HS group, histopathological injury was milder than other two groups. Before heat stress, histopathological injury in NS+HS group was more serious than other two groups (Fig. 5A). Histological grading of acute lung injury: Histological grading score became higher with increase of Tc in the three groups. Before heat stress, the score in NS+HS group was higher than the other two groups. When Tc of rats reached 41°C, the score in SOD+HS was lower than the other two groups (Fig. 5B).
Changes in average arterial pressure after heat stress
In HS and NS+HS groups, the MAP of rats started declining when their Tc increased from 36°C to 38°C (T2) during heat stress and recovered voluntarily after their Tc increased from 38°C to 39.5°C (T3). Following severe HS, i.e., after the Tc increased to 41°C, an irreversible decrease in MAP occurs until death. During the whole process, there was no significant difference between the two groups.
The rats in SOD+HS group displayed no significant MAP reduction when their Tc increased from 36°C to 38°C (T2). Their MAP was apparently higher than that in the two other groups (P < 0.05). At Tc of 38°C to 39.5°C, the MAP of the three groups was not significantly different. Severe heat stress, i.e., a Tc of 41°C, resulted in a sharp slump in MAP of the three groups followed by an irreversible shock until death. However, among the three groups, SOD+HS group displayed a higher MAP than the other two groups and the disparity was statistically significant, as shown in Figure 6.
Loss in body mass of three groups
There was a statistically significant body mass loss during heat stress process in all three groups (P < 0.05). However, no significant difference was seen among the three groups (Fig. 7).
After Tc = 41°C, the survival time of SOD+HS group was significantly higher than that of NS+HS and HS group (P < 0.01), and that of NS+HS group was longer than that in HS group (P < 0.05), as shown in Figure 8.
With the world becoming warmer and heat waves growing in severity and frequency (20), the incidence of life-threatening HS arising from high temperatures increases. Therefore, it is imperative to investigate the mechanisms underlying HS and develop preventative and remedial approaches.
In this study, we not only monitored the relationship between MAP and microcirculation, but also comprehensively assessed the altered microcirculation by evaluating the factors including microcirculatory blood flow rate, microvessel reactivity to NE, pulmonary permeability, and lung histopathological injury. In addition, via SOD intervention, we also probed into the mechanism of the oxidative stress functions in microcirculatory disorders triggered by severe HS.
The study results revealed that during the whole process of heat stress, the decrease in blood flow rate was mainly induced by reduced RBC flow velocity instead of the diameter. It can also be concluded that the infusion of fluid (NS) does not play an important role in protecting microcirculatory blood flow rate. Instead, antioxidative stress could slow down the decrease in microvascular RBC velocity, resulting in the preservation of blood flow rate. In the early stages, heat stress dilated the arteries to maintain circulatory blood flow rate. The arteriolar system showed the biggest reduction in blood flow rate occurred during the early stages of heat stress. It occurred when the rectal temperature of the subjects reached 41°C. Antioxidant pretreatment was powerful in protecting the blood flow rate.
During the development of severe HS, rats in HS and NS+HS groups demonstrated microvascular hyper-reactivity to NE. In SOD+HS group, in contrast, there was no increase or decrease of microvascular reactivity to NE. This result revealed the characteristic of vascular reactivity in the development of HS,whereas usually in advanced septic shock, the microvascular reactivity to NE appears to decline (21). The reason for this discrepancy might be that the short average survival time was not longer than 1 h, during which the sympathetic nervous system was presumably still at the state of excitation. The pretreatment of antioxidant attenuated the microvascular hyper-reactivity to NE, which has not yet to be reported in other studies. Therefore, we intend to further investigate the altered cellularmorphology, cytokine expression, and ion channel excitation in vascular smooth muscle cells in our future studies.
It was previously demonstrated by our research group (16) that heat stress induces explosive production of ROS and endothelial cell apoptosis, which may be correlated with changes in vascular permeability after heat stress. In this experiment, the content of EB in lung tissues of HS group was found to increase significantly with heat stress. Histopathological injury of lung tissue also became more serious in this group. However, the EB content in lung tissues was lower in SOD+HS group during heat stress. The histopathological injury of lung tissue in SOD+HS group was milder than that in the other two groups. The results indicate increased pulmonary permeability during HS. SOD helps to preserve vascular barrier in the early stages of heat stress. Before heat stress and at Tc = 38°C, the water content in SOD+HS and NS+HS group was higher than that in HS groups. And before heat stress, histopathological injury in NS+HS group was also more serious than that in the other two groups. This result might be caused by the injection of NS before initiation of heat stress, but antioxidant SOD was still effective in attenuating pulmonary edema following severe HS.
It was also demonstrated that microcirculatory disorders produce hypoperfusion of tissues and inadequate oxygen supply in systemic hemodynamics, which result in systemic circulatory shock and MODS (22). Microcirculatory disorders are inextricably correlated with pathological phenomena, including circulatory failure, edema, and bacterial translocations. Among critically ill patients (10), microcirculation disorder can lead to MODS which are associated with the decrease of survival rate. In this study, we demonstrated that there is obvious histopathological injury during heat stress, and SOD could have certain protective effects. This study also showed a decrease in microcirculatory blood flow rate and systemic circulatory blood pressure in the early stage of heat stress (Tc = 36°C–38°C). This result may be attributed to self-regulatory mechanisms such as body fluid redistribution. As the Tc rises to between 38°C and 39.5°C, the systemic circulatory blood pressure is restored to the original level, which is related to the excitation of sympathetic nerve system in heat stress response, but the microcirculatory blood flow rate is not restored at the same time. On the contrary, the microcirculation deteriorated continuously. The microcirculatory blood flow rate decreased after Tc rose to 41°C, which induced an extremely sharp reduction in blood flow and followed by irreversible shock and death. The study results demonstrated that in heat stress, microcirculatory disorders not only occur before systemic circulatory disturbances, but also sustain even when systemic circulation is transiently compensated, which induce shock with irreversible and poor systemic circulation and also adverse prognosis.
Previous studies suggest that dehydration was also one of the typical symptoms of HS (23). Our experiment shows that increasing temperatures induce approximately 3% body fluid loss in all three groups, the body weight loss mainly appeared at Tc = 41°C. There was no significant difference in body weight loss in three groups, indicating that different treatments in this study had not impacted on dehydration. On the contrary, this result also indicated the dehydration in this setting was not an interference factor for survival outcome.
In the model of ischemia/reperfusion, the production of oxygen free radicals during the interaction between endothelial cells and leukocytes was considered to be basis of early microcirculatory disorders, which was also demonstrated in hemorrhagic shock (13). In a recent study (11) involving an ischemia/reperfusion model, excessive oxidative stress triggers the production of inflammatory mediators, resulting in ischemia-reperfusion injury. In this study, at Tc = 41°C, the survival time in SOD+HS group was apparently longer than that in NS+HS and HS groups, and longer in NS+HS group than that in HS group, which suggests that pretreatment of antioxidative stress and fluid transfusion improves the prognosis of severe HS. Antioxidant therapy was more effective. Although this study only demonstrated that SOD had protective effect on this pretreatment setting, other studies have indicated that SOD and other antioxidant were also effective in posttreatment settings. In a study from Jin et al. (14), the administration of SOD at 2 h after hemorrhagic shock was able to recover the lower vasoreactivity as well as to increase the enhancing effect of dopamine on blood pressure of rats in severe hemorrhagic shock. Ke et al. (15) reported that in a rat model, antioxidant resveratrol were given 2 h after hemorrhagic shock. The study found that posttreatment of resveratrol could attenuate the intestinal injury induced by hemorrhagic shock.
Overall, this study shows for the first time that microcirculation disturbance occurs not only at the early stage, but also before systemic hemodynamic disorder. Monitoring microcirculation following HS is of prognostic value. Antioxidative stress improve microcirculation and results in favorable, prompt it may have certain protection effects in severe HS.
This research was underpinned by evidence from experimental groups of relatively small sample size; a further experiment of a larger sample size needs to be conducted. To assess the microvascular permeability, lung tissues were adopted in our experiment, but in the preexperiment other organs also exhibited an increase of EB exudation after heat stress. Therefore, the water content and the remaining amount of EB in other organs need to be evaluated in advanced experiments. The microvessels were found to overreact to vasoactive drugs in severe HS, which requires further research on vascular smooth muscle cells to figure out relevant mechanisms. Finally, it should be noted that our model involved pretreatment with SOD, and then subjected the animals to heat stress. Thus, it remains to be determined if posttreatment with SOD or other antioxidants following heat stress will also be effective in the treatment of HS.
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