INTRODUCTION

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HEATSTROKE is a complex clinical picture characterized by severe central nervous system disturbances (such as coma and delirium), hyperpyrexia, and hot, dry skin (18). The high mortality rate of heatstroke is believed to be related to its serious complications, including adult respiratory distress syndrome, disseminated intravascular coagulation, aspiration pneumonia, pulmonary edema, circulatory and renal failure, and severe electrolyte disturbances (1, 2, 10, 13, 20, 24, 25, 28).

There is little agreement on the hemodynamic status of patients or animals with heatstroke. For example, Clowes and O'Donnell (9) reported that seven of eight patients suffering from acute heatstroke had increased cardiac index and decreased peripheral vascular resistance. Sprung (30) reported that five of seven patients with heatstroke had decreased cardiac output and increased peripheral resistance. Recently, Dahmash et al. (10) reported that among 10 heatstroke patients, cardiac output was decreased in 1, normal in 5, and increased in 4. Systemic vascular resistance, on the other hand, was low in eight patients and normal in two patients. In experimental models, animals with heatstroke had decreased mean arterial pressure (19, 23, 24) and decreased peripheral vascular resistance (21).

Evidence has accumulated to indicate that the morbidity and mortality observed in heatstroke may be related to endotoxemia and release of interleukin (IL)-1 (3, 8, 14). Immunization against bacterial endotoxin (4) or administration of antibiotics before heat stress sharply reduced mortality in experimental animals (5, 6). The arterial hypotension associated with heatstroke could be attenuated by pretreatment of animals with an antagonist of IL-1 receptors (16, 22, 23). Therefore, a potential involvement of IL-1 receptor mechanisms in the development of circulatory failure in heatstroke is suggested.

The objective of this study was to clarify the relationship between IL-1 and hemodynamic changes associated with heatstroke. Rats, under general anesthesia, were exposed to a high ambient temperature to induce heatstroke (16, 22, 23) and to assess the hemodynamic changes associated with heatstroke in control rats and in rats pretreated with an IL-1-receptor antagonist. In addition, the effects of systemic administration of IL-1 on hemodynamic function parameters were assessed in normothermic controls. Furthermore, changes in the plasma levels of IL-1 were assessed in normothermic controls and in rats with heatstroke.

	MATERIALS AND METHODS

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Animals. Adult male Sprague-Dawley rats (Animal Resource Center, National Cheng-Kung University Medical College, Tainan, Taiwan), weighing 250-300 g at the start of the experiment, were housed individually in wire hanging cages in a temperature (24°C)-controlled animal colony, on a normal light-dark cycle (14:10 h; lights on at 6:00 AM). The animals had free access to food and water and were allowed to acclimatize to the light cycle and the room temperature for at least 2 wk before the experiment began.

Surgery and measurement of cardiovascular parameters. The right femoral artery and vein of rats under urethan (1.4 g/kg ip) anesthesia were cannulated with polyethylene tubing (PE-50) for monitoring of cardiovascular parameters, administration of drugs, and blood sampling. In addition, general procedures included trachea intubation for artificial ventilation, with the rate and tidal volume adjusted to maintain an end-expiration CO₂ concentration between 3.5 and 4.0% (monitored by a Gould Capnograph Mark IV), and keeping the colonic temperature (T_CO) at 38°C before heat exposure (32). T_CO was measured using a copper-constantan thermocouple enclosed in polyethylene tubing, sealed at one end, and inserted 6 cm into the colon. The animals were then put in a supine position.

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IL-1 bioassay. IL-1 concentration in the plasma was measured with the IL-1-dependent murine T cell line D10N4M (a kind gift from Dr. C. C. Chao, Neuroimmunology and Host Defense Laboratory, Minneapolis Medical Research Foundation, Minneapolis, MN), as previously described (7, 26). The D10N4M cells were maintained in RPMI-1640 (GIBCO BRL) with 10% fetal bovine serum (GIBCO BRL), recombinant human IL-2 (20 ng/ml, R & D), recombinant human IL-1 (40 pg/ml, R & D), 5 × 10⁵ M 2-mercaptoethanol (Serva, Heidelberg, Germany), and concanavalin A (3.0 µg/ml; Sigma), and were fed every 3 days before being assayed. Serial rabbit serum samples or recombinant human IL-1 (50 µl, as an internal reference) were added to each well of microplates (NUNC), followed by the addition of 50 µl of washed D10N4M cells (2 × 10⁵ cells/ml). After 72 h of incubation, the cells were pulsed with 0.5 µCi of [³H]thymidine (6.7 Ci/mmol, DuPont NEN), per well for 4 h. The cells were harvested on glass fiber filters with an automatic cell harvester (Cambridge, Watertown, MA). The radioactivity incorporated was assayed in a liquid scintillation counter (LS 5000 TA, Beckman, Fullerton, CA).

Induction of heatstroke. Four groups of animals were used. In the first group, rats were exposed for 70 min to heat and received a saline injection (1 ml/kg of 0.9% saline per 1 kg body wt iv) at the start of heat exposure. Heatstroke was induced by exposing the rats, under urethan anesthesia, to an ambient temperature (T_a) of 42°C (with a relative humidity of 60%); the moment at which MAP began to decrease from its peak level was taken as the onset of heatstroke. Our previous results show that, at the moment when MAP begins to decrease from its peak level, unanesthetized animals display heatstroke symptoms including loss of sensation, decreased cerebral perfusion pressure, and unconsciousness (29), whereas animals under general anesthesia display decreased cerebral perfusion pressure, cerebral ischemia, and neuronal injury (16, 22, 23). For humane reasons, in the present study, heatstroke was induced under general anesthesia. In the second group, rats under urethan anesthesia were exposed for 70 min to heat and received IL-1 receptor antagonist (IL-1ra, 200 µg/kg iv; Synergen) at the start of heat exposure. IL-1ra was expressed in Escherichia coli using a cDNA originally isolated from adherent monocytes (12). This protein is the nonglycosylated NH₂-terminal methionyl form of the naturally occurring protein and has a molecular mass of ~17 KDa. IL-1ra blocks binding of IL-1 as well as the naturally occurring glycosylated form does. In the third group, rats under urethan anesthesia were exposed to a T_a of 24°C for at least 90 min and were given an intravenous dose of 1 ml/kg of 0.9% saline at the start of testing. In the fourth group, rats exposed to a T_a of 24°C received an intravenous dose of recombinant murine IL-1 (30 µg/kg).

Data analysis. Numerical values cited are means ± SE. Repeated-measures analysis of variance was used for factorial experiments, whereas Duncan's multiple-range test (multiple time-point experiments) was used for post hoc multiple comparisons among means. Student's t-test was used when only two groups were compared. The criterion for statistical significance was set at P  0.05.

	RESULTS

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Tables 1, 2, and 3 summarize values for the various cardiovascular parameters collected from three groups of animals 70 min after the start of heat exposure or testing. The time-course data before, during, and after heat exposure are depicted in Figs. 1, 2, and 3. The hyperthermic rats that received saline injection 70 min after the start of heat exposure displayed higher values of T_CO (Table 1) and HR (Table 2) and lower values of MAP, CI, SI (Table 1), survival time (ST; interval between onset of heatstroke and death), R wave amplitude, and P-R and Q-T intervals QT (Table 2), and amplitude of systolic and diastolic waves, duration of systolic, diastolic, and dicrotic waves, and duration of whole BP cycle (Table 3), compared with normothermic controls that received saline treatment. However, there is an insignificant difference in amplitude of P, Q, S, and T waves and duration of P, Q, R, and T waves and QRS duration (data not shown) and TPRI (Table 1) between normothermic controls and hyperthermic animals that received saline treatment. These tables and figures also show that pretreatment of rats with an intravenous dose of IL-1ra (200 µg/kg), just at the start of heat exposure, significantly attenuated the reduction in MAP, CI, SI (Table 1), R wave amplitude and P-R, Q-T, and S-T intervals (Table 2), and amplitude of systolic and diastolic waves and duration of systolic, diastolic, and dicrotic waves and the whole BP cycle (Table 3) that occurred during onset of heatstroke.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effects of a high ambient temperature (Ta = 42°C) on colonic temperature (TCO), mean arterial pressure (MAP), cardiac index (CI), stroke index (SI), and total peripheral resistance index (TPRI) in 8 rats treated with saline or interleukin-1 receptor antagonist (IL-1ra, 200 µg/kg iv). Points represent means ± SE. * P < 0.05, significance of difference from control values (saline group), Student's t-test.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of high Ta (42°C) on amplitude of R wave (AR), P-R interval (PR), Q-T interval (QT), and duration of dicrotic wave (Ddic) in 8 rats receiving saline or IL-1ra (200 µg/kg iv). Points represent means ± SE. * P < 0.05, significance of difference from control values (saline group), Student's t-test.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of high Ta (42°C) on amplitude of systolic wave (Asys), amplitude of diastolic wave (Adia), duration of systolic wave (Dsys), duration of a whole BP cycle (Dw), and duration of diastolic wave (Ddia) in 8 rats receiving saline or IL-1ra (200 µg/kg iv). Points represent means ± SE. * P < 0.05, significance of difference from control values (saline group), Student's t-test.

Table 4 summarizes plasma IL-1 values in rats exposed for 70 min to heat (T_a 42°C) and in rats exposed to a T_a of 24°C. As can be seen from the table, the heat-exposed rats had a higher IL-1 concentration in the plasma compared with that of the controls.

In addition, as shown in Fig. 4, an intravenous dose of IL-1 (30 µg · ml¹ · kg¹) produced a progressive fall in MAP, CI, and SI in rats exposed to a T_a of 24°C. Again, TPRI was not affected by the intravenous administration of IL-1.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of intravenous administration (arrows) of saline or interleukin-1 (IL-1; 30 µg/kg) on MAP, CI, SI, and TPRI in 8 rats. IL-1 caused a significant reduction in MAP, CI, and SI from 0 to 140 min after injection. Points represent means ± SE.

	DISCUSSION

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This study provides the first experimental evidence supporting the hypothesis that a selective decline of stroke volume or ventricular depolarization may be an important mechanism signaling arterial hypotension in heatstroke. As demonstrated in the present results, some cardiovascular effects, such as tachycardia, depressed ventricular depolarization, decreased stroke volume, decreased cardiac output, arterial hypotension, and facilitated pacemaker signal conduction, were associated with heatstroke induction in rats. The reduction in ventricular depolarization during heatstroke induction might be reflected by a decrease in R wave amplitude, systolic wave amplitude, diastolic wave duration, and dicrotic wave duration. The facilitated pacemaker signal conduction might be demonstrated by a decrease in P-R and Q-T intervals in rat heatstroke. However, neither TPRI nor the hemodynamic parameters related to atrial depolarization or ventricular repolarization were associated with heatstroke induction. Historically, there have been two views about the pathogenesis of heatstroke, 1) direct thermal injury to the thermoregulatory centers in the brain causing thermoregulatory failure and shock (18) and 2) circulatory failure (1). From measurements of central venous pressure and splanchnic blood flow, Kielblock et al. (19) and Kregel et al. (21) concluded that a selective loss of compensatory splanchnic vasoconstriction may be an important mechanism signaling circulatory failure in heatstroke. However, the fall in splanchnic mesenteric artery resistance in itself may not be directly responsible for the fall in mean arterial pressure. The time course of change in splanchnic mesenteric artery resistance in relation to arterial hypotension indicates that the fall in mean arterial pressure is not caused by a decrease in total peripheral resistance (21); rather, pooling blood in the peripheral vasculature may reduce venous return and stroke volume and ultimately contribute to circulatory failure. Moreover, in the present results, we newly demonstrated that arterial hypotension was associated with a decline in ventricular depolarization or stroke volume in heatstroke. Thus it appears that heatstroke results in circulatory shock, and the mechanism initiating the circulatory impairment is more a myocardial mechanism (31) than a peripheral mechanism (such as pooling blood in the peripheral vasculature) (11).

The present results show that heatstroke was accompanied by increased plasma levels of IL-1. Animals injected intravenously with IL-1ra at the time of heatstroke induction were protected from some of the cardiovascular effects of heatstroke such as depressed ventricular depolarization, decreased stroke volume, decreased cardiac output, and arterial hypotension. It has also been shown that IL-1 activates a myocardial L-arginine-nitric oxide pathway that raises myocardial cyclic GMP and induces twitch abbreviation (15). Our present results further showed that intravenous administration of IL-1 decreased stroke volume, cardiac output, and mean arterial pressure in normothermic control animals. These observations tend to indicate that a selective decline in stroke volume or ventricular depolarization resulting from increased plasma levels of IL-1 may be an important mechanism signaling arterial hypotension or circulatory failure in rat heatstroke.

IL-1 has been implicated in the control of responses to systemic disease and injury and activation of fever, neuroendocrine, immune, and behavioral responses (27). Pretreatment with IL-1ra has also been shown to block the thermogenic, anorexic, and behavioral effects of recombinant human IL-1 (17). Both our previous (22-24, 29) and present results further show that some cardiovascular effects of heatstroke such as intracranial hypertension, arterial hypotension, and cerebral ischemia are attenuated by IL-1ra pretreatment. Therefore, the recent discovery of specific inhibitors of cytokine synthesis, release, or action may offer significant therapeutic benefit in a variety of inflammatory and ischemic conditions.

In summary, the results showed that 1) heatstroke was accompanied by increased plasma levels of IL-1; 2) animals injected with IL-1ra at the time of heatstroke induction were protected from some of the cardiovascular effects of heatstroke such as depressed ventricular depolarization, decreased stroke volume, and arterial hypotension; and 3) the hemodynamic changes associated with heatstroke could be mimicked by IL-1 injection. The data suggest that heatstroke stimulates synthesis and release of IL-1 in the plasma, depresses ventricular depolarization and stroke volume, and results in arterial hypotension.