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INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION
1Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, Mizuho, Nagoya 467-8601; 2Department of Molecular Physiology, National Institute of Physiological Sciences, Okazaki 444-8585; 3Departments of Surgery, Physiology, and Cardiology and Center for Regenerative Medicine, Tokai University School of Medicine, Isehara 259-1193; and 4Department of Preventive Nutriceutical Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe Dori, Nagoya 467-8603, Japan
Submitted 14 April 2003 ; accepted in final form 15 August 2003
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ABSTRACT |
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body temperature regulation; salivation; heat stress; rat model; genotypic adaptation; biodefense
Animal models with specific congenital traits of heat tolerance are essential for studying various adaptive changes at all levels of organism function. We have developed an inbred strain of heat-tolerant rats (FOK) through selection from several thousands of rats by acute heat exposure (42.5°C) once in each lifetime for many generations (9). The increased heat tolerance of the FOK strain has been documented (8, 9). In the present study we were concerned with clarifying the strategy of FOK rats for enduring heat, because the precise phenotype of the heat tolerance ability of this inbred rat has yet to be characterized.
The time course of core temperature (Tc) responses to heat exposure is an indicator of thermoregulatory ability. In common strains of rats, Tc either increases linearly to a lethal temperature or increases with an initial convex-shaped rise followed by a final rise (9). Somewhat heat-resistant rats [e.g., a part of the Sprague-Dawley (SD) strain] exhibit a triphasic Tc response to ambient heat: a rapid initial rise in Tc, a plateau for a short period of time, and then a further rise (8-10). FOK rats can endure ambient heat much longer than SD rats (9). The enhanced heat tolerance capacity of FOK rats is largely due to a heightened saliva spreading capacity (8).
Evaporation—the primary modality for heat loss when ambient temperature (Ta) exceeds body temperature (10)—is an efficient means for removing heat from an object (572.8 cal/g water = 43.2 kJ/mol, at 40°C). However, to be effective a thin layer of fluid must be spread across the interface between a body surface and the environment. In animals that neither sweat nor pant, saliva spreading is a means of laying a thin film of fluid over an exposed body surface. Because rats neither pant nor sweat, saliva spreading ability is a limiting factor for survival in hot environments (10). FOK rats can endure a standard heat exposure (Ta = 42.5°C) for 
5-6 h, 2.5-5 times longer than any other tested strain of rat (8, 9). In such a trial FOK rats can lose up to 14% of their body weight through evaporation (9). FOK rats secreted the largest amount of saliva among the four rat strains assessed and spread that saliva over up to more than half their body surface area under extreme heat stress (8).
Whether the heightened saliva spreading capacity of the FOK rats is due to enhanced secretory capacity or to changes in thermoregulatory control has yet to be determined. The submandibular glands of FOK rats are larger than those of control strains (although there was no difference in the parotid gland size among the rat strains) (8). Ligation experiments demonstrated that thermal salivation can be induced through the hypothalamus-superior salivatory nucleus-chorda tympani-submandibular glands pathway (8). ACh is a major secretagogue that evokes copious fluid secretion (20). Heat mainly evokes fluid secretion by the parasympathetic pathway (18), and the secretion rate is not reduced by sympathetic denervation (18). If strain differences in heat tolerance are attributable to glandular events, the secretion rates of extirpated submandibular glands from FOK rats should be higher than those from control strains. If, however, the strain differences are due to changes in thermoregulatory control, there should be negligible differences in secretion rates among glands isolated from the different strains.
The aim of the present study was to resolve several specific issues related to the FOK rat: 1) to clarify the basic strategy of FOK rats for enduring heat, 2) to determine the regulation mode of the evaporative cooling activity underlying the strategy, and 3) to determine whether differences in the secretion rate among four rat strains and between two temperatures were attributable to glandular responsiveness or central events.
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MATERIALS AND METHODS |
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The male rats used were 13 wk old in experiments 1-4 and 11 wk old in experiment 5. Five strains of rats with different heat tolerances were used: highly heat-tolerant FOK rats, moderately heat-tolerant SD rats, and heat-intolerant ACI, Donryu, and WKAH rats. ACI/Jcl rats were purchased from CLEA Japan; HOS:Donryu and WKAH/Hkm rats were purchased from Japan SLC. The former name of the WKAH rats was Wistar King-Aptakeman/MK. The SD rats used were Crj:CD from Charles River Japan. The FOK rats used were FOK/Ncu. All rats had been kept at a Ta of 25°C before experimentation. The FOK rats had not been exposed to heat for the last two generations. Sample Tc responses to a standard heat exposure (42.5°C) were recorded from one rat for each strain with a telemetric system. The research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School. The Institutional Animal Care and Use Committee of Nagoya City University Graduate School of Medical Sciences approved the experimental protocol.
Experimental Design
Experiment 1: level of hyperthermia and extent of saliva spreading at different ambient temperatures. Thirty-five conscious ACI and forty-two conscious FOK rats were divided into five and six groups, respectively. Rats were kept at rest at a Ta of 25°C until psychological stress-induced fever ceased. Each animal was then quickly placed in a stainless steel wire cage and immediately transferred to a climatic room (320 x 420 x 250 cm, width x height x depth) with a constant Ta of 35.0, 37.5, 38.5, 40.0, 42.5, or 45.0°C (FOK only) and 50 ± 5% relative humidity. Tc was continuously monitored via a thermocouple. After 60 min of heat exposure, Tc and the extent of saliva spreading were measured and recorded.
Experiment 2: strain difference in threshold Tc for onset of salivation. Six conscious ACI, WKAH, Donryu, SD, and FOK rats were individually caged. Tc was continuously monitored with a telemetric system. After 3 h of resting at 25°C the Ta was increased to 40.0°C within 1 h. Changes in Tc paralleled changes in Ta until evaporative heat dissipation mechanisms were activated. The threshold Tc for the onset of thermal salivation was determined when a drop of saliva was first observed between the lips.
Experiment 3: strain difference in relationship between salivation rate from submandibular glands in vivo and Tc. Six ACI, WKAH, Donryu, SD, and FOK rats were ligated at parotid ducts under pentobarbital sodium anesthesia. Seven days later, the rats were anesthetized with ketamine and transferred to a climatic room with a constant Ta of 38.0°C and relative humidity of 50 ± 5%. Tc was continuously monitored with a rectal thermocouple. Saliva was allowed to drip into a beaker. The saliva secretion rate was compared with Tc as Tc rose from 37.0 to 43.0°C.
Experiment 4: strain differences in secretion rate of submandibular glands in vitro among four rat strains. Five ACI, Donryu, SD, and FOK rats kept at a Ta of 25°C were anesthetized with pentobarbital (50 mg/kg body wt ip), and the submandibular glands were extirpated. The submandibular glands were perfused with 0.5 µM ACh at 37°C. The secretion rate was determined for the 20 min of static phase from 5 min to 25 min after the beginning of the ACh perfusion.
Experiment 5: effects of temperature on secretion rate of submandibular glands in vitro. The submandibular glands of five WKAH rats and five FOK rats were extirpated under pentobarbital anesthesia and perfused with 1.0 µM ACh. The secretion rates were compared between 37°C and 40°C for the 15 min of static phase from 5 min to 20 min after the beginning of the perfusion.
Methods
Scoring of saliva spreading. The extent of the saliva spreading was carefully observed and graded with a modified version of the scoring method of Maling and Koppanyi (Ref. 17; see Ref. 8). This was a semiquantitative method but was free from any additional stressful treatment. Saliva spreading was graded visually from 0 to 13 in terms of the body surface area covered with saliva: 1, between incisors and reaching the lower lip; 2, jaw; 3, neck; 4-5, chest; 6-9, abdomen; and 10, scrotum. An additional score of 1 each was allotted to one-sided outer hindleg, one-sided outer foreleg, and face up to eye level, allowing a maximum score of 11-13 points.
Tc measurement. In experiments 1 and 3 Tc was continuously monitored with a copper-constantan thermocouple inserted into the rectum 6 cm from the anal sphincter. In the sample tracing and experiment 2, Tc was monitored continuously with a telemetric system from Data Science International (St. Paul, MN). The transmitter (model TA10TA-F40) was surgically implanted in the abdomen per the manufacturer's instructions. The receiver was model RPC-1, the data acquisition instrument was MacLab 8/S (ADInstruments), and the data acquiring program was Chart v4.0 (ADInstruments).
Anesthesia. In experiment 3 ketamine HCl (120 mg/kg) was injected intraperitoneally 30 min before the heat exposure to determine the salivation rate as previously described (7). A tail press stimulation of 1 kg/9 mm2 was applied every 45 min after the first injection. If the rats responded to the tail press, a supplementary dose of ketamine HCl (42.5 mg/kg) was injected intraperitoneally (7). Ketamine is an antagonist of the N-methyl-D-aspartate subtype of excitatory glutamate receptor and does not strongly inhibit the center of the autonomic nervous system (6, 15). The ketamine dose used does not promote salivation but partially inhibits thermal salivation (7).
Quantification of saliva secretion rate from submandibular glands in vivo (experiment 3). Ketamine-anesthetized rats were kept in a prone position on a slanted wire net (6.8% head-down tilt) at a Ta of 38°C. The head was placed lower than the hips. The face of each rat protruded over the edge of the wire net with a beaker containing mineral oil placed directly below. As Tc was gradually increased, saliva dripped into the beaker. The parotid duct had been ligated 5-6 days before the experiment. The ratio between the output rate (µl/min) of the submandibular and sublingual glands was 13:1 (4). Therefore, the submandibular output and sublingual saliva were determined together.
Determination of secretion rate in isolated submandibular glands (experiments 4 and 5). The submandibular glands were surgically isolated under pentobarbital anesthesia, and the excretory ducts and glandular veins were cannulated to sample the saliva. The arteries were also cannulated and perfused at a rate of 2.0 ml/min with a bicarbonate-free solution buffered at pH 7.4 with 10 mM HEPES. The perfusate (composition in mM: 145 Na+, 4.3 K+, 1.0 Ca2+, 1.0 Mg2+, 153.3 Cl-, 5.0 glucose) was equilibrated with 100% O2. Ducts and the connective tissue surrounding the glands were omitted from glandular wet weight. The temperature of the perfusate and the gland was kept at 37.0 or 40.0°C by a circulated bath. Glands were stimulated by 0.5 or 1.0 µM ACh. Salts, glucose, and HEPES were obtained from Nacalai Tesque (Kyoto, Japan). The ACh used was ACh chloride from Daiichi Pharmaceutical (Kyoto, Japan). All pharmacological agents were dissolved in the perfusate immediately before the start of the experiment.
Statistics
The results are presented as means ± SD. The difference between two groups of results was analyzed by a nonparametric Mann-Whitney test. Differences among more than two groups were analyzed by the nonparametric Kruskal-Wallis test (ANOVA) and repeated-measures ANOVA. Fisher's protected least-significant difference test was also used in the case of pair combinations of more than two groups. The significance of the correlation coefficient was determined by the nonparametric Spearman rank correlation test. Correlation was also calculated with regression analysis.
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RESULTS |
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Early in the heat exposure the rats walked around slowly and exhibited searching behavior. The rats became less animated when the Tc increase began to slow. The FOK rats became relaxed, laid down, reared up, and rested on wire net wall for a long time during the steady-state hyperthermic period, although they sometimes rose and walked. Sample tracings of the Tc responses to prolonged exposure to high Ta (42.5°C, a standard heat stress protocol) are shown in Fig. 1. The FOK rats endured the heat substantially (2.5-5 times) longer than the other four rat strains. The SD rats endured heat longer (approximately double) than the three heat-intolerant strains [as previously reported (9)].
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Experiment 1: Tc and Extent of Saliva Spreading in FOK and ACI Rats at Different Ta
Tc of the FOK rats remained at an elevated stable plateau for several hours at Ta between 35.0 and 45.0°C, whereas the Tc in most ACI rats increased steadily at a Ta of 40 and 42.5°C. The Tc at 60 min of heat exposure was higher in ACI rats than in FOK rats over a Ta range of 37.5-42.5°C (Fig. 2). The correlation coefficient between the Ta and Tc in ACI rats at 60 min after the beginning of the heat exposure was 0.939 (P < 0.0001). In contrast, Tc of FOK rats, although elevated, was constant (39.5 ± 0.7°C) over the Ta range from 37.5 to 42.5°C (Tc increased after 60-min exposure to 45°C) (P < 0.0001). At Ta >40.0°C, it should be noted, the plateau Tc in FOK rats were lower than Ta.
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The extent of saliva spreading increased in FOK rats (P < 0.0001) as Ta increased over 37.5°C (Fig. 3). Saliva was spread over the entire ventral surface, the outside of the legs, and the face up to the eye level in FOK rats. The extensive saliva spreading of FOK rats at Ta >37.5°C likely contributed to the Tc plateau at high Ta.
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Experiment 2: Strain Difference in Threshold Tc for Onset of Salivation in FOK Rats
There was a significant difference in the Tc threshold for the onset of salivation among the five strains (P < 0.0001). The Tc threshold for salivation was <39.0°C in the Donryu, SD, and FOK rats (38.9 ± 0.2, 38.8 ± 0.3, and 38.7 ± 0.1°C respectively). These values were lower than those in the ACI rats (39.7 ± 0.4°C; P < 0.01) and the WKAH rats (40.1 ± 0.5°C; P < 0.0001). Thus the FOK rat was one of the strains with a low threshold Tc for the onset of thermal salivation.
Experiment 3: Strain Difference in Relationship Between Salivation Rate and Tc in FOK Rats In Vivo
The relationship between the saliva secretion rate and Tc differed among the rat strains (repeated-measures ANOVA: P < 0.0001; Fig. 4). The saliva secretion rates in Donryu and SD rats did not increase proportionally with Tc. Conversely, the saliva secretion rates of FOK rats increased proportionally with Tc [39.0-42.0°C (
= 0.883, P < 0.0001)]. The slope of salivation rate over Tc was steeper in FOK rats than those in other strains. The increase in the salivation rate per degree Celsius was 16.21 ± 3.01 mg·100 g body wt-1·min-1 in FOK rats but only 7.64 ± 1.14 mg·100 g body wt-1·min-1 in ACI rats. In the Tc range of 40.0-40.5°C, the saliva secretion rate of FOK rats was higher than those of ACI, WKAH, and Donryu rats (P < 0.05). In the Tc range of 40.5-42.0°C, the variance among the four rat strains was significant (P < 0.0002). In particular, the secretion rate of FOK rats was much higher than those of the other strains (P < 0.001). There was no difference in salivation rate between ACI and SD rats at Tc of 40.5-42.0°C. At Tc >42.0°C, the salivation rate decreased in all animals.
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Experiment 4: Differences in Saliva Secretion Rates of Submandibular Glands In Vitro Among Four Rat Strains
There were no significant differences among the strains in the secretion rates per gram of submandibular gland at 37.0°C (Table 1). There were also no significant differences in the secretion rates per 100 g of body weight among the strains (Table 1). The secretion rate of isolated submandibular glands from the four rat strains in vitro corresponded to those in FOK rats in vivo with a Tc of 40.0-41.0°C.
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Experiment 5: Effects of Temperature on Secretion Rates of Submandibular Glands In Vitro
There were no significant differences in the secretion rates per 100 g of body weight between 37.0°C and 40.0°C in submandibular glands extirpated from WKAH and FOK rats and perfused with ACh (Table 2).
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DISCUSSION |
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Two general categories of adaptation have been recognized: capacity adaptation and resistance adaptation (23). Resistance adaptations are those that permit a shift of internal functions (for example, Tc) and expand the environment that can be survived. The strategy of FOK rats was to shift Tc from the normal level (near 37°C) to the hyperthermic level of 39.5 ± 0.7°C. Thus the strategy of the FOK rat for enduring heat was a case of resistance adaptation. This strategy of FOK rats for enduring hot environments is beneficial to health in two respects. First, the FOK rats can escape the multisystem disorders that would be caused by heatstroke, because their steady-state hyperthermic level (39.5 ± 0.7°C) is 3°C lower than the upper lethal limit (43.1-43.3°C) (9). Severe hyperthermia induces disseminated intravascular coagulation and excess of inflammatory responses (2). Severe hyperthermia is also accompanied by increases in blood-brain permeability, brain water content, and glial fibrillary acidic protein level in rats and by decreases in mean arterial pressure (3), cerebral blood flow, and myelin basic protein (24). Second, serious dehydration/hyperosmolality, which results from long-term evaporative cooling (8, 19), is reduced in FOK rats because during heat exposure the regulated (39.5 ± 07°C) is 2-3°C higher than the normal Tc (near 37°C). Serious dehydration/hyperosmolality attenuates evaporative heat loss (11) and the firing rate of the warm neurons in the median preoptic nucleus (21), which promote evaporative heat loss (25). Therefore, severe dehydration/hyperosmolality could cause further worsening of hyperthermia. Although species with sufficient sweat capacity such as human beings (26) and patas monkeys (12) reduce Tc to normal level (near 37°C), FOK rats do not. Thus FOK rats are able to delay serious dehydration and subsequent heatstroke because they retain body fluid that would be lost if Tc were depressed to 37.0°C.
It was demonstrated that the efficacious contribution of a negative-feedback loop between the thermo-regulation system and the salivary glands underlies the strategy of FOK rats (Fig. 4). Negative-feedback regulation was not proven in the other rat strains. Warm signals from the whole heated body are integrated in thermoregulation centers in the hypothalamus and medulla oblongata (13, 25). As a result, increased efferent impulses from the hypothalamus via the superior salivatory nucleus in the medulla oblongata can stimulate the submandibular glands proportionally to the Tc increase from the threshold (8, 25). A Tc reaching 42.0°C is the range where heat shock protein 70 is gradually synthesized and is an adverse condition for the synthesis of general proteins (5). Nevertheless, it can be speculated that unknown components in the thermoregulation system are especially activated in proportion to the Tc increase from the threshold. The increased saliva spreading dissipates body heat and consequently decreases Tc. Thus heating can be canceled out by the evaporative heat dissipation, and consequently Tc can be maintained within a narrow range (39.5 ± 0.7°C) despite a wide Ta range.
The saliva secretion rates in vitro did not differ among the rat strains or between the two temperatures tested under perfusion of a sufficient amount of ACh. Thus strain difference in the negative-feedback regulation is attributable to strain difference in central events. Nevertheless, these findings do not exclude the possibility of a minor contribution of alterations in the submandibular glands of FOK rats, such as enlargement (8) and secretion of a high concentration of chromogranin A (a Ca2+ storage protein coupled with the inositol 1,4,5-trisphosphate receptor/Ca2+ channel in secretory granules; Refs. 1, 27).
The FOK rat is available for quantitative trait loci analysis and positional cloning (14) because it is an inbred species. It can be expected that knowledge of physiological genomics will be obtained in the areas of thermoregulation, adaptation, osmoregulation, circulation, electrolyte and fluid balance, resistance to environment, and cellular stresses. In addition, the FOK rat is also cold resistant as a result of the repeated heat selection over generations (28). The amount of docosahexanoic acid is high in FOK rats, as in cold-adapted rats (8, 22). Nonshivering thermogenesis in FOK rats is higher than that in the other strains but is not mediated by the well-known pathway via 
-adrenergic receptor (16). Thus FOK rats are also available for studies of cross-adaptation/cross-resistance.
The strategy of the FOK rat for enduring extreme heat is to keep Tc within a moderate steady-state hyperthermic level (39.5 ± 0.7°C). The strategy mainly depends on a low threshold and highly functional negative-feedback regulation between the thermoregulation center and the salivary glands. Heat tolerance in FOK rats is mainly attributable not to salivary glands but to characteristics of the thermoregulation center.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of E. Tanaka: Dept. of Nutritional Sciences, Tokyo University of Agriculture.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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