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INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION
Department of Psychology and Program in Neuroscience, University of Delaware, Newark, Delaware 19716-2590
Submitted 1 May 2003 ; accepted in final form 25 July 2003
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ABSTRACT |
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aging; heat production; heat loss; ambient temperature; lipopolysaccharide
To develop a fever, an animal must increase its heat production and decrease its heat loss in a coordinated effort to raise its body temperature (Tb). Studies in young rats have reported a 12-20% increase in oxygen (O2) consumption (a measure of heat production) after pyrogen administration at Ta values from 21 to 25°C (3, 4, 10, 18). Others studies have reported decreases in heat loss in young rats after LPS (9, 15).
In the present study, we investigated thermoregulatory responses in old and young rats after peripheral administration of LPS. We measured whole body heat production and total heat loss at two different Tas, 21 and 31°C. We found that heat production decreased dramatically in the old rats at 21°C. These results suggest that the blunted fever seen in old rats at lower Tas is most likely due to a decrease in heat production.
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METHODS AND MATERIALS |
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Experimental design. At least 1 wk postoperatively, a rat was placed in the calorimeter (see below) without food or water for at least 4 h for acclimatization. The next day, after 3 h in the calorimeter (at 10:00 AM), the rat was injected (ip) with either LPS or saline at a Ta of either 21 or 31 ± 1°C. The rat remained in the calorimeter for a further 7 h. At least 3 days after the first injection, the procedure was repeated with the alternate injection at the same Ta. At least 3 wk later, the experiment was repeated with the same rat at the other Ta. Injection and Ta exposure were counterbalanced to account for any order effects. Rats were weighed before being placed in the calorimeter on each injection day. At the time of injection, vaginal smears were obtained from the young female rats to determine the stage of the estrous cycle.
All 12 young rats were tested at both Tas. Three old male and four old female rats received injections at both Tas. Four old males and two old females received injections at only one Ta. Old rats at a Ta of 21°C include the seven old rats that received injections at both Tas plus two old males and one old female that were tested at a Ta of 21°C only. Old rats at a Ta of 31°C include the seven old rats that received injections at both Tas plus two old males and one old female that were tested at a Ta of 31°C only.
LPS. Purified lyophilized phenol extract of Escherichia coli endotoxin (0111:B4; Sigma, St. Louis, MO) was dissolved in sterile saline, aliquoted, and frozen at -20°C. LPS was injected (ip) at a dose of 50 µg/kg. Injection of an equivalent volume of saline was used as a control.
Measurement of Tb. Tb of rats in their home cages was monitored using a peripheral processor (Datacol III System) connected to a personal computer (PC). Temperature-dependent transmitter pulse frequencies were converted to Tb by the Datacol system and stored on hard disk. When animals were in the calorimeter, the transmitter frequencies were received via a loop antenna and collected in the same manner. Tb was recorded every 5 min until the end of the experiments.
Measurement of heat loss and heat production. Nonevaporative heat loss was measured in a calorimeter (30 x 15 x 15 cm, model Sec A; Thermonetics, La Jolla, CA). Heat generated by the animal passed through the walls of the calorimeter and was measured by thermoelectric sensors distributed throughout the walls of the calorimeter. The sensors operate on the gradient layer principle in which the flow of heat through a solid results in a measurable temperature gradient in the direction of heat flow. The temperature difference across the gradient layer was measured by thermopiles, the output of which is a measure of the heat flow. The output signal (a direct current potential) was recorded on a PC and then converted to calories per hour. Evaporative heat loss was calculated from the airflow and the temperature and humidity of air that were measured by inlet and outlet micropsychrometers (Thermonetics). Airflow, humidity, and temperature values were entered into Akton Psychrometric Chart software (Akton, Concord, CA) to yield evaporative heat loss in British thermal units.
Heat production was estimated by O2 consumption and calculated from measurements of O2 content and airflow. Room air was drawn through the calorimeter at 2 ml/min. Expired air was desiccated by drawing it through a canister of 8-mesh Drierite (W.A. Hammond Drierite, Xenia, OH). Airflow was controlled with a Matheson mass flow controller. A fraction of the dried air was drawn through an O2 analyzer (Applied Electrochemistry, S-3A) that provided continuous recording of percent O2 values. At 1-min intervals, these values were fed into a PC and converted into milliliters of O2 per minute using the formula
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where O2in is the baseline O2 percentage going into the chamber, O2out is the O2 percentage coming out of the chamber, and F is flow rate of air into the chamber (in ml/min). This value was then multiplied by the caloric equivalent of O2 (0.4825 cal/ml) to yield calories per hour.
All heat production and heat loss values were converted into watts and divided by kg0.75. Nonevaporative and evaporative heat loss were added together to yield total heat loss.
Data analysis. Results are presented as means ± SE; 1-min O2 consumption and heat loss and 5-min Tb data were averaged over 30 min. O2 consumption and heat loss data are presented as percent change from time 0. Baseline measurements were designated as the 30-min average before each injection. Statistical differences were analyzed using analysis of variance (ANOVA) followed by Tukey's post hoc tests. Separate ANOVAs with repeated measures were run on the rats that received injections at both Tas. Results from analyses with and without rats that received treatment at only one Ta were similar. Unless otherwise stated, statistical significance was taken from the analysis that included all animals. Significance level was set at P < 0.05.
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RESULTS |
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None of the young female rats was in estrus on the days of injection. Because there were no significant sex differences at either Ta, data from males and females were grouped for both ages.
Young vs. old rats after LPS at 21°C. Starting about 90 min after LPS injection, young rats showed febrile responses leading to a maximum fever of 38.3 ± 0.2°Cby 330 min (Fig. 1A; P < 0.001). After LPS, old rats showed a small but significant rise in Tb compared with saline (P < 0.001). However, this rise did not significantly differ from baseline Tb. O2 consumption rose immediately in response to handling and injection in both groups of rats. In the young rats, O2 consumption stayed elevated for 6 h, whereas in the old rats, it fell by a maximum of 
15% and only returned to baseline levels toward the end of the time in the calorimeter (Fig. 1B; P < 0.001 compared with saline, P < 0.001 compared with young). Heat loss decreased in the same way in both groups in response to LPS (Fig. 1C). In summary, at 21°C, old rats had a blunted fever response to LPS that was accompanied by a drop in O2 consumption.
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Young vs. old rats after LPS at 31°C. The fever response of the young and old rats did not differ at this Ta (Fig. 2). After LPS, young rats showed febrile responses leading to a maximum fever of 38.7 ± 0.2°C by 300 min (P < 0.001). Old rats showed febrile responses leading to a maximum fever of 38.4 ± 0.2°C by 300 min (P < 0.001). O2 consumption was not significantly different between young and old rats at this Ta, nor was heat loss. In summary, at 31°C there were no metabolic differences between young and old rats and their fevers were similar.
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DISCUSSION |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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O2 consumption is a measure of whole body heat production. It reflects the contribution of both shivering and nonshivering thermogenesis (accomplished through activation of brown adipose tissue; BAT). It is generally lower in warm environments, as was seen here in the young rats but not in the old ones (Table 1). After LPS injections, young rats increased their O2 consumption by >15% at 21°C and by 12% at 31°C. These results support findings from other laboratories in which O2 consumption at Tas between 22 and 26°C increased 12-20% during fever (3, 4, 10).
In contrast, old rats decreased their O2 consumption by 15% at a Ta of 21°C. This was completely unexpected, and several reasonable hypotheses fail to explain it. For instance, the amount of functional BAT declines with age, which could result in a decrease in nonshivering heat production (5, 11). Scarpace et al. (17) reported that blunted fevers in old rats might be due to a failure to increase BAT thermogenesis after pyrogen. We did not measure BAT activity directly, but it would necessarily be reflected in the O2 consumption values, and, indeed, resting O2 consumption levels were lower in our old rats. However, a failure on the part of old rats to increase BAT activity would not explain the decrease in O2 consumption we report here. At best, it should leave O2 consumption unchanged, not reduced, after LPS.
Given that old rats prefer warm Tas (6), 21°Cismost likely a mild cold stress. One might assume, therefore, that the old rats may already be close to their maximum heat-producing capabilities. However, from our own studies (unpublished data) as well as others (8, 12), we know that old rats exposed to cold can increase O2 consumption by as much as 41%. Therefore, the possibility that old rats simply cannot increase heat production at 21°C is not tenable.
Another possibility is that old rats are more sensitive to LPS than are young rats. This would mean that a dose that generally causes fever in young rats could cause endotoxin shock in old rats. Endotoxin shock is characterized by a decrease in O2 consumption and increased peripheral vasodilation, hypothermia, and cold-seeking behavior (16). However, despite showing decreased O2 consumption after 50 µg/kg of LPS, our old rats showed no other symptom of shock. None of the old rats became hypothermic, none preferred a cooler Ta (6), and none showed increased heat loss (reflecting peripheral vasodilation). Indeed, old rats decreased heat loss as well as did young rats after LPS. Further, a lower dose of 25 µg/kg of LPS did not cause fever in old rats at a Ta of either 21 or 31°C (unpublished data).
A last possibility for the decreased O2 consumption is that the cytokines induced peripherally by LPS injection are not penetrating the blood-brain barrier as well in old rats and that they therefore have much higher levels of peripheral cytokines than do young rats. LPS induces macrophages to produce and release proinflammatory cytokines (specifically, IL-1
, IL-6, and tumor necrosis factor-
) that are responsible for a number of peripheral and central effects. McLay et al. (13) reported that IL-1 does not cross the blood-brain barrier in aged mice nearly as readily as it does in young ones. In turn, this may lead to increased levels of peripheral cytokines. Indeed, there are several reports of increased peripheral cytokines after LPS in old rodents compared with young (2, 7, 19, 20). We do not know whether high levels of peripheral cytokines can cause a decrease in metabolic rate, but this is certainly a question worth investigating.
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DISCLOSURES |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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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REFERENCES |
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TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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and nitric oxide production in endotoxic mice. J Immunol 156: 1525-1530, 1996.[Abstract]
This article has been cited by other articles:
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  J. B. Buchanan, E. Peloso, and E. Satinoff A warmer ambient temperature increases the passage of interleukin-1{beta} into the brains of old rats Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R361 - R368. [Abstract] [Full Text] [PDF]
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A. A. Romanovsky Anorexia: the toll for lipopolysaccharide recognition Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R274 - R275. [Full Text] [PDF]
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