Journal of Neuroscience Methods 147 (2005) 2935

Baker yeast-induced fever in young rats: Characterization and validation of an animal model for antipyretics screening
a,b , Ana Paula Oliveira Ferreira a,b , Jorgete Tomazetti a,b , Daiana Silva Avila Juliana Saibt Martins a,b , Fabiane Rosa Souza a,b , Carine Royer a,b , Maribel Antonello Rubin a , Marl Redin Oliveira a,b , H elio Gauze Bonacorso a , a a Marcos Ant onio Pinto Martins , Nilo Zanatta , Carlos Fernando Mello b,
a

Departamento de Qu mica, Centro de Ci encias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil b Departamento de Fisiologia e Farmacologia, Centro de Ci encias da Sa ude, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 11 August 2004; accepted 3 March 2005

Abstract In this study we describe a low-cost and reliable method for inducing fever in young male rats (2830 days of age, 7590 g), which seems suitable for the screening of new antipyretics. The effects of temperature measuring procedure-induced stress on the basal rectal temperature and on Baker yeast-induced hyperthermia was assessed. Rectal temperature (TR ) was recorded every hour for 12 h (07:0019:00 h) with a lubricated thermistor probe. The animals were injected intraperitoneally with baker yeast (0.25, 0.135, 0.05 g/kg) or the equivalent volume of saline at 7:00 h. The administration of 0.135 g/kg baker yeast induced a sustained increase in rectal temperature for 4 h. Classical (dipyrone and acetaminophen) and novel (MPCA and FPCA) antipyretics, at doses that had no effect per se, reverted baker yeast-induced fever. The method presented induces a clear-cut fever, which is reverted by antipyretics commonly used in human beings and selected novel antipyretics in small animals. The method also allows antipyretic evaluation with low amount of drugs, due to the use of small animals and to the small variability of the pyretic response, which ultimately causes a signicant reduction in the number of animals necessary for antipyretic evaluation. Therefore, this study describes an animal model of fever that is not only advantageous from the economical and technical point of view, but that also bears ethical concerns. 2005 Elsevier B.V. All rights reserved.
Keywords: Fever; Baker yeast; Rat; Animal model; Antipyretics screening

1. Introduction Although a signicant progress for the understanding of the mechanisms of thermoregulation has been achieved in the past 30 years (Kluger, 1991), the number of safe and effective antipyretics available in the clinics remained practically unaltered during this period. Different factors may have accounted for this fact, but it is particularly remarkable the inherent difculty to induce fever in small experimental animals (Kluger, 1991). In fact, it has been fairly well known

Corresponding author. Tel.: +55 55 2208432; fax: +55 55 2208031. E-mail address: cf.mello@smail.ufsm.br (C.F. Mello).

that rabbits develop fever in response to lipopolysaccharide (LPS) more easily than rats (Morimoto et al., 1990; Stitt et al., 1985), and that mice barely express this sign (Kluger, 1991). The fact that small animals do not present reliable pyrogen-induced fever makes antipyretics screening difcult and expensive, since the amount of drug necessary for these tests increases proportionally to the size of the animal. Moreover, breeding of larger rodents also implies increasing accommodation and other maintenance costs. Besides these economical features, there are also other factors that make antipyretics screening difcult in small rodents, such as the well-known stress-induced hyperthermia (Endo and Shiraki, 2000; Kluger et al., 1987; Thompson et al., 2003), which

0165-0270/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.03.002

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usually mask pyrogen-induced fever by increasing control temperature values (Briese and Cabanac, 1991) and the use of different pyrogenic agents (Winter et al., 1963). To use different pyrogens to cause fever in a given work may be sometimes advantageous, since it may indicate possible mechanisms of action (Shimada et al., 1994). However, the use of different pyrogens in the literature certainly have also provided an additional source of variation for antipyretic screening results, since it has been reported that LPS-induced fever is dependent on the serotype of its source (Dogan et al., 2000). In addition, there are several reports of hypothermia, instead of fever, after LPS and yeast administration to rats and mice (Dogan et al., 2002; Renetti et al., 1990). Therefore, it seems that a reliable experimental protocol for antipyretics screening using small animals is still lacking in the literature. Moreover, the development of a reliable model of pyrexia in small animals would facilitate the study of fever mechanisms and thermoregulation. In this study we describe a protocol of fever induction in young rats by baker yeast that seems suitable for the screening of new antipyretics, considering both the economical and technical aspects of drug screening, since it uses small animals and is reliable in the sense that it induces long-lasting fever to the vast majority of the animals. Importantly, such a fever is sensitive to antipyretics that are used in the clinics and is induced by an easily available nontoxic pyrogen.

of eight to a cage at controlled temperature (23 1 C) with a 12 h light:12 h dark cycle (lights on at 07:00 h) and with standard lab chow and tap water ad libitum. The animals were transferred to the experimental room 2 h before the experiments for acclimation to the environment. All measures temperatures were taken between 08:00 and 19:00 h and room temperature was kept at 23 (1) C. Each animal was used only once, and no more than one animal per litter was assigned to each group. The experiments were approved by the Committee on the Use and Care of Laboratory Animals of our University. 2.3. Rectal temperature measurement Rectal temperature (TR ) was measured by inserting a lubricated thermistor probe (external diameter: 3 mm) 2.8 cm into the rectum of the animal. The probe was linked to a digital device, which displayed the temperature at the tip of the probe with a 0.1 C precision. The values displayed were manually recorded. 2.4. Effect of baker yeast on rectal temperature Immediately after measuring the initial basal rectal temperature, the animals were injected with baker yeast (0.050.25 g/kg, i.p.) or 0.9% NaCl (10 ml/kg). TR changes were recorded every hour up to 12 h, and expressed as the difference from the basal value. Since it has been previously reported that handling and temperature measuring-related stress alter rectal temperature, these animals were habituated to the injection and measuring procedure for 2 days before experiments were carried out. In these sessions, the animals were subjected to the same temperature measuring procedure described above, and were injected intraperitoneally (i.p.) with 0.9% NaCl (10 ml/kg). 2.5. Effect of habituation on basal rectal temperature and on baker yeast-induced fever After determining the effective pyrogenic dose of yeast in previously habituated animals, we evaluated whether habituation was necessary for the appearance and for the reliability of the pyrogenic response. The animals were subjected to two, one or no habituation sessions and, in the day of the experiment, injected with the pyrogenic dose of yeast (0.135 g/kg). TR changes were recorded every hour up to 12 h, and expressed as the difference from the basal value. 2.6. Effect of antipyretics on basal rectal temperature The animals had their TR measured for 4 h, and after the fourth TR measurement they were subcutaneously (s.c.) injected with vehicle (5% Tween 80 in 0.9% NaCl, 5 ml/kg), dipyrone (0.15, 0.3, 0.5 or 1.5 mmol/kg), acetaminophen (1, 1.25 or 1.5 mmol/kg) MPCA (0.15, 0.3, 0.5 or 1.5 mmol/kg),

2. Materials and methods 2.1. Drugs Commercially available dried baker yeast (Saccharomyces cerevisiae, Saf do Brasil Produtos Aliment cios Ltda, Brazil) was suspended in pyrogen-free 0.9% NaCl in a water bath at 37 C for 5 min. Dipyrone (Hoechst) was prepared in 0.9% NaCl. 3-Methyl-5-hydroxy-5trichloromethyl-4,5-dihydro-1H-pyrazole-1-carboxyamide (MPCA) and 3-phenyl-5-hydroxy-5-trichloromethyl4,5-dihydro-1H-pyrazole-1-carboxyamide (PPCA) were synthesized by the NUQUIMHE, as reported elsewhere (Bonacorso et al., 1999) and were suspended in 5% Tween 80 and 0.9% NaCl (Souza et al., 2002). Acetaminophen was purchased from Sigma (Saint Louis, MO) and was suspended in 5% Tween 80 and 0.9% NaCl. All other reagents were of analytical grade and were purchased from local suppliers. Drug doses used in those experiments designed to evaluate the effectiveness of antipyretics in the presently reported experimental model were chosen based on doseeffect curves, and had no effect per se in the assay. 2.2. Subjects and maintenance Male Wistar rats (2830 days of age, 7590 g) bred in our animal house were used. The animals were housed in groups

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or PPCA (0.05, 0.15, 0.5 or 1.5 mmol/kg). TR was recorded every hour up to 8 h after the drug injections. 2.7. Effect of antipyretics on baker yeast-induced hyperthermia The animals had their basal TR measured and were injected with a pyrogenic dose of baker yeast (0.135 g/kg). TR changes were recorded every hour up to 4 h, when antipyretics dipyrone (0.3 mmol/kg), acetaminophen (1.25 mmol/kg), MPCA (0.15 mmol/kg) or PPCA (0.05 mmol/kg) were administered. The TR was monitored over the following 8 h. 2.8. Statistical analysis Basal rectal temperature and changes in rectal temperature were expressed as means S.E.M. of the differences from TR at 07:00 h. Data were analysed by two- or three-way analysis of variance (ANOVA), with time of measures treated as within subject factor, depending on the experimental design. Post hoc analysis was carried out by the F-test for simple effect and the StudentNewmanKeuls test, when appropriate. A value of P < 0.05 was considered statistically signicant.

action: F(33,660) = 2.34; P < 0.05. Post hoc analysis (F-test for simple effect) showed that animals receiving 0.135 g/kg varied TR differently from saline-treated animals along time [F(11,660) = 3.77; P < 0.001]. Such an effect was caused by a sustained increase in TR 4 h after yeast administration. The administration of higher amounts of yeast (0.50 g/kg, i.p.) caused severe hypothermia and death (data not shown), while lower doses (0.05 g/kg) were not as effective to cause hyperthermia as the 0.135 g/kg dose. 3.2. Effect of habituation on basal rectal temperature The effect of the number of habituation sessions on the basal rectal temperature and on yeast-induced fever (0.135 g/kg) was investigated. For the sake of clarity, data are presented in three separate graphics (Fig. 2), though statistical analysis was carried out using all data. Statistical analysis revealed a signicant habituation levels (0, 1 or 2 days of habituation) by treatment (saline or yeast) by time of measures interaction: F(22,594) = 1.56; P < 0.05. Post hoc analysis revealed that only animals subjected to habituation sessions presented yeastinduced hyperthermia. This occurred because nonhabituated animals presented higher rectal temperature values than those habituated, making difcult the observation of yeast-induced hyperthermia. Although statistical analysis of basal rectal temperatures at testing did not differ between habituation conditions [F(2,126) = 1.94; P > 0.1Fig. 3], statistical analysis (ANOVA with repeated measures) of basal rectal temperature of animals subjected to two habituation sessions revealed that animals decreased basal rectal temperature along the habituation sessions [F(2,126) = 5.84; P < 0.005Fig. 4]. Data from yeast- and NaCl-injected animals were pooled for this analysis, since animals were injected with yeast or NaCl only after the basal temperature measuring. Therefore, at the time of basal rectal temperature measuring (day 3), all the animals were subjected to the same experimental conditions. 3.3. Effect of antipyretics on basal rectal temperature of young rats The effect of classical (dipyrone and acetaminophen) and of novel (MPCA and PPCA) antipyretics on the basal rectal temperature of young rats was evaluated. For each antipyretic a doseeffect curve was performed, and the maximal dose that caused no effect per se on rectal temperature was determined to test, in a subsequent set of experiments, if each antipyretic could revert yeast-induced fever. Dipyrone altered basal rectal temperature of young rats, as revealed by the signicant dipyrone doses time of measures interaction [F(28,210) = 6.99; P < 0.001Fig. 5] in the twoway ANOVA with repeated measures. Post hoc analysis (Ftest for simple effect) showed that while 0.3 mmol/kg dipyrone did not alter basal rectal temperature [F(7,210) = 2.7; P > 0.05], higher doses (0.5 and 1.5 mmol/kg) caused signicant hypothermia compared to control [F(7,210) = 6.81;

3. Results 3.1. Effect of increasing baker yeast doses on rectal temperature Fig. 1 shows the effect of the intraperitoneal injection of increasing amounts of baker yeast on TR (for the sake of clarity, 0.050 g/kg data were omitted in the graphic). Statistical analysis (two-way ANOVA) revealed a signicant baker yeast dose (0, 0.05, 0.135, 0.250 g/kg) time of measures inter-

Fig. 1. Effect of the i.p. administration of 0.9% NaCl, 0.135 or 0.250 g/kg baker yeast on rectal temperature change ( TR ) along time. Values represent mean S.E.M. change from baseline rectal temperature (n = 1517 animals per group). Statistical analysis (two way ANOVA) showed that 0.135 g/kg baker yeast curve (black triangle) differs from vehicle curve (open circle). For the sake of clarity, 0.050 g/kg data were omitted in the graphic. F values are given in the text.

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Fig. 3. Effect of the number of habituation sessions on basal rectal temperature at testing. 0, naive animals; 1, animals subjected to one session of habituation; 2, animals subjected to two sessions of habituation. Data are reported as means S.E.M. (n = 1517 animals per group). No two groups differ statistically (one way ANOVA).

Acetaminophen, like dipyrone, altered basal rectal temperature of young rats [signicant acetaminophen doses measures interaction F(21,175) = 1.77; P < 0.05]. Post hoc analysis (F-test for simple effect) showed that while 1.5 mmol/kg acetaminophen caused some degree of hypothermia [F(7,175) = 4.87; P < 0.05], the dose of 1.25 mmol/kg had no effect on rectal temperature [F(7,175) = 1.00; P < 0.05Fig. 6). Therefore, the dose of 1.25 mmol/kg was chosen to test whether acetaminophen reverted yeast-induced fever in rats in a subsequent set of experiments. The novel antipyretic pyrazolines MPCA and PPCA, similarly to all other antipyretics tested in this study, altered the basal rectal temperature of young rats [signicant drug doses time of measures interaction for MPCA: F(28,217) = 2.77; P < 0.05Fig. 7 and for PPCA: F(28,252) = 2.01; P < 0.05Fig. 8]. Post hoc analyses (F-test for simple effect) revealed that high doses of

Fig. 2. Rectal temperature change ( TR ) along time induced by 0.135 g/kg baker yeast in animals subjected to zero (A) one (B) or two (C) habituation sessions. Values represent mean S.E.M. change from baseline rectal temperature (n = 1217 animals per group). For the sake of clarity, data are presented in three separate graphics, though statistical analysis (two way ANOVA) was carried out using all data. F values are given in the text.

P < 0.001, and F(7,210) = 13.38; P < 0.001, respectively]. Therefore, the dose of 0.3 mmol/kg was selected to test whether dipyrone reverted yeast-induced fever in rats in a subsequent set of experiments.

Fig. 4. Basal rectal temperature of animals subjected to two habituation sessions. Values represent mean S.E.M. basal rectal temperature (n = 1517 animals per group).

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Fig. 5. Effect of subcutaneous administration of 0.9% NaCl, 0.3 or 1.5 mmol/kg dipyrone (Dip) on rectal temperature change ( TR ) along time. Values represent mean S.E.M. change from baseline rectal temperature (n = 68 animals per group). The arrow indicates the time of injection of drugs or vehicle. Post hoc analysis (F-test for simple effect) showed that 1.5 dipyrone curve (black triangle) differs from vehicle curve (open circle). For the sake of clarity, 0.15 and 0.5 mmol/kg dipyrone data were omitted.

Fig. 7. Effect of subcutaneous administration of vehicle (5% Tween 80), 0.15 or 0.5 mmol/kg MPCA on rectal temperature change ( TR ) along time. Values represent mean S.E.M. change from baseline rectal temperature (n = 68 animals per group). The arrow indicates the time of injection of drugs or vehicle. Post hoc analysis (F-test for simple effect) showed that 1.5 mmol/kg MPCA curve (black triangle) differs from vehicle curve (open circle) (P < 0.05). For the sake of clarity, 0.3 and 1.5 mmol/kg MPCA data were omitted.

MPCA (1.5 mmol/kg: F(7,217) = 6.39; P < 0.05) and of PPCA (0.5 and 1.5 mmol/kg: F(7,252) = 2.77; P < 0.05 and F(7,252) = 3.7; P < 0.05, respectively) caused signicant hypothermia. The doses of 0.15 mmol/kg of MPCA and 0.05 mmol/kg of PPCA had no effect on basal rectal temperature [F(7,217) = 1.21; P > 0.05 and F(7,252) = 0.94; P > 0.05, respectively], and were chosen to test whether these compounds reverted yeast-induced fever in rats in a subsequent set of experiments.

3.4. Classic and novel antipyretics revert yeast-induced fever in young rats Fig. 9AD show the effect of classic antipyretics and of novel antipyretics on the yeast-induced fever in young rats. All antipyretics, at doses that had no effect per se on rectal temperature, reverted baker yeast-induced fever [F(7,189) = 14.45; P < 0.001, for dipyrone, F(7,189) = 5.54; P < 0.001, for acetaminophen,

Fig. 6. Effect of subcutaneous administration of vehicle (5% Tween 80), 1.25 or 1.5 mmol/kg acetaminophen (Acet) on rectal temperature change ( TR ) along time. Values represent mean S.E.M. change from baseline rectal temperature (n = 49 animals per group). The arrow indicates the time of injection of drugs or vehicle. Post hoc analysis (F-test for simple effect) showed that 1.5 acetaminophen curve (black triangle) differs from vehicle curve (open circle). For the sake of clarity, 1.0 mmol/kg acetaminophen data were omitted.

Fig. 8. Effect of subcutaneous administration of vehicle (5% Tween 80), 0.05 or 0.5 mmol/kg PPCA on rectal temperature change ( TR ) along time. Values represent mean S.E.M. change from baseline rectal temperature (n = 78 animals per group). The arrow indicates the time of injection of drugs or vehicle. Post hoc analysis (F-test for simple effect) showed that 0.5 mmol/kg PPCA curve (black triangle) differs from vehicle curve (open circle) (P < 0.05). For the sake of clarity, 0.15 and 1.5 mmol/kg PPCA data were omitted.

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Fig. 9. Effect of subcutaneous administration of (A) 0.9% NaCl or 0.3 mmol/kg dipyrone (Dip), (B) 5% Tween 80 or 1.25 mmol/kg acetaminophen, (C) 5% Tween 80 or 0.5 mmol/kg MPCA and (D) 5% Tween 80 or 0.15 mmol/kg PPCA on baker yeast-induced fever. The arrow indicates the time of injection of drugs or vehicle. Data are reported as means S.E.M. (n = 612 animals per group) of change from baseline rectal temperature. * Indicates that pharmacological treatment altered variation of rectal temperature along time, compared to 0.9% NaCl. F values are given in the text.

F(7,210) = 3.81; P < 0.01, for MPCA and F(7,175) = 3.11; P < 0.005, for PPCA].

4. Discussion In this study we describe a low-cost and reliable method for inducing fever in young rats. The present method of fever induction seems to be advantageous compared to other methods that use large animals and other pyrogenic agents, considering different aspects of animal and drug testing, which will be discussed in detail below. First of all, we shall establish some conditions or properties that would be particularly desirable in a good animal model of fever. A good pyrogenic response should (1) occur in all or the vast majority of the animals; (2) be high enough to be unequivocally detected; (3) install promptly or in a few hours, in order to minimize the duration of experiments and animal discomfort; (4) last enough to allow pharmacological manipulations; (5) be sensitive to classic antipyretics, partic-

ularly those considered effective in human beings. We believe that if these conditions were met, a reliable animal model of fever that bears ethical concerns would result. The presently reported method of fever induction seems to meet all these conditions, since it causes an easily detectable increase in the rectal temperature of young rats, which achieves statistical signicance with a relatively small number of animals. It is worth remarking that such a difference between group means was easily detected because it was high enough, and because temperature variability within groups was small, regardless of whether they received yeast or not. Therefore, as described in the ANOVA results, most of the variability observed was due to yeast effects. Another condition that seems to be satised by this model is that fever installs in a few hours (45 h) after yeast injection. This characteristic is particularly important because it has been described that adult rats develop fever usually 918 h after intraperitoneal yeast injection (Santos and Rao, 1998). Therefore, the fact that in our model animals develop fever sooner could abbreviate the duration of the experiments

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and animal discomfort, a remarkable advantage when body temperature monitoring is manual. The fourth condition seems also to be met, since yeast-induced fever lasted at least 4 h, which is quite enough time to evaluate if different compounds may revert yeast-induced fever. However, it is worth remarking that the presently reported duration of yeast-induced fever (45 h) contrasts with previous reports which have described long lasting fever episodes (up to 24 h) elicited by yeast in adult rats (Bruguerolle and Roucoules, 1994; Rouveix, 1980). Such a discrepancy may be due to age differences, which may account for different immune responses or other factors, such as habituation to the pyrogenic stimulus (Balmagiya and Rozovski, 1983; Chorinchath et al., 1996; Ferguson et al., 1985; Florez-Duquet et al., 2001). Finally, our model seems to meet the fth criterion, since yeast-induced fever was reverted by classic (dipyrone and acetaminophen) and novel (MPCA and PPCA) antipyretics, at doses that had no effect per se in the assay. Regarding this point it is particularly interesting that the pyrazole derivatives (dipyrone, MPCA and PPCA) were more effective than acetaminophen to cause antipyresis. Such a more effective antipyretic response of dipyrone compared to acetaminophen has been fairly well known in the clinics (Borne, 1995), and such a similarity with the human condition tempt us to propose that the present model may be useful to screen effective antipyretics for human beings. Nevertheless, caution regarding this point is advisable, and further in-depth studies with other antipyretics and with human beings will dene whether the antipyretic activity (and potency) of different drugs in this model possesses predictive value to human beings. In summary, in this study we present and characterize a reliable protocol to induce fever in young rats that seems particularly suitable to evaluate new antipyretics, since the fever induced by the present protocol is sensitive to classic and novel antipyretic drugs. One of the main advantages of the present model is that it allows antipyretic evaluation with low amount of drugs, due to the use of small animals and to the small variability of the pyretic response, which ultimately causes a signicant reduction in the number of animals necessary for such an evaluation. Therefore, this protocol seems to be not only advantageous from the economical and technical point of view, but also an animal model of fever that bears ethical concerns.

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