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INNOVATIVE METHODOLOGY
CALL FOR PAPERS
Physiology and Pharmacology of Temperature Regulation
Systemic Inflammation Laboratory, Trauma Research, St. Joseph's Hospital and Medical Center, Phoenix, Arizona
Submitted 26 May 2005 ; accepted in final form 28 July 2005
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ABSTRACT |
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3 h) hypothermic response in mice, the typical early (latency, 10–30 min) hypothermic response seen in rats did not occur. The present investigation identifies experimental conditions to study LPS-induced mono-, bi-, and polyphasic fevers and late hypothermia in mice and provides detailed characteristics of these responses. body temperature; fever; hypothermia; febrile phases; thermoneutrality; systemic inflammation; stress; mice; rats
Over the last decade, genetically engineered mice have been increasingly used as a tool for studying the mechanisms of LPS-induced systemic inflammation and its thermoregulatory manifestations (2, 3, 9, 12–14, 18, 21, 37). However, the thermoregulatory responses to LPS have not been fully characterized in this species due to the following potential pitfalls.
First, a stressful, painful procedure of LPS injection was used in the majority, if not all, studies. Typically (for recent examples, see Refs. 3, 6, 13, 16, 21), a mouse that had not been habituated to the injection procedure was removed from its cage and pricked with the injection needle in its abdomen (for an intraperitoneal injection) or tail (for an intravenous injection). Such a stressful procedure resulted in a highly variable, often profound (1–3°C) and long-lasting (30–120 min), elevation in Tb. Because the febrile response starts 10–30 min after LPS administration in several species, including the rat (35) and rabbit (19), the stress hyperthermia was likely to override the initial febrile rise of Tb. That stress hyperthermia masks the first phase of LPS-induced polyphasic fever in the rat has been shown in a direct experiment (27). Not only does needle pricking-induced hyperthermia hide some parts of the thermoregulatory response to LPS, but it also may alter the dynamics of the response at its later stages. For example, Sugimoto et al. (34) have reported that needle pricking transforms the monophasic febrile response of rats to LPS into a qualitatively different, biphasic, response.
The second pitfall is that many experiments in mice were performed at a poorly controlled Ta: it fluctuated within 2°C in a single study and varied by almost 10°C among studies (6, 13, 16, 21). For example, Ta was 22–25°C in the studies by Li et al. (16) and Oka et al. (21), but it was 29–31°C in the studies by Gourine et al. (6) and Kozak and Kozak (13). Whereas the former range is clearly below the thermoneutral zone of the mouse, the latter range is neutral or near-neutral (5). In several species, especially small rodents such as the rat, the thermoregulatory response to LPS strongly depends on Ta, and a difference of a few degrees centigrade is sufficient to change the response drastically, e.g., from hypothermia to fever (27). Hence, poor control of Ta was likely responsible for the overall inconsistency in the Tb patterns observed in mice.
The third pitfall is that the thermoregulatory responses to LPS strongly depend on its dose. As explained above, both rats (27) and rabbits (19) mount qualitatively different responses to different LPS doses. However, with the exception of a few studies (e.g., Refs. 12 and 21), such dependence was largely ignored in the mouse.
The present study was aimed at identifying and characterizing the murine thermoregulatory responses to LPS under experimental conditions free of the three pitfalls described above. In this study, the procedure of LPS injection was painless and nonstressful, Ta was tightly controlled, and a wide range of LPS doses was tested.
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MATERIALS AND METHODS |
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Surgery. Each mouse was subjected to either one or both of the following surgical procedures: chronic jugular vein catheterization and implantation of a temperature transmitter in the peritoneal cavity. These procedures were performed under ketamine-xylazine-acepromazine (42.0, 4.8, and 0.6 mg/kg, respectively, ip) anesthesia and antibiotic (enrofloxacin, 3.75 mg/kg sc) protection. The mouse was kept on a heating pad and periodically (every 5 min) ventilated with oxygen through a custom-made mask. For jugular catheterization, a 5-mm longitudinal incision was made on the ventral surface of the neck, 5 mm right to the trachea. The right jugular vein was exposed, freed from its surrounding connective tissue, and ligated. The vein was then catheterized according to the procedure by Harms and Ojeda (8) with minor modifications. In brief, a silicone catheter (ID, 0.5 mm; OD, 0.9 mm) was attached to the hub end of a bent (L-shaped) 21-gauge injection needle with its hub removed. The needle (with the catheter attached) was thrust into the lumen of the vein and pulled out through the chest muscles. The catheter was then disconnected from the needle, filled with heparinized (50 U/ml) saline, pulled back into the jugular vein, and pushed forward to a premeasured distance. As a result, the tip of the catheter was placed in the superior vena cava. The catheter was fixed in place with ligatures, and its free end was knotted, tunneled under the skin, and exteriorized at the nape. The surgical wound on the neck was sutured. For implantation of a miniature (25 x 8 x 8 mm) temperature transmitter (series 4000 E-Mitter; Mini Mitter, Bend, OR), a midline laparotomy was performed, the probe was inserted in the peritoneal cavity, and the surgical wound was sutured. To prevent postsurgical hypothermia, all mice were allowed to recover from anesthesia at Ta of 32°C. On day 1 postsurgery, the catheter was flushed with heparinized (10 U/ml) pyrogen-free saline. The mice tolerated the surgical procedures well: they showed only a minor (5–10%) loss of body mass on day 1 and regained mass on day 2. The experiments were performed on day 3 by using either telemetric or thermocouple thermometry.
Telemetric thermometry. This experimental setup permitted monitoring the abdominal temperature (an index of Tb) of freely moving mice implanted with temperature transmitters. Telemetry receivers (model ER-4000; Mini Mitter, Bend, OR) were positioned inside a climatic chamber (model 3940; Forma Scientific, Marietta, OH) and connected to a computer. The chamber had dimension of 79 x 152 x 69 cm and was equipped with fluorescent lamps that provided regular lighting conditions. Air humidity was set to 50%. According to the manufacturer's specifications, the precision of the temperature control in the chamber is ±0.1°C and the uniformity is ±0.4°C. The home cage of each mouse was placed on top of a receiver, a Plexiglas enclosure was placed inside the cage, and the mouse was left in the enclosure. The jugular catheter was extended with a length of polyethylene-50 (PE-50) tubing filled with the drug of interest (for details, see LPS preparation and administration below). The extension was passed through a hanger on the top of the enclosure and then through a wall port of the climatic chamber. The extension was connected to a syringe filled with saline. This telemetry setup (Fig. 1) permitted intravenous administration of the drug to the mouse from outside the chamber without disturbing the animal.
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0.5 µl air bubble. At the time of injection, LPS (100–104 µg/kg) was flushed into the mouse's circulation by replacing the LPS suspension at the mouse end of the extension with 0.3 ml of saline injected from the syringe end. In a control, the extension was prefilled with saline only, and the injection was performed by the same procedure. This procedure was executed without touching the animal or causing pain or stress. In a separate experiment, LPS (10 ml/kg; 103–104 µg/kg) or saline was injected intraperitoneally by a common, stressful procedure that involved pricking the abdominal wall with a needle. Data processing and analysis. The absolute values of Tb and, when available, Tsk were used to evaluate the thermoregulatory responses to LPS. In most cases, the responses were compared across treatments and time points by a two-way ANOVA, and F and P values (for the entire observation period/response duration) were reported. When the effect of treatment was evaluated on two parameters simultaneously (the effect of LPS dose on the latency and amplitude of the first phase of polyphasic fever and the effect of LPS on Tb and Tsk in the thermocouple experiment), one-way MANOVA was used, and R and P values were reported. The analyses were performed using Statistica AX'99 (StatSoft, Tulsa, OK). Compared responses were considered significantly different at P < 5.0 x 10–2. Results are reported as means ± SE.
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RESULTS |
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0.5°C) decrease in Tb over the course of the experiment (Fig. 5A). Such a decrease often occurs in untreated or saline-treated mice during the light phase of the day at a subneutral or neutral Ta, presumably reflecting the circadian rhythm of Tb (15, 20, 21, 31, 36). Compared with saline, all doses of LPS (100–104 µg/kg) induced a significant rise in Tb (100 µg/kg: F1,429 = 1.6 x 101, P = 9.4 x 10–5; 101 µg/kg: F1,390 = 1.3 x 102, P = 1.0 x 10–7; 101.5 µg/kg: F1,429 = 7.2 x 101, P = 1.0 x 10–7; 101.75 µg/kg: F1,507 = 3.6 x 102, P = 1.0 x 10–7; 102 µg/kg: F1,507 = 1.1 x 103, P = 1.0 x 10–7; 103 µg/kg: F1,507 = 7.3 x 103, P = 1.0 x 10–7; 104 µg/kg: F1,468 = 9.5 x 103, P = 1.0 x 10–7). The dynamics of the response was dose dependent. LPS induced a monophasic fever at 100–101 µg/kg (Fig. 5, B and C) and a biphasic fever at 101.5 µg/kg (Fig. 5D). Similar to what occurs in rats at a narrow dose range around 101 µg/kg (24), there was a drastic change in the response of the mice when the dose increased from 101.5 to 101.75–102 µg/kg: the biphasic fever was replaced by a qualitatively different polyphasic fever (Fig. 5, E and F). Three distinct febrile phases were observed over the time of the experiment; the third phase was the most pronounced (Fig. 6, A and C). When the data were plotted in a phase plane (rate of Tb change vs. Tb; for further explanations, see Ref. 29), these three phases were visualized as three separate loops (Fig. 6, B and D). As the dose of LPS was further increased (103–104 µg/kg), the second and third phases merged together and became indistinguishable (Fig. 5, G and H). Similar to LPS fever in the rat (26, 27, 30, 35), both the latency and amplitude (R6,38 = 1.3 x 101; P = 5.9 x 10–8) of the first phase of polyphasic fever in the mouse decreased as the LPS dose increased (Fig. 7).
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34°C to a near-Ta value (R16,52 = 3.3 x 100, P = 6.0 x 10–4; Fig. 8B), thus indicating tail skin vasoconstriction.
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0.5°C) decrease in Tb over the course of the experiment (Fig. 9A). Compared with saline, every dose of LPS tested induced significant changes in Tb (100 µg/kg: F1,468 = 2.2 x 102, P = 1.0 x 10–7; 101.75 µg/kg: F1,507 = 2.1 x 102, P = 1.0 x 10–7; 104 µg/kg, fever: F1,299 = 1.2 x 102, P = 1.0 x 10–7; 104 µg/kg, hypothermia: F1,221 = 9.2 x 102, P = 1.0 x 10–7). These changes differed from those observed at a neutral Ta in two ways. First, the animals appeared more sensitive to LPS in a cool environment. Indeed, the first febrile phase caused by low doses (100 and 101.75 µg/kg) had a substantially shorter (by 20 min) latency at a subneutral Ta (Fig. 9, B and C) than at a neutral Ta (Fig. 5, B and E). Furthermore, the response to 100 µg/kg was transformed from a monophasic to biphasic fever (compare Figs. 5B and 9B). Second, the febrile response in a cool environment was followed by a late decrease in Tb. At higher doses (>102 µg/kg), this decrease led to hypothermia, which started at 180 min postinjection and was pronounced (3°C at 104 µg/kg; Fig. 9D). At lower doses (e.g., 101.75 µg/kg), this decrease resulted in the disappearance of the otherwise prominent third febrile phase (compare Figs. 5E and 9C).
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30 min) hypothermia, although such hypothermia reportedly (4, 7, 21, 23) occurs in the same species in response to a stressful injection of moderate-to-high doses of LPS. The mice in the present study failed to develop early hypothermia even when high doses of LPS (103–104 µg/kg) were injected at Tas as low as 20, 18, or even 16°C (data not shown). It was, therefore, important to test whether a stressful intraperitoneal injection of LPS would cause early hypothermia in the setup used. At a subneutral Ta of 26°C (telemetry setup), the mice were injected with intraperitoneal LPS (103 or 104 µg/kg) or saline via a common procedure involving animal handling and needle pricking. When injected this way, saline caused an immediate small increase in Tb (stress hyperthermia) followed by a slow decline in Tb. In response to LPS, the mice developed a similar small increase in Tb followed by hypothermia; compared with saline, hypothermia was significant only in response to the highest dose of LPS (F1,96 = 8.7 x 100, P = 4.1 x 10–3; Fig. 10). In contrast to the late hypothermic response to stress-free intravenous administration of LPS (Fig. 9), the response to stressful intraperitoneal administration was early (latency, 30 min).
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DISCUSSION |
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We have found that mice exposed to a neutral Ta respond to LPS with fever in a dose-dependent fashion: as the dose is increased from 100 to 102 µg/kg, the fever is transformed from monophasic to biphasic to polyphasic. The polyphasic character of the febrile response of mice to LPS has been recognized by Oka et al. (21). By assessing thermoeffector activity (vasomotor tone of the tail skin) and using several modes of data presentation, including the phase-plane plot, the present study has identified and characterized three phases of the polyphasic response. The present study has also established that many characteristics of LPS fever in mice are similar to those reported for rats. For example, the second and third phases of the febrile response in mice (Fig. 5) and rats (27) become less distinct and fuse when the dose of LPS approaches 103–104 µg/kg. Another similarity is that both the amplitude and the latency of the first phase of polyphasic fever in mice (Fig. 7) and rats (26, 35) are inversely proportional to the LPS dose. Whereas it is not surprising that the latency of the response decreases as the dose of LPS increases, the fact that the amplitude of the first phase in both species decreases with an increase in the dose is counterintuitive and remains unexplained.
Thermoregulatory responses of mice to LPS in a cool environment.
The present study has found that exposure of mice to a cool environment changes the thermoregulatory responses to LPS in two ways. First, it has a sensitizing effect on the febrile response. Indeed, responses to low (100 µg/kg) and intermediate (101.75 µg/kg) doses of LPS start earlier in a cold environment. Furthermore, what is a monophasic fever in a neutral environment turns into a biphasic fever in the cold. Such a paradoxical, sensitizing effect has not been described before. Second, the febrile response of mice in a cool environment is followed by a late (starts at 180 min postinjection) decrease in Tb. At lower doses of LPS (e.g., 101.75 µg/kg), this decrease is seen as the disappearance of the otherwise prominent third febrile phase. At higher doses (>102 µg/kg), this decrease results in a pronounced (3°C) hypothermic response. In other words, Tb-decreasing mechanisms are turned on at later stages of the response to LPS in a cool environment. These mechanisms seem to compete with Tb-increasing mechanisms, and such a competition may result in the disappearance of late febrile phases and/or in the development of hypothermia. A late (latency > 120 min) hypothermic response to LPS in mice has been previously reported (1, 12, 21, 22). Such a response may last for up to 48 h and seems to be associated with ongoing inflammation, as evident from the sensitivity of late hypothermia to both dexamethasone and inhibitors of eicosanoid biosynthesis (22).
The most surprising observation of the present study is that mice do not respond to LPS with an early hypothermia, whereas, in rats, the early (10–30 min, latency; 90 min, nadir) hypothermia is a highly reproducible and pronounced (up to several degrees centigrade) response to moderate or high doses of intravenous LPS in a subthermoneutral environment (27, 28, 30, 32, 33). The present study shows that C57BL/6 mice do not develop early hypothermia even in response to very high doses of LPS (up to 104 µg/kg) at Tas as low as 16°C. Furthermore, another strain of mice (wild-type mice with a mixed B6;129P2 background; Taconic, Germantown, NY) has also been found to be incapable of responding with early hypothermia to LPS (103–104 µg/kg iv) at a subneutral Ta of 26°C (A. A. Steiner, A. Y. Rudaya, and A. A. Romanovsky, unpublished observations). However, the early hypothermic response did occur in C57BL/6 mice in the present study when LPS was administered by using a common, stressful procedure of intraperitoneal injection. Such a response has also been observed by others in different mouse strains when LPS was administered by a stressful procedure involving either pricking the abdomen (21, 23) or tail (4, 7) with an injection needle for intraperitoneal or intravenous administration, respectively. That early hypothermia occurs in the mouse exclusively under stressful conditions suggests that this is not an intrinsic response to LPS in this species. This finding may be another example of how stress interferes with thermoregulatory responses (10, 27, 34).
In summary, the present investigation identifies experimental conditions to study LPS-induced mono-, bi-, and polyphasic fevers and late hypothermia in mice and provides detailed characteristics of these responses. It also highlights the importance of methodological issues in thermoregulation research. While studying the febrile and hypothermic responses of mice to LPS, caution should be taken to administer LPS in a stress-free fashion, to account for the intricate dependence of the response on LPS dose and to run experiments at a controlled Ta.
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ACKNOWLEDGMENTS |
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Present address of A. Y. Rudaya: Institute of Physiology, National Academy of Sciences, Minsk 220072, Belarus.
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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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  A. A. Steiner, D. L. Oliveira, J. L. Roberts, S. R. Petersen, and A. A. Romanovsky Nicotine administration and withdrawal affect survival in systemic inflammation models J Appl Physiol, October 1, 2008; 105(4): 1028 - 1034. [Abstract] [Full Text] [PDF]
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 A. A. Steiner, V. F. Turek, M. C. Almeida, J. J. Burmeister, D. L. Oliveira, J. L. Roberts, A. W. Bannon, M. H. Norman, J.-C. Louis, J. J. S. Treanor, et al. Nonthermal Activation of Transient Receptor Potential Vanilloid-1 Channels in Abdominal Viscera Tonically Inhibits Autonomic Cold-Defense Effectors J. Neurosci., July 11, 2007; 27(28): 7459 - 7468. [Abstract] [Full Text] [PDF]
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A. A. Romanovsky Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R37 - R46. [Abstract] [Full Text] [PDF]
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Z. Szelenyi Neuronal CCK and thermoregulation: two receptors with different functions Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R109 - R111. [Full Text] [PDF]
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W. Kozak, S. Wrotek, and A. Kozak Pyrogenicity of CpG-DNA in mice: role of interleukin-6, cyclooxygenases, and nuclear factor-{kappa}B Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R871 - R880. [Abstract] [Full Text] [PDF]
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T. Hubschle, J. Mutze, P. F. Muhlradt, S. Korte, R. Gerstberger, and J. Roth Pyrexia, anorexia, adipsia, and depressed motor activity in rats during systemic inflammation induced by the Toll-like receptors-2 and -6 agonists MALP-2 and FSL-1 Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R180 - R187. [Abstract] [Full Text] [PDF]
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A. A. Steiner, A. Y. Rudaya, J. R. Robbins, A. S. Dragic, R. Langenbach, and A. A. Romanovsky Expanding the febrigenic role of cyclooxygenase-2 to the previously overlooked responses Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1253 - R1257. [Abstract] [Full Text] [PDF]
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