INTRODUCTION

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INCREASING EVIDENCE from the disciplines of neuroscience and immunology indicate that cytokines can engage the nervous system. For example, intravenous and intraportal administration of interleukin-1 (IL-1) in anesthetized rats increases afferent vagal nerve activity (3, 22), intravenous and intracerebroventricular administration of IL-1 alters efferent sympathetic nerve discharge (SND) (15, 16, 23, 28, 32), and plantar injection of IL-1 induces transient spontaneous discharges in primary afferent fiber filaments (12). Important relative to the current study, IL-1 sensitizes somatic sensory nerve endings to mechanical and thermal stimulation (12) and sensitizes visceral afferents to ischemia and histamine (11).

Changing the level of activity in peripheral sympathetic nerves in response to external and internal stimuli is an important way that mammals maintain physiological homeostasis. Importantly, SND responses to a given stimulus can be substantially modulated when a second stimulus is combined with the first. For example, muscle SND responses evoked by rhythmic exercise are potentiated by isocapnic hypoxia (29), hypertonic but not normal saline infusion produces renal sympathoexcitation after hemorrhage in anesthetized rabbits (30), and elevated cerebrospinal fluid osmolality produces vasoconstriction of the skin in rabbits exposed to heat (34).

Does IL-1 modulate SND responses to acute physical stress? Because IL-1 enhances the responsiveness of primary afferents to various experimental paradigms (including mechanical and thermal stimuli) (11, 12) and because sympathetic neural circuits are affected by alterations in the physiological status of the animal (17), we reasoned that IL-1 might play a neuromodulatory role in sympathetic regulation to acute environmental stress. The aim of this study was to test the hypothesis that IL-1 alters interscapular brown adipose tissue (IBAT) and renal SND responses to mild hypothermia in chloralose-anesthetized rats. IBAT nerve recordings were completed because activation of this nerve enhances heat production through nonshivering thermogenesis (8-10, 14, 19). Renal nerve recordings were completed because hypothermia reduces the level of renal SND (17) and because the sympathetic innervation to the kidney influences renal blood flow, renin release, and salt and water retention by the renal tubules (2), responses that are part of the integrative physiological changes associated with hypothermia and sickness behavior.

	METHODS

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General procedures. The surgical procedures and experimental protocols used in the current study were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (345 ± 2 g) were initially anesthetized with methohexital sodium (Brevital, 50-60 mg/kg ip) (17, 18, 28). Catheters were placed in the femoral vein for administration of -chloralose (initial dose of 50 mg/kg and maintenance doses of 35-45 mg · kg¹ · h¹) (17, 18, 28), maintenance doses of methohexital sodium (10-20 mg/kg during surgical interventions) (17, 18, 28), and IL-1 or saline. The rats were intubated, paralyzed with gallamine triethiodide (initial dose of 5-10 mg/kg iv and maintenance doses of 10-15 mg · kg¹ · h¹) (17, 18), and artificially ventilated. Femoral arterial pressure and heart rate (HR) were recorded using standard procedures. Colonic temperature (T_c) was measured with a thermistor probe inserted ~5 cm into the colon and was kept at 38°C during surgical interventions by a heating plate located beneath the animal and by a heat lamp.

Neural recordings. Activity was recorded biphasically with a platinum bipolar electrode after capacity-coupled preamplification (band pass, 30-3,000 Hz) from the central end of cut renal and IBAT sympathetic nerves. The renal nerve was isolated retroperitoneally (17, 18, 28), and the IBAT nerve was isolated after visualization of the IBAT after a nape incision (13). The nerve-electrode preparations were covered with a silicone gel. The sympathetic nerve potentials were full-wave rectified and integrated (time constant, 10 ms), which produced a smooth tracing of the synchronized discharges (28). The level of activity in sympathetic nerves was quantified after integration as volts × seconds and corrected for background noise after ganglionic blockade (15 mg/kg trimethaphan camsylate) or nerve crush (17, 18, 28).

Experimental protocols. Mean arterial pressure (MAP), HR, and SND (IBAT and renal) were continuously recorded during four experimental protocols, three of which included the intravenous administration of IL-1 (290 ng/kg). IL-1 was dissolved in phosphate-buffered saline, and rats that received this cytokine were administered a single dose. Protocol I determined the combined effect of IL-1 and acute cold stress that produced mild hypothermia on renal and IBAT SND (n = 8). IBAT and renal SND were recorded before (control) and for 60 min after IL-1 administration (T_c maintained at 38°C during these periods). Sixty minutes after IL-1, T_c was decreased from 38 to 36.1 ± 0.3°C (time to reduce T_c, 11 ± 2 min) by turning off the heat sources and placing packaged ice on the metal heating plate in close proximity to the animal. As the target T_c was neared, the ice was removed from the table, allowing T_c to reach a steady state so that SND measurements could be completed. IL-1 was administered 60 min before initiation of acute cold stress because in chloralose-anesthetized rats IL-1 produces progressive (peak increases 45-60 min after IL-1) and significant increases in splenic and lumbar SND but does not change the level of renal and IBAT SND (28). Therefore, although it was expected that IL-1 alone would not change the level of IBAT and renal SND, we waited an extended period of time after IL-1 administration before inducing hypothermia to allow time for this cytokine to interact with central neural circuits. Protocol II determined the effect of mild hypothermia (no prior IL-1 administration) on renal and IBAT SND (n = 17). A 60-min control period was completed (300 µl iv saline, administered at the beginning of this period) before T_c was decreased from 38 to 36.0 ± 0.1°C (time to reduce T_c, 11 ± 1 min) using the same cooling protocol as described above. After the level of SND at 36°C was measured, the cold stimulus was maintained in five experiments until T_c reached 31°C. Protocol III determined the effect of IL-1 administration without subsequent hypothermia (T_c maintained at 38°C) on IBAT and renal SND (n = 5). SND recordings were maintained for 75 min after IL-1 to control for the time required to complete protocol I. Protocol IV also examined the combined effect of IL-1 and mild hypothermia on IBAT and renal SND; however, the sequence of interventions was different than that in protocol I. IL-1 (n = 8) or saline (n = 8) was administered 5 min before T_c was decreased from 38 to 35.5°C (time to reduce T_c, 10-15 min; same cooling protocol as described above). T_c was maintained at this level for 120 min (sustained hypothermia). At this time, T_c was reduced an additional 0.5°C from 35.5 to 35°C. A brief summary of the experimental protocols is shown in Table 1.

Data and statistical analysis. Values are means ± SE. Control values of SND were taken as 100%. Results were analyzed using analysis of variance techniques with a repeated measures design followed by Bonferroni post hoc tests. P < 0.05 indicated statistical significance.

	RESULTS

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Protocol I: effect of IL-1 and acute hypothermia on IBAT and renal SND. Figure 1A shows traces from a representative experiment of simultaneously recorded renal and IBAT SND bursts during control (T_c = 38°C; left), 60 min after IL-1 administration (T_c = 38°C; middle), and after application of acute cold stress, which was initiated 60 min after IL-1 and produced mild hypothermia (T_c = 36.5°C; right). In contrast with the renal nerve, there was little spontaneous IBAT nerve activity during control. Renal and IBAT SND were unchanged from control 60 min after IL-1. However, IBAT SND was markedly increased, whereas renal SND remained unchanged, when T_c was reduced to 36.5°C. Mean data for the group (n = 8) are presented in Fig. 1B. IBAT SND remained unchanged 60 min after IL-1 (T_c = 38°C) but was significantly increased 460 ± 144% from control after acute cold stress, which was initiated 60 min after IL-1 and produced mild hypothermia (T_c from 38 to 36.1 ± 0.3°C in 11 ± 2 min). Renal SND was not significantly changed after IL-1 (8 ± 9%) or during hypothermia (25 ± 10%). MAP (control, 110 ± 4 mmHg; IL-1, 100 ± 7 mmHg; hypothermia, 98 ± 7 mmHg) and HR (control, 345 ± 15 beats/min; IL-1, 336 ± 8 beats/min; hypothermia, 318 ± 12 beats/min) were not significantly changed after IL-1 or hypothermia.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: traces of integrated renal sympathetic nerve discharge (SND) and interscapular brown adipose tissue (IBAT) bursts recorded before (control; left), 60 min after interleukin-1 (IL-1) administration (right), and after acute cold stress (right), which was initiated 60 min after IL-1 and produced mild hypothermia [colonic temperature (Tc) = 36.5°C]. B: average IBAT and renal SND responses 60 min after IL-1 administration and after acute cold stress, which produced mild hypothermia (Tc = 36.1 ± 0.3°C). SND is expressed as a percentage of control activity. *Significantly different from control.

Protocol II: effect of acute hypothermia on IBAT and renal SND. The effect of acute cold stress, which produced mild hypothermia (T_c from 38°C to 36 ± 0.1°C in 11 ± 1 min) without prior administration of IL-1 on the level of IBAT and renal SND, was determined in 17 experiments. IBAT (+36 ± 29%) and renal (5 ± 5%) SND did not change from control during hypothermia. MAP (control, 118 ± 4 mmHg; hypothermia, 108 ± 4 mmHg) and HR (control, 370 ± 8 beats/min; hypothermia, 350 ± 9 beats/min) were significantly reduced during hypothermia. IBAT SND was increased 341 ± 47% (P < 0.05) and renal SND was decreased 27 ± 8% (P < 0.05) in five experiments in which T_c was reduced to 31°C.

Protocol III: effect of IL-1 on IBAT and renal SND. The effect of IL-1 without subsequent hypothermia on IBAT and renal SND was determined in five experiments. IBAT SND (14 ± 10%) was unchanged, whereas renal SND was reduced (22 ± 9%, P < 0.05), from control 75 min after IL-1. MAP (control, 108 ± 5 mmHg; IL-1, 110 ± 6 mmHg) and HR (control, 395 ± 12 beats/min, IL-1, 398 ± 13 beats/min) were unchanged from control levels after IL-1.

Protocol IV: effect of IL-1 and acute and sustained hypothermia on IBAT and renal SND. Figure 2 summarizes IBAT (A) and renal (B) SND responses to IL-1 (n = 8) and saline (n = 8) administration at the following experimental points: acute hypothermia (T_c = 35.5°C) produced by cold stress, which was initiated 5 min after IL-1 and saline administration; at 60 and 120 min of sustained mild hypothermia (T_c = 35.5°C); and after a second period of cold stress, which reduced T_c from 35.5 to 35°C. Reducing T_c from 38 to 35.5°C immediately after IL-1 or saline administration did not affect IBAT SND. Increases in IBAT SND at 60 and 120 min of sustained hypothermia were significantly higher in IL-1-treated rats than in saline-treated rats. Reducing T_c from 35.5 to 35°C after sustained hypothermia increased (P < 0.05) IBAT SND from levels recorded at 35.5°C in both IL-1- and saline-treated rats; however, increases in IBAT SND were significantly higher in rats that received IL-1. Renal SND was significantly reduced from control during each experimental intervention in rats pretreated with IL-1 and saline; however, responses did not differ among groups. MAP (IL-1: control, 136 ± 4 mmHg; sustained hypothermia, 113 ± 7 mmHg; saline: control, 124 ± 10 mmHg; sustained hypothermia, 98 ± 7 mmHg) and HR (IL-1: control, 385 ± 12 beats/min; sustained hypothermia, 312 ± 16 beats/min; saline: control, 368 ± 13 beats/min; sustained hypothermia, 285 ± 7 beats/min) were significantly reduced from control during sustained hypothermia (at 60 and 120 min, only 60-min data included) in IL-1- and saline-treated rats.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Average IBAT (A) and renal (B) SND responses to IL-1 and saline administration at the following experimental points: after an initial bout of cold stress, which produced acute hypothermia (Tc = 35.5°C); at 60 and 120 min of sustained hypothermia, with Tc maintained at 35.5°C; and after a second bout of cold stress, which reduced Tc from 35.5 to 35.0°C. SND is expressed as a percentage of control activity. *Significantly different from levels of IBAT SND recorded at 35.5°C (120 min). Significantly different from levels of IBAT SND recorded in saline-treated rats.

	DISCUSSION

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This study examined the effects of IL-1 and mild hypothermia on renal and IBAT SND regulation in -chloralose-anesthetized rats. The following observations were made: First, induction of acute mild hypothermia (T_c = 36°C) 60 min after IL-1 produced marked IBAT sympathoexcitation, whereas application of either stimulus alone did not alter the level of IBAT SND. Renal SND responses to the combination of IL-1 and mild hypothermia, IL-1 alone, and mild hypothermia alone did not differ. Second, increases in IBAT SND were significantly higher during sustained hypothermia (T_c = 35.5°C for 120 min) and in response to an additional reduction in T_c (from 35.5 to 35°C) after sustained hypothermia in IL-1-treated rats compared with saline-treated rats. In contrast, renal SND responses to IL-1 and sustained hypothermia were similar in rats treated with IL-1 and saline. These results demonstrate that IL-1 alters IBAT (but not renal) SND responses to acute and sustained hypothermia.

IL-1 plays a key role in mediating many of the diverse physiological responses of the acute phase reaction, including fever, aphagia, activation of the hypothalamic-pituitary-adrenal axis, and activation of the sympathetic nervous system (1). Changing the level of activity in peripheral sympathetic nerves in response to various stimuli is an important way that physiological homeostasis is maintained. Importantly, the function of central sympathetic neurons responsible for efferent SND depends on the continuous modulation of neuronal excitability in response to different physiological states. Because immune system products influence numerous physiological responses that require the integrative action of the central nervous system (1, 4, 27) and because the sympathetic nervous system can be substantially modulated by changes in the physiological status of the animal, we hypothesized that IL-1 might play a role as a neuromodulator in sympathetic nerve regulation. The current results support this hypothesis and demonstrate target organ selectivity in the neuromodulatory effect of IL-1 on efferent SND.

Because the neuromodulatory effect of IL-1 on IBAT SND was demonstrated using direct recordings of IBAT SND, the current results do not address any potential interaction among IL-1, hypothermia, and the sympathetic nervous system at the level of the tissue or neuroeffector junction. However, because peripheral SND recordings provide a window into the functional status of central sympathetic neural circuits, we speculate that the observed neuromodulatory effect is mediated by interactions among IL-1, hypothermia, and sympathetic premotor neurons. It must be noted, however, that modulation at the level of the sympathetic ganglion cannot be discounted because postganglionic IBAT SND recordings were completed. Although sympathetic neural circuits are contained in spinal, brain stem, and forebrain sites (31, 33), recent studies by Morrison (20, 21) demonstrate that the rostral raphe pallidus is involved in regulation of IBAT SND in anesthetized rats. The role of this brain stem nucleus in mediating the neuromodulatory role of IL-1 on IBAT SND remains to be determined. In addition, how this nucleus or others contained in sympathetic neural networks interact with central sites involved in mediating the effects of cytokines on the hypothalamic-pituitary-adrenal axis (5, 6, 35) is not known.

IL-1 was administered in the current study because this cytokine alters the level of efferent SND (16, 28, 32), increases splenic blood flow (26), and has been used to elucidate the central neural pathways subserving cytokine-induced effects on neuroendocrine neurons (5-7). The dose of IL-1 used in the present study was similar to that used in previous studies (5, 6, 16, 26, 28, 32) that have documented physiological effects of intravenous IL-1. On the basis of the body weight and the estimated blood volume (25) of the rats used, we estimate that the dose of IL-1 in the present study produced peak plasma concentrations of 4,000-5,000 pg/ml, similar to those produced after intraperitoneal administration of lipopolysaccharide (36), a widely accepted model for systemic bacterial infection. However, circulating levels of IL-1 remain elevated for up to 24 h after systemic lipopolysaccharide administration (36), whereas the half-life for elimination of IL-1 after intravenous injection has been reported to be <1 h (24). We suggest that the acute intravenous IL-1 injection paradigm is relevant in some aspects, but is not identical to an established animal model of systemic bacterial infection. Nonetheless, the current results demonstrate that, in addition to altering the level of sympathetic nerve activity, an additional important mechanism by which IL-1 influences sympathetic nerve regulation is to modulate SND responses to acute stress.