INTRODUCTION

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INTERLEUKIN-1 (IL-1) is a proinflammatory cytokine that initiates a diverse array of immune and physiological responses, including responses mediated by central neural circuits, such as fever, activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, and behavioral depression (2, 5, 6). IL-1 induction in the central nervous system (CNS) occurs after peripheral (3, 17, 25, 27, 34) and central (32) immune challenge. Peripheral cytokines gain access to cells in the CNS through circumventricular organs that are devoid of or possess a leaky blood-brain barrier (33) and by crossing the blood-brain barrier with the use of active transport mechanisms (1). Taken together, it is well established that CNS IL-1 increases in response to immune stimuli, thereby initiating a variety of host defense responses.

Although baseline CNS production of IL-1 is low, IL-1 (20) and IL-1 mRNA (9, 35) are present in the rat CNS under nonactivated (no peripheral or central immune challenge) conditions. Does endogenous CNS IL-1 play a role in physiological regulation under basal conditions? The results of several studies suggest that this might be the case. Microinjection of IL-1 antibody into the hypothalamus (paraventricular and arcuate nuclei) attenuates the hypertensive response induced by microinjection of glutamate into the central amygdaloid nucleus (21). Intracerebroventricular infusion of IL-1 antibody increases norepinephrine secretion from the posterior hypothalamus and decreases nitric oxide (NO) synthase (NOS) mRNA in this nucleus (35), suggesting a role for endogenous IL-1 in regulation of CNS neurotransmitters/neuromodulators. Important relative to the current study, intracerebroventricular infusion of IL-1 antibody increases mean arterial pressure (MAP) more than 20 mmHg in anesthetized Sprague-Dawley rats (35), suggesting that endogenous IL-1 may tonically influence cardiovascular regulation. Does endogenous CNS IL-1 influence regulation of efferent sympathetic nerve outflow? One potential mechanism mediating the substantial increase in MAP to central infusion of IL-1 antibody may be that under basal conditions endogenous central neural IL-1 tonically inhibits sympathetic nerve outflow, suggesting a role for central IL-1 in sympathetic nerve discharge (SND) regulation.

In the present study, we tested the hypothesis that lateral ventricular infusion of IL-1 antibody increases the level of SND in chloralose-anesthetized rats. SND (renal and splenic), MAP, and heart rate (HR) responses to IL-1 antibody were determined in baroreceptor-intact (intact) and sinoaortic-denervated (SAD) rats.

	METHODS

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General procedures. The surgical procedures and experimental protocols were approved by the Institutional Animal Care and Use Committee. Experiments were performed on male Sprague-Dawley rats (300-350 g). Anesthesia was initially induced with an intraperitoneal injection of methohexital sodium (Brevital; 50-60 mg/kg). Catheters placed in the femoral vein were used for the administration of Brevital (10-20 mg/kg during surgical procedures) and -chloralose (initial dose, 50-60 mg/kg; maintenance dose, 35-45 mg · kg¹ · h¹) (14, 15, 29). The trachea was cannulated with a polyethylene-240 catheter. Femoral arterial pressure was monitored with the use of a pressure transducer that was connected to a blood pressure analyzer (model BPA, Digi-Med; Louisville, KY). HR was derived from the pulsatile arterial pressure output of the blood pressure analyzer. Colonic temperature was maintained between 37.7°C and 38.0°C during surgical and experimental procedures with the use of a temperature-controlled table.

Lateral ventricular cannulation. Lateral ventricular cannulas used for the intracerebroventricular administration of IL-1 antibody, saline, and IgG were surgically implanted after completion of the general surgical procedures. Anesthetized rats were placed in a stereotaxic frame, the head was leveled between the lambda and bregma, and a small hole was made in the skull (1.2-1.4 mm lateral to the midline and 0.8-1.0 mm posterior to the bregma). A 10-mm stainless steel guide cannula (22 gauge) was lowered 4 mm below the surface of the skull and fixed in place with the use of cranioplastic cement. A stainless steel injector was introduced through the guide cannula to protrude 0.5 mm beyond the tip of the guide cannula.

Sinoaortic denervation. Bilateral denervation of the aortic arch was completed in anesthetized rats by cutting the superior laryngeal nerve near its junction with the vagus nerve and by removing the superior cervical ganglion (19). Bilateral carotid sinus denervation was completed by removal of the adventitia from the carotid sinus bifurcation and by application of 10% phenol to this area (19). Sinoaortic denervation was considered complete by the loss of coherence between the arterial pulse and SND (11, 16). Sinoaortic denervation procedures were completed before lateral ventricle cannulation (n = 11).

Neural recordings. After completion of the lateral ventricular cannulation and sinoaortic denervation (selected experiments) procedures, the anesthetized rats were prepared for SND recordings. Activity was recorded biphasically with a platinum bipolar electrode after preamplification (bandpass 30-3,000 Hz, model p511, Grass Instruments; W. Warwick, RI) from renal and splenic sympathetic nerves. The left renal and splenic nerves were isolated from a lateral approach, and nerve-electrode preparations were covered with silicone gel. For monitoring during the experiment and for subsequent data analysis, the filtered neurogram was routed to an oscilloscope (model 54602B, Hewlett-Packard; Palo Alto, CA) and a nerve traffic analyzer (model 662C-3, University of Iowa Bioengineering; Iowa City, IA). Sympathetic nerve potentials were full-wave rectified, integrated (time constant 10 ms) and quantified as volts × seconds (14, 15, 29). The level of activity in sympathetic nerves was corrected for background noise after administration of the ganglionic blocker trimethaphan camsylate (10-15 mg/kg iv) (14, 15, 29).

Central and systemic administration of IL-1 antibody, saline, and IgG. After completion of the nerve-electrode preparations, the anesthetized rats were allowed to stabilize for up to 60 min before initiation of the experimental protocols. For intracerebroventricular infusions the injector was connected via polyethylene tubing to a 100-µl microsyringe driven by a micropump (2 µl/min, 25 min). Goat anti-rat IL-1 antibody (15 µg dissolved in 50 µl of PBS solution, R&D Systems; Minneapolis, MN), saline (50 µl), or IgG (15 µg dissolved in 50 µl of PBS solution, goat IgG) was infused into the lateral ventricle for a period of 25 min. MAP, SND (renal and splenic), and HR were recorded continuously before (preinfusion period, noted as 10 min in Figs. 1 and 2), during (25 min), and for 45 min after intracerebroventricular infusion. Single-dose administrations of IL-1 antibody (15 µg dissolved in 5 µl of PBS solution administered over 2 min, followed by a 1 µl saline flush) and saline (5 µl administered over 2 min, followed by a 1 µl saline flush) were completed by connection of the injector via polyethylene tubing to a 10-µl microsyringe. MAP, SND, and HR were recorded continuously before (preinjection period, noted as 10 min in Fig. 3) and for 53 min after single-dose intracerebroventricular injections. In two experiments MAP and SND were recorded before and for 70 min after intravenous (femoral vein) IL-1 antibody administration (15 µg, 5 µl, for 2 min).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Mean arterial pressure (MAP) recorded before (preinfusion, 10 min), during (0-25 min, indicated by horizontal bar at bottom), and for 45 min after intracerebroventricular infusion of interleukin-1 (IL-1) antibody (15 µg, 50 µl), saline (50 µl), or IgG (15 µg, 50 µl) in baroreceptor-intact (intact) and sinoaortic-denervated (SAD) rats. * Significantly different from preinfusion, 10 min; intact IL-1 antibody significantly different from saline and IgG; SAD IL-1 antibody significantly different from intact IL-1 antibody.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Renal (A) and splenic (B) sympathetic nerve discharge (SND) recorded before (preinfusion, 10 min), during (0-25 min, indicated by horizontal bars at bottom) and for 45 min after infusion of IL-1 antibody (15 µg, 50 µl icv), saline (50 µl), or IgG (15 µg, 50 µl) in intact and SAD rats. * Significantly different from preinfusion, 10 min; intact IL-1 antibody significantly different from saline and IgG; SAD IL-1 antibody significantly different from intact IL-1 antibody.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Renal (A) and splenic (B) SND recorded before (preinjection, 10 min), during (0-2 min, indicated by horizontal bars at bottom), and for 53 min after administration of a single dose of IL-1 antibody (15 µg, 5 µl icv) or saline (5 µl) in intact rats. * Significantly different from preinjection, 10 min; significantly different from saline.

Brain histology. At the end of each experiment, fluorescent latex microspheres (50 nm diameter, Lumafluor; Naples, FL) were injected into the lateral ventricle. The rats received an overdose of methohexital sodium (150 mg/kg iv) and were transcardially perfused with 0.15 M NaCl (containing 3 IU/ml heparin), followed by a fixative solution consisting of 10% buffered neutral formalin (pH 7.4). The brains were removed, blocked, postfixed in buffered neutral formalin for at least 2 h, and placed in 20% sucrose for cryoprotection. The brains were frozen sectioned at 40 µm in the coronal plane, collected into PBS, and mounted on slides in serial sequence. The sections were rinsed in distilled water, air dried, and cleared in xylenes. Lateral ventricular injection sites were confirmed by observing fluorescent microspheres in the ventricular system with brightfield or epifluoresence.

Data and statistical analysis. Values are means ± SE. Control values of SND were taken as 0%. Statistical analysis was completed using repeated-measures analysis of variance with Bonferroni post hoc tests. The overall level of statistical significance was P < 0.05.

	RESULTS

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MAP, SND, and HR responses to intracerebroventricular infusion of IL-1 antibody, saline, and IgG. Experiments were completed in intact and SAD rats. Preinfusion (10 min) levels of MAP did not differ between groups (Fig. 1). MAP was progressively and significantly increased from preinfusion levels during IL-1 antibody infusion in intact (n = 9) and SAD (n = 6) rats and remained increased after cessation of infusion in SAD but not intact rats (Fig. 1). MAP was significantly higher in SAD compared with intact rats after cessation of IL-1 antibody infusion. MAP remained unchanged from preinfusion levels during and after intracerebroventricular infusion of saline (n = 6, intact; n = 5, SAD; n = 1, combined for presentation) and IgG (n = 5, intact; n = 1, SAD; n = 4, combined for presentation).

View this table: [in this window] [in a new window]   Table 1.   Heart rate recorded before, during, and after intracerebroventricular infusion of IL-1 antibody, saline, or IgG in intact and SAD hearts

MAP, SND, and HR responses to single-dose intracerebroventricular administration of IL-1 antibody and saline in intact rats. MAP remained unchanged from preinjection levels for >50 min after single-dose administration of IL-1 antibody and saline (Table 2). Renal (Fig. 3A) and splenic (Fig. 3B) SND were progressively and significantly increased from preinjection (10 min) levels after single-dose intracerebroventricular IL-1 antibody (renal, n = 4; splenic, n = 4) but not saline (renal, n = 5; splenic, n = 5) administration (Fig. 3). HR remained unchanged from preinjection levels after single-dose administration of IL-1 antibody and saline (Table 2).

View this table: [in this window] [in a new window]   Table 2.   MAP and HR recorded before (preinjection) and after (53-min postinjection) intracerebroventricular injection of a single dose of IL-1 antibody or saline in intact rats

	DISCUSSION

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This study determined renal and splenic SND responses to lateral ventricular administration of IL-1 antibody in chloralose-anesthetized rats. Our results provide experimental support for three findings that contribute to understanding the role of central neural IL-1 in cardiovascular and SND regulation. First, intracerebroventricular infusion of IL-1 antibody significantly increased MAP and the magnitude of the pressor response was higher in SAD compared with intact rats. In contrast, MAP remained unchanged during and after intracerebroventricular infusion of saline and an isotype control. Second, intracerebroventricular infusion of IL-1 antibody significantly increased renal and splenic SND and the sympathoexcitatory responses were significantly higher in SAD compared with intact rats. In contrast, SND remained unchanged during and after intracerebroventricular infusion of saline and an isotype control. Third, intracerebroventricular administration of a single dose of IL-1 antibody significantly increased splenic and renal SND. These results support a role for central neural IL-1 in the tonic regulation of SND and arterial blood pressure.

IL-1 provides a signaling pathway to sympathetic neural circuits. Intravenous administration of IL-1 produces nonuniform SND responses (26, 28, 29) and modulates interscapular brown adipose tissue sympathetic nerve responses to hypothermia (14). Central administration of IL-1 alters SND and cardiovascular regulation (12, 13, 35, 36); however, divergent results have been reported. For example, intracerebroventricular administration of IL-1 has been shown to increase renal and splenic SND with no effect on arterial blood pressure (12, 13), whereas Ye et al. (35) reported dose-dependent decreases in arterial blood pressure and norepinephrine release from the posterior hypothalamus after intracerebroventricular IL-1. Although peripheral administration of IL-1 provides an experimental advantage to that of a broadly acting cytokine stimulant like lipopolysaccharide, one limitation to its experimental use is difficulty in determining whether exogenous administration of IL-1 produces concentrations in central sympathetic neural circuits that are similar to those observed during pathophysiological states. To obviate this limitation, this study and other studies (21, 35) have used the exogenous administration of IL-1 antibody to study the role of IL-1 in SND and cardiovascular regulation. Ye et al. (35) reported significant increases in arterial blood pressure and norepinephrine secretion from the posterior hypothalamus after intracerebroventricular administration of IL-1 antibody, whereas Lu et al. (21) reported that hypothalamic microinjection of IL-1 antibody attenuates the hypertensive response induced by microinjection of glutamate into the central amygdaloid nucleus. The current results extend these findings by demonstrating that intracerebroventricular IL-1 antibody, administered either as an infusion or a single dose, significantly increased renal and splenic SND, providing evidence that under the conditions of the present experiments central neural IL-1 influences SND regulation. Importantly, intravenous administration of IL-1 antibody (peripheral control), intracerebroventricular infusion of saline (volume control), and intracerebroventricular infusion of IgG (isotype control) had no effect on SND or MAP, confirming the specific effect of IL-1 antibody on SND regulation.

MAP and SND responses to intracerebroventricular IL-1 antibody infusion were significantly higher in SAD compared with intact rats, demonstrating that sympathoexcitatory responses to IL-1 antibody infusion were opposed by activation of the arterial baroreceptors secondary to increases in MAP induced by intracerebroventricular IL-1 antibody. This finding provides additional support for the concept that endogenous central neural IL-1 impacts SND regulation.

The CNS contains the substrate for mediating IL-1-induced physiological responses. For example, neuronal and nonneuronal cells in the mouse and rat brain express mRNA for IL-1 receptors (4, 7), IL-1 receptors are located in the rat brain (8), and IL-1 mRNA is expressed (albeit in low levels) in the hypothalamus and brain stem under basal or nonactivated conditions (9, 35). Although the present results suggest that IL-1 exerts a tonic inhibitory effect on SND, the mechanisms mediating this response remain undefined. Because of the known interactions among IL-1, NO, and the sympathetic nervous system (24, 35), one possible mediator may be NO. Central administration of IL-1 increases neuronal NOS (nNOS) mRNA abundance (35), whereas central administration of anti-rat IL-1 antibody decreases nNOS mRNA expression in the posterior hypothalamus, paraventricular nucleus, and the locus ceruleus while increasing arterial blood pressure (35). The results of several studies (10, 31) suggest that nNOS is a component of brain stem transduction pathways that tonically inhibit sympathetic outflow. Moreover, NOS inhibition increases arterial blood pressure, an effect that is attenuated by sympathectomy (30) and renal denervation (22). It is tempting to speculate that the delayed onset of MAP and SND responses to intracerebroventricular IL-1 antibody infusion may be a function of the time required to reduce CNS NO expression secondary to decreased levels of IL-1.

There are at least three limitations to the present study. First, because the sympathetic nervous system is capable of generating heterogenous response profiles (23), the current results are applicable to renal and splenic SND only. Second, anesthesia may influence SND responses to intracerebroventricular IL-1 antibody infusion. Although this cannot be entirely discounted, an anesthetized preparation was used because we have previously documented the effect of IL-1 on SND regulation in anesthetized rats (14, 15, 29) and because we wanted to complete recordings in sympathetic nerve pairs, a technique that is difficult to complete in conscious rats. In addition, rats were anesthetized with -chloralose, an anesthesia that is widely used in studies concerned with autonomic and cardiovascular regulation. Third, intracerebroventricular injections provide limited information concerning specific central sites mediating SND responses to IL-1 antibody. Although this is the case, Konsman et al. (18) demonstrated that cerebrospinal fluid provides an important diffusion medium whereby cytokines act as volume transmission signals to the brain. With this in mind, we chose to use intracerebroventricular administration of IL-1 antibody as an initial experimental strategy to determine whether IL-1 antibody affects SND regulation. On the basis of the current findings, additional studies can be completed to determine specific central sites mediating the observed responses.