Divergent effects of brain interleukin-1ß in mediating fever, lethargy, anorexia and conditioned fear memory.

The influence of brain interleukin-1 (IL-1ß) on memory processes includes both detrimental and beneficial effects. To further explore the dynamics of brain IL-1ß in mediating learning and memory during acute sickness, we injected species-homologous rat IL-1ß (100ng/5µl) or vehicle (0.1% bovine serum albumin, 5µl) directly into the cisterna magna (i.c.m.) of male Sprague-Dawley rats. We measured, in parallel, body temperature, food intake, body mass, cage activity, as well as learning and memory using contextual fear conditioning. To investigate the effects of IL-1ß on learning and memory processes we used: (1) a retrograde experiment that involved injecting rats i.c.m. with IL-1ß immediately after training in the novel context, and (2) an anterograde experiment that involved injecting rats i.c.m. with IL-1ß two hours before training in the novel context. In addition, hypothalamic and hippocampal concentrations of IL-1ß were measured at several time points following injection. Administration of IL-1ß induced fever, lethargy and anorexia for∼two-to-three days and increased the concentration of IL-1ß in the hippocampus and hypothalamus for at least eight hours. Training in the context immediately before IL-1ß administration (retrograde experiment), did not impair contextual and auditory fear memory. However, when training in the context occurred concurrently with elevated hippocampal IL-1ß levels, two hours after IL-1ß administration (anterograde experiment), contextual, but not auditory, fear memory was impaired. Our results show that there are instances where memory consolidation can occur concurrently with elevated levels of IL-1ß in the hippocampus, fever, anorexia and lethargy during acute short-term sickness.

Simulated acute central Mycoplasma infections in rats induce fever, anorexia, body mass stunting and lethargy but spare memory.

Despite the documented post-infectious neurological complications of a central nervous system (CNS) Mycoplasma infection in humans, very few studies have investigated the acute inflammatory responses and sickness behaviours induced by CNS Mycoplasma infections. We therefore determined the effect of acute central administration of fibroblast-stimulating lipopeptide-1 (FSL-1), derived from Mycoplasma salivarium, and FAM-20 from a more pathogenic species, namely Mycoplasma pneumoniae, on behavioural and inflammatory responses in rats. Male Sprague-Dawley rats had radiotransmitters implanted, intra-abdominally, to measure body temperature and cage activity continuously. After recovery from surgery, rats were conditioned in a fear conditioning task and then immediately received an intra-cisterna magna (i.c.m.) injection of either: (1) FSL-1 (10 or 100µg/5µl) or its vehicle (phosphate-buffered saline, 5µl), or (2) FAM-20 (10 or 100µg/5µl) or its vehicle (dimethyl sulfoxide, 5µl). Body mass and food intake were measured daily. Memory was assessed seven days after injection using fear conditioning tests. A single, i.c.m. injection of either FSL-1 or FAM-20 induced profound, dose-dependent fever, anorexia, lethargy and body mass stunting in rats. Moreover, rats that received an i.c.m. injection of 100µg/5µl FAM-20 had a significant increase in the concentration of IL-1ß in both the hypothalamus and the hippocampus for ~27h after injection. Seven days after FSL-1 or FAM-20 injection, when body mass of rats still was stunted, they maintained their memory for fear of the context and for fear of the tone, despite the increase in hippocampal IL-1ß concentration after FAM-20 administration. Thus, acute simulated CNS Mycoplasma infections caused pronounced sickness responses and brain inflammation in rats, but spared fear memory.

Critical role for peripherally-derived interleukin-10 in mediating the thermoregulatory manifestations of fever and hypothermia in severe forms of lipopolysaccharide-induced inflammation.

Although peripherally released interleukin (IL)-10 has a critical regulatory role in limiting fever in mild-to-moderate forms of inflammation, its role in regulating the more complex thermoregulatory manifestations of hypothermia and fever noted during severe inflammation is less clear. Using cytokine antagonism, we therefore investigated the involvement of peripherally released IL-10 in mediating hypothermia, fever and inflammation induced by intraperitoneal (IP) administration of a large dose of lipopolysaccharide (LPS). Male Wistar rats (200-250 g) were anaesthetized and implanted intra-abdominally with temperature-sensitive radiotelemeters. Rats were randomly assigned to receive IL-10 antiserum (IL-10AS) or normal sheep serum IP, 4 h before receiving an IP injection of LPS (10 mg/kg) or phosphate-buffered saline (PBS). Inflammatory responses were measured in plasma and tissue samples (spleen, liver and brain) at 90 min and 6 h after the IP injection of LPS or PBS. Administration of LPS induced an initial period of hypothermia (~90 min) after which fever developed. Pre-treating rats with IL-10AS abolished the LPS-induced increase in plasma IL-10 levels, attenuated the hypothermia and increased the amplitude of the fever. Moreover, IL-10AS pre-treatment augmented the LPS-induced increase in plasma levels of tumor necrosis factor-alpha (90 min and 6 h), IL-1ß (90 min), prostaglandin E2 (90 min) and IL-6 (6 h), in the periphery, but not the hypothalamus, over the duration of hypothermia and fever. Via its action on the synthesis of inflammatory mediators in the spleen and liver, endogenous IL-10 plays a crucial regulatory role in mediating hypothermia and fever during severe aspectic (LPS-induced) systemic inflammation.

Simulated systemic recurrent Mycoplasma infection in rats induces recurrent sickness responses without residual impairment in spatial learning and memory.

In spite of their prevalence and importance, recurrent acute infections seldom have been investigated in the laboratory. We set out to measure fever and sickness behaviour in simulated recurrent Mycoplasma infection; Mycoplasma is a common clinical cause of recurrent acute infection. Male Sprague-Dawley rats had radiotransponders implanted to measure abdominal temperature and cage activity. After recovery, rats received three intraperitoneal (I.P.) injections, 10 days apart, of either fibroblast-stimulating lipopeptide-1 (FLS-1), a pyrogenic moiety of Mycoplasma salivarium, at a dose of 500 µg.kg(-1) in 1 ml.kg(-1) phosphate-buffered saline (PBS), or vehicle (PBS, 1 ml.kg(-1)). Body mass and food intake were measured daily. For measurement of learning and memory, training in a Morris Water Maze commenced 10 days after the last of the three successive injections and continued daily for 4 days. Spatial memory was assessed on the following day. Hippocampal tissue of rats was collected on the day of the last exposure to the maze. Recurrent FSL-1 administration induced recurrent fevers (~1°C) for about 9h, recurrent lethargy (~40-60%) for 1 day, recurrent anorexia (~16-30%) for 1 day, and recurrent reductions in the rate of mass gain (~112%) for 1 day, but did not induce persistent stunting. Recurrent FSL-1 administration did not result in tolerance to fever, lethargy or anorexia. There was no residual histological damage to the hippocampus and no residual detrimental effect in learning or memory in rats. Though we cannot extrapolate our results directly to humans, clinical recurrent acute Mycoplasma infection may not impose a high risk of stunting or impaired spatial learning and memory.

Dissociation between learning and memory impairment and other sickness behaviours during simulated Mycoplasma infection in rats.

To investigate potential consequences for learning and memory, we have simulated the effects of Mycoplasma infection, in rats, by administering fibroblast-stimulating lipopepide-1 (FSL-1), a pyrogenic moiety of Mycoplasma salivarium. We measured the effects on body temperature, cage activity, food intake, and on spatial learning and memory in a Morris Water Maze. Male Sprague-Dawley rats had radio transponders implanted to measure abdominal temperature and cage activity. After recovery, rats were assigned randomly to receive intraperitoneal (I.P.) injections of FSL-1 (500 or 1000 µg kg(-1) in 1 ml kg(-1) phosphate-buffered saline; PBS) or vehicle (PBS, 1 ml kg(-1)). Body mass and food intake were measured daily. Training in the Maze commenced 18 h after injections and continued daily for four days. Spatial memory was assessed on the fifth day. In other rats, we measured concentrations of brain pro-inflammatory cytokines, interleukin (IL)-1ß and IL-6, at 3 and 18 h after injections. FSL-1 administration induced a dose-dependent fever (∼1°C) for two days, lethargy (∼78%) for four days, anorexia (∼65%) for three days and body mass stunting (∼6%) for at least four days. Eighteen hours after FSL-1 administration, when concentrations of IL-1ß, but not that of IL-6, were elevated in both the hypothalamus and the hippocampus, and when rats were febrile, lethargic and anorexic, learning in the Maze was unaffected. There also was no memory impairment. Our results support emerging evidence that impaired learning and memory is not inevitable during simulated infection.

Differences in the relative involvement of peripherally released interleukin (IL)-6, brain IL-1ß and prostanoids in mediating lipopolysaccharide-induced fever and sickness behavior.

Although peripherally released interleukin (IL)-6 is critical for fever, its role in sickness behaviors, in particular anorexia and lethargy, induced by lipopolysaccharide (LPS) administration appears to be less important. Using quantifiable measures of fever, anorexia and lethargy, that is, body temperature, food intake and voluntary wheel-running, we investigated whether the less-than-essential role for IL-6 in mediating sickness behaviors compared to fever implies important roles for other inflammatory mediators, particularly IL-1ß and prostanoids, in these responses. Male Sprague-Dawley rats were randomly assigned to receive one of the following three injections before receiving a subcutaneous (SC) injection of LPS (250 µg/kg) or saline: (1) intraperitoneal injection of pre-immune serum or antiserum to IL-6 (IL-6AS), to reduce the biological activity of peripherally released IL-6; (2) intracerebroventricular injection of vehicle or a caspase-1 inhibitor, to inhibit the production of mature IL-1ß; or (3) intraperitoneal injection of vehicle or one of the two doses (1 or 10 mg/kg) of diclofenac, a nonselective cyclooxygenase inhibitor shown to block the formation of prostanoids. LPS administration induced fever, anorexia and lethargy with an accompanying increase in IL-6 and IL-1ß concentrations in the circulation and IL-1ß in the brain. Rats pre-treated with: (1) IL-6AS had reduced plasma levels of bioactive IL-6, no fever and attenuated sickness behaviors; (2) the caspase-1 inhibitor had reduced concentrations of IL-1ß in the pre-frontal cortex, hypothalamus and hippocampus, and attenuated fever and sickness behaviors; (3) diclofenac had a dose-dependent attenuation in fever and sickness behaviors. Doses of diclofenac which completely abolished fever however had lesser effects on anorexia and lethargy. Our results confirm a difference in the sensitivity of sickness responses to IL-6 antagonism and identify that it may be related to different levels of sensitivity or responsiveness in brain regions and/or mechanisms, to prostanoids, IL-1ß, or IL-6 itself.

Interleukin (IL)-6 and IL-1 beta act synergistically within the brain to induce sickness behavior and fever in rats.

Pro-inflammatory cytokines interleukin (IL)-6 and IL-1 beta can act in the brain (centrally) to cause fever. Sickness behaviors which accompany fever also appear to involve the central action of IL-1 beta. We injected species-homologous rat IL-6 and IL-1 beta directly into the brains of conscious rats to examine the effect of these cytokines on fever, and two behaviors affected by sickness, voluntary wheel-running and food intake. Male Sprague-Dawley rats selected for their predisposition to spontaneously run on running wheels were used in the experiment. Each rat was anaesthetized and had a temperature-sensitive radiotransmitter implanted intra-abdominally, and a 23-gauge stainless steel guide cannula inserted stereotaxically over the lateral cerebral ventricle. Rats were randomly assigned to receive intracerebroventricular injections of three doses of either IL-1 beta or IL-6 (100 ng, 1 ng or 0.1 ng IL-1 beta and 200 ng, 20 ng or 2 ng IL-6), or one of three different combinations of IL-1 beta and IL-6. Rats receiving either IL-1 beta or IL-6 showed a dose-dependent increase in body temperature and decrease in wheel-running (ANOVA, p<0.0001). Only rats receiving the highest dose of IL-1 beta significantly decreased food intake and body mass compared to rats receiving vehicle (ANOVA, p<0.001). Doses of IL-1 beta and IL-6 which, when injected on their own were non-pyrogenic and did not affect food intake and body mass, induced fever and anorexia when they were co-injected centrally. These results show that species-homologous rat IL-6 and IL-1 beta can act directly within the brain to decrease voluntary activity and suggest they also can act synergistically to induce anorexia and fever.

c-Fos immunoreactivity in selected brain regions of rats after heat exposure and pyrogen administration.

We determined c-Fos immunoreactivity (Fos-IR) in selected hypothalamic nuclei, the organum vasculosum of the laminae terminals (OVLT) and somatosensory cortex of rats after hyperthermia induced by exogenous heat exposure, Gram-negative or Gram-positive pyrogen administration. The magnitude of Fos-IR was similar in thermoregulatory hypothalamic nuclei of rats after heat exposure or lipopolysaccharide (LPS) injection, despite the different origins of the hyperthermias. Heat-induced hyperthermia was associated with increased Fos-IR in the somatosensory cortex. LPS, but not heat exposure or injection of killed Staphylococcus aureus cells activated OVLT neurons. The OVLT may thus not be a port of entry for humoral mediators of Gram-positive bacterial fevers.

Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior.

Pro-inflammatory cytokines, interleukin (IL)-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) synthesized by activated macrophages and monocytes in response to administration of lipopolysaccharide (LPS), are considered important mediators of fever and sickness behavior. We administered rat-specific antisera for TNF-alpha, IL-1beta, IL-6 and leptin, to determine the involvement of peripherally released cytokines in LPS-induced fever and sickness behavior, measured as suppression of voluntary wheel-running and food intake. Male Sprague-Dawley rats (approximately 200 g) selected for their predisposition to spontaneously run on running wheels were anaesthetized with a combination of ketamine hydrochloride (80 mg/kg i.m.) and xylazine (4 mg/kg i.m.) and implanted intra-abdominally with temperature-sensitive radiotelemeters. Rats were injected intraperitoneally with anti-rat sera to one of the following, TNF-alpha, IL-1beta, IL-6 or leptin or with pre-immune sheep serum, followed by a subcutaneous injection of either LPS (250 microg/kg) or sterile saline. Lipopolysaccharide administration induced a approximately 1.3 (0.2) degrees C fever lasting approximately 10 h and reduced voluntary running by 93 (8.6)% and food intake by 51 (21.3)% compared to the saline response (ANOVA, P<0.05). Injection of anti-IL-6 serum or anti-leptin serum abolished the LPS-induced fever, anti-TNF-alpha serum affected only the early phase of fever and anti-IL-1beta serum had no effect on fever (ANOVA, P<0.05). LPS-induced suppression of voluntary running and food intake were attenuated in rats receiving anti-IL-6 serum, while the decrease in food intake was totally abolished in rats receiving anti-leptin serum (ANOVA, P<0.05). Injection of anti-TNF-alpha or anti-IL-1beta serum had no effect on LPS-induced sickness behavior. Peripherally released IL-6 and leptin therefore appear to be important in regulating LPS-induced fever and sickness behavior.

Inhibitors of nitric oxide synthesis block cold-induced thermogenesis in rats.

N-nitro-arginine methyl ester (L-NAME), an unspecific nitric oxide synthase inhibitor, was administered to individually caged Sprague-Dawley rats exposed to cold (18 degrees C) and thermoneutral (30 degrees C) environmental temperatures during the active phase of the animals' circadian cycle. Unrestrained rats were administered intraperitoneal injections of 100 mg x kg-1 L-NAME or 1 mL x kg-1 saline. Telemetry was used to measure abdominal temperature. On a separate occasion, metabolic rate and evaporative water loss were measured using indirect calorimetery, before and after the injection of 100 mg x kg-1 L-NAME, in rats exposed to the two environments. Injection of L-NAME had no significant effect on body temperature, metabolic rate, or evaporative water loss in rats exposed to the 30 degrees C environment. In the 18 degrees C environment, L-NAME injection caused a prolonged fall in body temperature ( F(1,12) = 17.43, P = 0.001) and a significant decrease in metabolic rate (Student's t test, P = 0.001) and evaporative water loss (one-sample t test, P = 0.04). Therefore, the effects that systemic injection of L-NAME has on body temperature are dependent on environmental temperature, with nitric oxide synthase inhibition seemingly preventing the metabolic component of cold defence.

Adaptive heterothermy and selective brain cooling in arid-zone mammals.

Adaptive heterothermy and selective brain cooling are regarded as important thermal adaptations of large arid-zone mammals. Adaptive heterothermy, a process which reduces evaporation by storing body heat, ought to be enhanced by ambient heat load and by water deficit, but most mammals studied fail to show at least one of those attributes. Selective brain cooling, the reduction of brain temperature below arterial blood temperature, is most evident in artiodactyls, which possess a carotid rete, and traditionally has been considered to protect the brain during hyperthermia. The development of miniature ambulatory data loggers for recording body temperature allows the temperatures of free-living wild mammals to be measured in their natural habitats. All the African ungulates studied so far, in their natural habitats, do not exhibit adaptive heterothermy. They have low-amplitude nychthemeral rhythms of temperature, with mean body temperature over the night exceeding that over the day. Those with carotid retes (black wildebeest, springbok, eland) employ selective brain cooling but zebra, without a rete, do not. None of the rete ungulates, however, seems to employ selective brain cooling to prevent the brain overheating during exertional hyperthermia. Rather, they use it at rest, under moderate heat load, we believe in order to switch body heat loss from evaporative to non-evaporative routes.

Effects on fetal and maternal body temperatures of exposure of pregnant ewes to heat, cold, and exercise.

We exposed Dorper-cross ewes at approximately 120-135 days of gestation to a hot (40 degrees C, 60% relative humidity) and a cold (4 degrees C, 90% relative humidity) environment and to treadmill exercise (2.1 km/h, 5 degrees gradient) and measured fetal lamb and ewe body temperatures using previously implanted abdominal radiotelemeters. When ewes were exposed to 2 h of heat or 30 min of exercise, body temperature rose less in the fetus than in the mother, such that the difference between fetal and maternal body temperature, on average 0.6 degrees C before the thermal stress, fell significantly by 0.54 +/- 0.06 degrees C (SE, n = 8) during heat exposure and by 0.21 +/- 0.08 degrees C (n = 7) during exercise. During 6 h of maternal exposure to cold, temperature fell significantly less in the fetus than in the ewe, and the difference between fetal and maternal body temperature rose to 1.16 +/- 0.26 degrees C (n = 9). Thermoregulatory strategies used by the pregnant ewe for thermoregulation during heat or cold exposure appear to protect the fetus from changes in its thermal environment.