The hyperthermic effect of central cholecystokinin is mediated by the cyclooxygenase-2 pathway.

Cholecystokinin (CCK) increases core body temperature via CCK2 receptors when administered intracerebroventricularly (icv). The mechanisms of CCK-induced hyperthermia are unknown, and it is also unknown whether CCK contributes to the fever response to systemic inflammation. We studied the interaction between central CCK signaling and the cyclooxygenase (COX) pathway. Body temperature was measured in adult male Wistar rats pretreated with intraperitoneal infusion of the nonselective COX enzyme inhibitor metamizol (120 mg/kg) or a selective COX-2 inhibitor, meloxicam, or etoricoxib (10 mg/kg for both) and, 30 min later, treated with intracerebroventricular CCK (1.7 µg/kg). In separate experiments, CCK-induced neuronal activation (with and without COX inhibition) was studied in thermoregulation- and feeding-related nuclei with c-Fos immunohistochemistry. CCK increased body temperature by ∼0.4°C from 10 min postinfusion, which was attenuated by metamizol. CCK reduced the number of c-Fos-positive cells in the median preoptic area (by ∼70%) but increased it in the dorsal hypothalamic area and in the rostral raphe pallidus (by ∼50% in both); all these changes were completely blocked with metamizol. In contrast, CCK-induced satiety and neuronal activation in the ventromedial hypothalamus were not influenced by metamizol. CCK-induced hyperthermia was also completely blocked with both selective COX-2 inhibitors studied. Finally, the CCK2 receptor antagonist YM022 (10 µg/kg icv) attenuated the late phases of fever induced by bacterial lipopolysaccharide (10 µg/kg; intravenously). We conclude that centrally administered CCK causes hyperthermia through changes in the activity of "classical" thermoeffector pathways and that the activation of COX-2 is required for the development of this response.NEW & NOTEWORTHY An association between central cholecystokinin signaling and the cyclooxygenase-prostaglandin E pathway has been proposed but remained poorly understood. We show that the hyperthermic response to the central administration of cholecystokinin alters the neuronal activity within efferent thermoeffector pathways and that these effects are fully blocked by the inhibition of cyclooxygenase. We also show that the activation of cyclooxygenase-2 is required for the hyperthermic effect of cholecystokinin and that cholecystokinin is a modulator of endotoxin-induced fever.

Cold-Induced Thermogenesis and Inflammation-Associated Cold-Seeking Behavior Are Represented by Different Dorsomedial Hypothalamic Sites: A Three-Dimensional Functional Topography Study in Conscious Rats.

In the past, we showed that large electrolytic lesions of the dorsomedial hypothalamus (DMH) promoted hypothermia in cold-exposed restrained rats, but attenuated hypothermia in rats challenged with a high dose of bacterial lipopolysaccharide (LPS) in a thermogradient apparatus. The goal of this study was to identify the thermoeffector mechanisms and DMH representation of the two phenomena and thus to understand how the same lesion could produce two opposite effects on body temperature. We found that the permissive effect of large electrolytic DMH lesions on cold-induced hypothermia was due to suppressed thermogenesis. DMH-lesioned rats also could not develop fever autonomically: they did not increase thermogenesis in response to a low, pyrogenic dose of LPS (10 µg/kg, i.v.). In contrast, changes in thermogenesis were uninvolved in the attenuation of the hypothermic response to a high, shock-inducing dose of LPS (5000 µg/kg, i.v.); this attenuation was due to a blockade of cold-seeking behavior. To compile DMH maps for the autonomic cold defense and for the cold-seeking response to LPS, we studied rats with small thermal lesions in different parts of the DMH. Cold thermogenesis had the highest representation in the dorsal hypothalamic area. Cold seeking was represented by a site at the ventral border of the dorsomedial nucleus. Because LPS causes both fever and hypothermia, we originally thought that the DMH contained a single thermoregulatory site that worked as a fever-hypothermia switch. Instead, we have found two separate sites: one that drives thermogenesis and the other, previously unknown, that drives inflammation-associated cold seeking.SIGNIFICANCE STATEMENT Cold-seeking behavior is a life-saving response that occurs in severe systemic inflammation. We studied this behavior in rats with lesions in the dorsomedial hypothalamus (DMH) challenged with a shock-inducing dose of bacterial endotoxin. We built functional maps of the DMH and found the strongest representation of cold-seeking behavior at the ventral border of the dorsomedial nucleus. We also built maps for cold-induced thermogenesis in unanesthetized rats and found the dorsal hypothalamic area to be its main representation site. Our work identifies the neural substrate of cold-seeking behavior in systemic inflammation and expands the functional topography of the DMH, a structure that modulates autonomic, endocrine, and behavioral responses and is a potential therapeutic target in anxiety and panic disorders.

Hyperbilirubinemia exaggerates endotoxin-induced hypothermia.

Systemic inflammation is accompanied by an increased production of reactive oxygen species (ROS) and by either fever or hypothermia (or both). To study aseptic systemic inflammation, it is often induced in rats by the intravenous administration of bacterial lipopolysaccharide (LPS). Knowing that bilirubin is a potent ROS scavenger, we compared responses to LPS between normobilirubinemic Gunn rats (heterozygous, asymptomatic; J/+) and hyperbilirubinemic Gunn rats (homozygous, jaundiced; J/J) to establish whether ROS mediate fever and hypothermia in aseptic systemic inflammation. These two genotypes correspond to undisturbed versus drastically suppressed (by bilirubin) tissue accumulation of ROS, respectively. A low dose of LPS (10 µg/kg) caused a typical triphasic fever in both genotypes, without any intergenotype differences. A high dose of LPS (1,000 µg/kg) caused a complex response consisting of early hypothermia followed by late fever. The hypothermic response was markedly exaggerated, whereas the subsequent fever response was strongly attenuated in J/J rats, as compared to J/+ rats. J/J rats also tended to respond to 1,000 µg/kg with blunted surges in plasma levels of all hepatic enzymes studied (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase), thus suggesting an attenuation of hepatic damage. We propose that the reported exaggeration of LPS-induced hypothermia in J/J rats occurs via direct inhibition of nonshivering thermogenesis by bilirubin and possibly via a direct vasodilatatory action of bilirubin in the skin. This hypothermia-exaggerating effect might be responsible, at least in part, for the observed tendency of J/J rats to be protected from LPS-induced hepatic damage. The attenuation of the fever response to 1,000 µg/kg could be due to either direct actions of bilirubin on thermoeffectors or the ROS-scavenging action of bilirubin. However, the experiments with 10 µg/kg strongly suggest that ROS signaling is not involved in the fever response to low doses of LPS.

Modulation of body temperature and LH secretion by hypothalamic KNDy (kisspeptin, neurokinin B and dynorphin) neurons: a novel hypothesis on the mechanism of hot flushes.

Despite affecting millions of individuals, the etiology of hot flushes remains unknown. Here we review the physiology of hot flushes, CNS pathways regulating heat-dissipation effectors, and effects of estrogen on thermoregulation in animal models. Based on the marked changes in hypothalamic kisspeptin, neurokinin B and dynorphin (KNDy) neurons in postmenopausal women, we hypothesize that KNDy neurons play a role in the mechanism of flushes. In the rat, KNDy neurons project to preoptic thermoregulatory areas that express the neurokinin 3 receptor (NK3R), the primary receptor for NKB. Furthermore, activation of NK3R in the median preoptic nucleus, part of the heat-defense pathway, reduces body temperature. Finally, ablation of KNDy neurons reduces cutaneous vasodilatation and partially blocks the effects of estrogen on thermoregulation. These data suggest that arcuate KNDy neurons relay estrogen signals to preoptic structures regulating heat-dissipation effectors, supporting the hypothesis that KNDy neurons participate in the generation of flushes.

Contributions of different modes of TRPV1 activation to TRPV1 antagonist-induced hyperthermia.

Transient receptor potential vanilloid-1 (TRPV1) antagonists are widely viewed as next-generation pain therapeutics. However, these compounds cause hyperthermia, a serious side effect. TRPV1 antagonists differentially block three modes of TRPV1 activation: by heat, protons, and chemical ligands (e.g., capsaicin). We asked what combination of potencies in these three modes of TRPV1 activation corresponds to the lowest potency of a TRPV1 antagonist to cause hyperthermia. We studied hyperthermic responses of rats, mice, and guinea pigs to eight TRPV1 antagonists with different pharmacological profiles and used mathematical modeling to find a relative contribution of the blockade of each activation mode to the development of hyperthermia. We found that the hyperthermic effect has the highest sensitivity to the extent of TRPV1 blockade in the proton mode (0.43 to 0.65) with no to moderate sensitivity in the capsaicin mode (-0.01 to 0.34) and no sensitivity in the heat mode (0.00 to 0.01). We conclude that hyperthermia-free TRPV1 antagonists do not block TRPV1 activation by protons, even if they are potent blockers of the heat mode, and that decreasing the potency to block the capsaicin mode may further decrease the potency to cause hyperthermia.

Neural substrate of cold-seeking behavior in endotoxin shock.

Systemic inflammation is a leading cause of hospital death. Mild systemic inflammation is accompanied by warmth-seeking behavior (and fever), whereas severe inflammation is associated with cold-seeking behavior (and hypothermia). Both behaviors are adaptive. Which brain structures mediate which behavior is unknown. The involvement of hypothalamic structures, namely, the preoptic area (POA), paraventricular nucleus (PVH), or dorsomedial nucleus (DMH), in thermoregulatory behaviors associated with endotoxin (lipopolysaccharide [LPS])-induced systemic inflammation was studied in rats. The rats were allowed to select their thermal environment by freely moving in a thermogradient apparatus. A low intravenous dose of Escherichia coli LPS (10 microg/kg) caused warmth-seeking behavior, whereas a high, shock-inducing dose (5,000 microg/kg) caused cold-seeking behavior. Bilateral electrocoagulation of the PVH or DMH, but not of the POA, prevented this cold-seeking response. Lesioning the DMH with ibotenic acid, an excitotoxin that destroys neuronal bodies but spares fibers of passage, also prevented LPS-induced cold-seeking behavior; lesioning the PVH with ibotenate did not affect it. Lesion of no structure affected cold-seeking behavior induced by heat exposure or by pharmacological stimulation of the transient receptor potential (TRP) vanilloid-1 channel ("warmth receptor"). Nor did any lesion affect warmth-seeking behavior induced by a low dose of LPS, cold exposure, or pharmacological stimulation of the TRP melastatin-8 ("cold receptor"). We conclude that LPS-induced cold-seeking response is mediated by neuronal bodies located in the DMH and neural fibers passing through the PVH. These are the first two landmarks on the map of the circuitry of cold-seeking behavior associated with endotoxin shock.

Microsomal prostaglandin E synthase-1, ephrins, and ephrin kinases as suspected therapeutic targets in arthritis: exposed by "criminal profiling".

Feeding information obtained in one criminal case into the profile of another crime often helps to solve the latter. The literature on two different "crimes," namely, acute systemic inflammation and arthritis (including osteoarthritis [OA] and rheumatoid arthritis [RA] deals largely with the same "gang" of inflammatory mediators, such as prostaglandin (PG) E2. Early investigations suggested that microsomal PGE synthase-1 (mPGES-1; a terminal PGE2-synthesizing enzyme) plays a pivotal role in bacterial lipopolysaccharide (LPS)-induced systemic inflammation, but overlooked the possibility that the same enzyme could be involved in OA or RA. Later studies showed that mPGES-1 is indeed a key perpetrator in arthritic diseases, a fact that could have been predicted earlier by pooling the new knowledge about mPGES-1 into the profile of arthritic diseases. In this review, we analyze our recent study on the expression of erythropoietin-producing hepatocellular (Eph) receptor kinases and their ligands, ephrins, in LPS-induced systemic inflammation. By pooling these results together with literature data into the profile of RA, we conclude that Eph kinases and ephrins are prime suspects for being involved in the pathogenesis of RA. We further conjecture that the involvement of Eph kinases and ephrins may be realized via the induction of angiogenesis in the inflamed joint, promotion of leukocyte infiltration, and activation of the infiltrated cells. Studies to test this new hypothesis seem warranted, and our prediction is that the "smoking gun" will be found.

The organum vasculosum laminae terminalis in immune-to-brain febrigenic signaling: a reappraisal of lesion experiments.

The organum vasculosum laminae terminalis (OVLT) has been proposed to serve as the interface for blood-to-brain febrigenic signaling, because ablation of this structure affects the febrile response. However, lesioning the OVLT causes many "side effects" not fully accounted for in the fever literature. By placing OVLT-lesioned rats on intensive rehydration therapy, we attempted to prevent these side effects and to evaluate the febrile response in their absence. After the OVLT of Sprague-Dawley rats was lesioned electrolytically, the rats were given access to 5% sucrose for 1 wk to stimulate drinking. Sucrose consumption and body mass were monitored. The animals were examined twice a day for signs of dehydration and treated with isotonic saline (50 ml/kg sc) when indicated. This protocol eliminated mortality but not several acute and chronic side effects stemming from the lesion. The acute effects included adipsia and gross (14% of body weight) emaciation; chronic effects included hypernatremia, hyperosmolality, a suppressed drinking response to hypertonic saline, and previously unrecognized marked (by approximately 2 degrees C) and long-lasting (>3 wk) hyperthermia. Because the hyperthermia was not accompanied by tail skin vasoconstriction, it likely reflected increased thermogenesis. After the rats recovered from the acute (but not chronic) side effects, their febrile response to IL-1beta (500 ng/kg iv) was tested. The sham-operated rats developed typical monophasic fevers ( approximately 0.5 degrees C), the lesioned rats did not. However, the absence of the febrile response in the OVLT-lesioned rats likely resulted from the untreatable side effects. For example, hyperthermia at the time of pyrogen injection was high enough (39-40 degrees C) to solely prevent fever from developing. Hence, the changed febrile responsiveness of OVLT-lesioned animals is given an alternative interpretation, unrelated to febrigenic signaling to the brain.

Expression of genes controlling transport and catabolism of prostaglandin E2 in lipopolysaccharide fever.

Prostaglandin (PG) E(2) is a principal downstream mediator of fever and other symptoms of systemic inflammation. Its inactivation occurs in peripheral tissues, primarily the lungs and liver, via carrier-mediated cellular uptake and enzymatic oxidation. We hypothesized that inactivation of PGE(2) is suppressed during LPS fever and that transcriptional downregulation of PGE(2) carriers and catabolizing enzymes contributes to this suppression. Fever was induced in inbred Wistar-Kyoto rats by intravenous LPS (50 microg/kg); the controls received saline. Samples of the liver, lungs, and hypothalamus were harvested 0, 0.5, 1.5, and 5 h postinjection. The expression of the two principal transmembrane PGE(2) carriers (PG transporter and multispecific organic anion transporter) and the two key PGE(2)-inactivating enzymes [15-hydroxy-PG dehydrogenase (15-PGDH) and carbonyl reductase] was quantified by RT-PCR. All four genes of interest were downregulated in peripheral tissues (but not the brain) during fever. Most remarkably, the expression of hepatic 15-PGDH was decreased 26-fold 5 h post-LPS, whereas expression of pulmonary 15-PGDH was downregulated (as much as 18-fold) throughout the entire febrile course. The transcriptional downregulation of several proteins involved in PGE(2) inactivation, first reported here, is an unrecognized mechanism of systemic inflammation. By increasing the blood-brain gradient of PGE(2), this mechanism likely facilitates penetration of PGE(2) into the brain and prevents its elimination from the brain.

Prostaglandin E(2)-synthesizing enzymes in fever: differential transcriptional regulation.

The febrile response to lipopolysaccharide (LPS) consists of three phases (phases I-III), all requiring de novo synthesis of prostaglandin (PG) E(2). The major mechanism for activation of PGE(2)-synthesizing enzymes is transcriptional upregulation. The triphasic febrile response of Wistar-Kyoto rats to intravenous LPS (50 microg/kg) was studied. Using real-time RT-PCR, the expression of seven PGE(2)-synthesizing enzymes in the LPS-processing organs (liver and lungs) and the brain "febrigenic center" (hypothalamus) was quantified. Phase I involved transcriptional upregulation of the functionally coupled cyclooxygenase (COX)-2 and microsomal (m) PGE synthase (PGES) in the liver and lungs. Phase II entailed robust upregulation of all enzymes of the major inflammatory pathway, i.e., secretory (s) phospholipase (PL) A(2)-IIA --> COX-2 --> mPGES, in both the periphery and brain. Phase III was accompanied by the induction of cytosolic (c) PLA(2)-alpha in the hypothalamus, further upregulation of sPLA(2)-IIA and mPGES in the hypothalamus and liver, and a decrease in the expression of COX-1 and COX-2 in all tissues studied. Neither sPLA(2)-V nor cPGES was induced by LPS. The high magnitude of upregulation of mPGES and sPLA(2)-IIA (1,257-fold and 133-fold, respectively) makes these enzymes attractive targets for anti-inflammatory therapy.