ETA receptors are involved in the febrile response induced by high dose of bacterial endotoxin.

Previous studies have demonstrated that endothelin-1 (ET-1) is involved in the febrile response induced by lipopolysaccharide (LPS) in male and female rats. This peptide induces fever acting on ETB receptors in the central nervous system. However, during sepsis, endothelinergic ETA receptors in the brain also exert an important role reducing the mortality of the animals. The present study evaluated the participation of ETA receptors in the febrile response induced by different doses LPS in rats. Male Wistar rats were treated with the ETA receptor antagonist BQ123 before or after the injection of a low dose (10 µg/kg) or a high dose (200 µg/kg) of LPS intraperitoneally. The febrile response was evaluated. The treatment with BQ123, in both protocols did not change the febrile response induced by the lower dose of LPS. The pre-treatment with BQ123 also did not significantly change the febrile response induced by a higher dose of LPS but the post-treatment with the antagonist abolished the febrile response induced by this dose of LPS. These results suggest that even though ETA receptors are not recruited in the febrile response induced by lower doses of LPS, they are involved in the febrile response induced by high doses of this stimulus.

Subchronic effects of dipyrone on the fish species Rhamdia quelen.

The use of the non-steroidal anti-inflammatory drugs such as dipyrone is so widespread that this drug and its metabolites have been detected in effluents and surface water. This study aimed to evaluate the potential toxic effects of dipyrone on the aquatic environment, using a native fish species, Rhamdia quelen. Fish were exposed to three concentrations of dipyrone, 0.5, 5 and 50 µg/L, in the water for 15 days, and hematological, biochemical, genetic and morphological biomarkers were evaluated. The glutathione S-transferase activity decreased in the highest concentration in relation to the control group. In addition, hematocrit, red blood cells and thrombocyte counts were decreased in all three exposed groups in relation to the control group. The comet assay showed DNA damage at the lowest concentration of dipyrone and significant kidney damage. Those results suggest that a constant exposure of aquatic organisms to dipyrone presents potential toxic effects.

Change in prostaglandin signaling during sickness syndrome hyperalgesia after ovariectomy in female rats.

The present study investigated hyperalgesia during sickness syndrome in female rats. Hyperalgesia was induced by an intraperitoneal injection of lipopolysaccharide (LPS) or an intracerebroventricular injection of prostaglandin E2 (PGE2). No differences were found in basal mechanical and thermal thresholds or in LPS-induced hyperalgesia in sham-operated animals in the diestrus or proestrus phase or in ovariectomized (OVX) animals. However, higher levels of PGE2 where found in the cerebrospinal fluid of OVX animals compared to sham-operated females. Intracerebroventricular injection of PGE2 produced rapid mechanical hyperalgesia in sham-operated rats while these responses were observed at later times in OVX animals. The protein kinase A (PKA) inhibitor H-89 reduced mechanical PGE2-induced hyperalgesia in OVX female rats, whereas no effect was observed in sham-operated animals. In contrast, the exchange protein activated by cyclic adenosine monophosphate (cAMP; Epac) inhibitor ESI-09 reduced mechanical PGE2-induced hyperalgesia, whereas no effect was observed in OVX animals. PGE2 also induced thermal hyperalgesia in sham-operated and OVX female rats and a similar effect of ESI-09 was observed. These results suggest that PGE2-induced hyperalgesia that is observed during sickness syndrome has different signaling mechanisms in cycling and OVX female rats involving the activation of the cAMP-Epac or cAMP-PKA pathways, respectively.

Intermittent binge-like ethanol exposure during adolescence attenuates the febrile response by reducing brown adipose tissue thermogenesis in rats.

Ethanol (EtOH) consumption is a primary health risk worldwide, which generally starts during adolescence in a binge pattern (i.e., the episodic consumption of high amounts). Binge EtOH consumption can lead to modifications of the innate and adaptive immune responses, including fever. The present study evaluated the febrile response that was induced by lipopolysaccharide (LPS) and prostaglandins E2 (PGE2) and the mechanisms of thermoregulation in adolescent rats that were exposed to EtOH in a binge-like pattern. Male Wistar rats were treated with an intraperitoneal (i.p.) injection of EtOH or saline on postnatal days (PND) 25, 26, 29, 30, 33, 34, 37, and 38. On PND 51, they received a pyrogenic challenge with LPS (i.p.) or PGE2 (intracerebroventricular) to induce a febrile response. Interscapular brown adipose tissue (BAT) mass and uncoupling protein (UCP) activity in isolated mitochondria were evaluated on PND 51. The rats were then subjected to cold challenges to analyze adaptive thermogenesis. Intermittent EtOH exposure during adolescence impaired the LPS- and PGE2-induced febrile response 12 days after the end of EtOH exposure. Ethanol exposure decreased interscapular BAT mass, oxygen consumption, and UCP activity in isolated mitochondria, resulting in an impairment in thermogenesis at 5 °C. No morphological changes in BAT were observed. These findings indicate that binge-like EtOH exposure during adolescence impairs thermoregulation by reducing BAT mass and function. This reduction may last for a prolonged period of time after the cessation of EtOH exposure and may affect both cold defenses and the febrile response during the development of infectious diseases.

Cannabinoid CB1 Receptor Antagonist Rimonabant Decreases Levels of Markers of Organ Dysfunction and Alters Vascular Reactivity in Aortic Vessels in Late Sepsis in Rats.

Sepsis is a life-threatening condition with high mortality rates that is caused by dysregulation of the host response to infection. We previously showed that treatment with the cannabinoid CB1 receptor antagonist rimonabant reduced mortality rates in animals with sepsis that was induced by cecal ligation and puncture (CLP). This improvement in the survival rate appeared to be related to an increase in arginine vasopressin (AVP) levels 12 h after CLP. The present study investigated the effects of rimonabant on organ dysfunction, hematologic parameters, and vascular reactivity in male Wistar rats with sepsis induced by CLP. Intraperitoneal treatment with rimonabant (10 mg/kg, 4 h after CLP) abolished the increase in the plasma levels of lactate, lactate dehydrogenase, glucose, and creatinine kinase MB without altering hematological parameters (i.e., leukopenia and a reduction of platelet counts). CLP increased plasma levels of nitrate/nitrite (NOx) and induced vasoconstriction in the tail artery. The treatment of CLP rats with rimonabant did not alter NOx production but reduced the vasoconstriction. Rimonabant also attenuated the hyperreactivity to AVP induced by CLP without affecting hyporesponsiveness to phenylephrine in aortic rings. These results suggest that rimonabant reduces organ dysfunction during sepsis, and this effect may be related to AVP signaling in blood vessels. This effect may have contributed to the higher survival rate in rimonabant-treated septic animals.

Peripheral kinin B(1) and B(2) receptor-operated mechanisms are implicated in neuropathic nociception induced by spinal nerve ligation in rats.

The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.

Female Sex Hormones Influence the Febrile Response Induced by Lipopolysaccharide, Cytokines and Prostaglandins but not by Interleukin-1ß in Rats.

There are differences in the immune response, and particularly fever, between males and females. In the present study, we investigated how the febrile responses induced by lipopolysaccharide (LPS) and different endogenous pyrogens were affected by female gonadal hormones. The febrile response to i.p. injection of LPS (50 µg/kg) was 40% lower in female rats compared to male or ovariectomised (OVX) female rats. Accordingly, oestrogen replacement in OVX animals reduced LPS-induced fever. Treatment with the prostaglandin synthesis inhibitor indomethacin (2 mg/kg, i.p. 30 min before) reduced the febrile response induced by LPS in both OVX (88%) and sham-operated (71%) rats. In line with the enhanced fever in OVX rats, there was increased expression of cyclooxygenase-2 (COX-2) in the hypothalamus and elevated levels of prostaglandin E2 (PGE2 ). In addition, OVX rats were hyper-responsive to PGE2 injected i.c.v. By contrast to the enhanced fever in response to LPS and PGE2 , the febrile response induced by i.c.v. injection of interleukin (IL)-1ß was unaffected by ovariectomy, whereas the responses induced by tumour necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-1α were completely abrogated. These results suggest that the mediators involved in the febrile response in females are similar to males, although the reduction of female hormones may decrease the responsiveness of some mediators such as TNF-α and MIP-1α. Compensatory mechanisms may be activated in females after ovariectomy such as an augmented synthesis of COX-2 and PGE2 .

Central mediators involved in the febrile response induced by polyinosinic-polycytidylic acid: lack of involvement of endothelins and substance P.

The present study evaluated the involvement of interleukin(IL)-1ß, tumor necrosis factor-α (TNF-α), IL-6, interferon(IFN)-γ, prostaglandins of the E2 series, endothelins, substance P and opioids within the central nervous system in polyinosinic:polycytidylic acid (Poly I:C)-induced fever in rats. Poly I:C injection induced a febrile response which was reduced by intracerebroventricular administration of the antibodies against TNF-α, IL-6, or IFN-γ, or by IL-1 or µ receptor antagonists. Intraperitoneal injection of indomethacin or oral administration of celecoxib also reduced Poly I:C-induced fever. Poly I:C increased prostaglandin E2 levels in the cerebrospinal fluid of the animals which was also reduced by indomethacin. The intracerebroventricular injection of ETB or NK1 receptor antagonists did not alter Poly I:C-induced fever. These data suggest the involvement of IL-1ß, TNF-α, IL-6, IFN-γ, prostaglandin E2, and opioids but not endothelins and substance P on Poly I:C-induced fever.

Interleukin (IL)-6 release and fever induced by a pre-formed pyrogenic factor (PFPF) derived from LPS-stimulated macrophages.

A novel pre-formed pyrogenic factor (PFPF), released by LPS-stimulated macrophages, has been identified, that induces an indomethacin-resistant fever. Its activity has to date not been found to match that of any described cytokine. In this study we observed that PFPF induced the release of large amounts of IL-6 from rat peritoneal macrophages. A combination of anti-cytokine antibodies and heat treatment excluded IL-1, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) as being responsible for this effect. PFPF also induced interleukin (IL)-1, IL-6 and TNF-alpha in a subcutaneous air pouch, as well as increasing plasma IL-6, and induced a fever of 0.58 +/- 0.07 degrees C (3 hours) that was not reduced by indomethacin (2 mg/kg, ip). Preparative isoelectric focusing (IEF) showed that the material responsible for inducing IL-6 release had a pI between 4.7 and 5.8 and corresponded to the IEF pool that induced fever when injected intracerebroventricularly.

Etanercept reduces thermal and mechanical orofacial hyperalgesia following inflammation and neuropathic injury.

BACKGROUND: This study evaluated the involvement of tumour necrosis factor α (TNF-α) in orofacial thermal and mechanical hyperalgesia induced by an inflammatory stimulus or by chronic constriction of the infraorbital nerve (CION) using etanercept (Eta), a TNF-receptor fusion protein that inhibits TNF-α action. METHODS: Animals were treated with Eta (0.5 and 5.0 mg/kg, s.c.) or dexamethasone (Dex, 0.5, 1.0 and 2.0 mg/kg, s.c.) and orofacial thermal (cold and heat) and mechanical hyperalgesia induced by an inflammatory stimulus (carrageenan, Cg 50 and 100 µg/lip) or by chronic CION, a model of neuropathic pain in the orofacial region was evaluated. Treatments with Dex or Eta were carried out before Cg or before or after CION. RESULTS: Eta or Dex abolished inflammatory thermal and mechanical hyperalgesia. Also, each drug, when given at the day of the surgery and the subsequent day, was effective to abolish thermal and mechanical hyperalgesia induced by CION, assessed on day 4 and on day 13 after the surgery, respectively. However, Eta, but not Dex, given after the CION, abolished thermal and mechanical hyperalgesia and reduced TNF-α level in the trigeminal ganglion. CONCLUSIONS: These results suggest that TNF-α has an important role in cold, heat and mechanical hyperalgesia induced by inflammation or neuropathy in the orofacial region and this may contribute for the establishment of new therapeutic strategies to treat orofacial pain.

Central substance P NK1 receptors are involved in fever induced by LPS but not by IL-1ß and CCL3/MIP-1α in rats.

Substance P (SP) is a neuropeptide that can modulate inflammatory mediator release through activation of NK(1) receptors (NK(1)R). Some studies have also suggested the involvement of SP in lipopolysaccharide (LPS)-induced fever. However, the precise contribution of this neuropeptide to the pathways activated during fever is unknown. In this study we investigated the effect of a selective NK(1)R antagonist, SR140333B, on the febrile response induced by LPS and cytokines. Our results show that the systemic injection of SR140333B did not modify the fever induced by LPS at a dose that is able to reduce protein extravasation induced by SP in the skin. On the other hand, intracerebroventricular administration of SR140333B significantly reduced the fever induced by peripheral injection of LPS. These data emphasize an important role for SP in the central nervous system during the febrile response to LPS, and are reinforced by the fact that intracerebroventricular injection of SP also induced fever in a dose-dependent manner in captopril-treated rats. Considering that the febrile response can result from the generation of several endogenous pyrogens, among them interleukin (IL)-1ß and macrophage inflammatory protein-1α (CCL3/MIP-1α), we also examined the effect of SR140333B on the fever induced by these cytokines which act through prostaglandin-dependent and -independent mechanisms, respectively. Surprisingly, SR140333B did not modify the febrile response to IL-1ß or CCL3/MIP-1α. Altogether these data suggest that the central action of SP is essential for LPS-, but not for IL-1ß- or CCL3/MIP-1α-induced fever.

Involvement of CRH in fever induced by a distinct pre-formed pyrogenic factor (PFPF).

OBJECTIVE: This study investigated the role of corticotrophin-releasing hormone (CRH) in mediating the fever induced by a novel pre-formed pyrogenic factor (PFPF), using a CRF antagonist in vivo and evaluating the capacity of PFPF to stimulate CRH release from the hypothalamus in vitro. MATERIALS AND METHODS: Male Wistar rats were used. The PFPF, induced following brief incubation of rat peritoneal macrophages with LPS and retained on 10 or 20 kDa MW cut-off membranes, was injected intracerebroventricularly. Fever was monitored using a rectal probe. Hypothalamus tissue was incubated with PFPF to establish its ability to induce CRH release. The CRH was measured by ELISA. RESULTS: PFPF induced a dose-dependent fever that was abolished by boiling or pronase treatment. Whereas both dexamethasone and indomethacin were effective in reducing interleukin- (IL) 1beta-induced fever, only dexamethasone abolished the fever induced by PFPF. The CRH antagonist, a-helical CRH9-41, abolished the fever induced by synthetic CRH, IL-8 and PFPF but not tumour necrosis factor-a (TNF-alpha). Like IL-1, PFPF was able to induce the release of CRH from rat hypothalamic tissue in vitro. CONCLUSIONS: These results suggest that the fever induced by PFPF depends on CRH release but not prostaglandin synthesis.

A pre-formed Pyrogenic Factor Released by Lipopolysaccharide Stimulated Macrophages.

The aim of this study was to investigate the pyrogenic activity of factor(s) released by rat peritoneal macrophages following a brief stimulation with LPS. The effect of this factor on the number of circulating leukocytes and serum Fe, Cu and Zn levels, was also evaluated. The possibility that the content of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF) in the supernatant could explain the observations was investigated. Supernatant produced over a period of 1 h by peritoneal macrophages, following a 30 min incubation with LPS at 37 degrees C, was ultrafiltered through a 10 000 MW cut-off Amicon membrane, sterilized, and concentrated 2.5, 5, 10 and 20 times. The intravenous (i.v.) injection of this supernatant induced a concentration-dependent fever in rats with a maximal response at 2 h. The pyrogenic activity was produced by macrophages elicited with thioglycollate and by resident cells. The supernatants also induced neutrophilia and reduction in Fe and Zn 6 h after the injection. Absence of activity in boiled supernatants, or supernatants from macrophages incubated at 4 degrees C with LPS, indicates that LPS was not responsible for the activity. In vitro treatment with indomethacin (Indo), dexamethasone (Dex), or cycloheximide (Chx) did not modify the release of pyrogenic activity into the supernatant or its effects on the reduction in serum metal levels. Although Chx abolished the production of mediator(s) inducing neutrophilia, and Dex reduced the induction of IL-1beta, TNF and IL-6, injection of the highest concentration of these cytokines detected in the supernatants did not induce fever. In vivo treatment with Dex, but not Indo, abolished the fever induced by the supernatant. These results suggest that macrophages contain pre-formed pyrogenic mediator(s), not related to IL-1beta, IL-6 or TNF, that acts indirectly and independently of prostaglandtn. It also seems likely that the pyrogenic activity is related to the factor responsible for the reduction of serum Fe and Zn levels, but not the neutrophilia.

Interleukin-8 induces fever by a prostaglandin-independent mechanism.

We have studied the mechanism by which interleukin-8 (IL-8) induces fever in rats. Intracerebroventricular injections of IL-8 (5.5-50 ng) evoked dose-dependent increases in body temperature, which reached a plateau 5 h after injection, i.e., later than intracerebroventricular interleukin-1 beta (IL-1 beta; 2 h). The pyrogenic activity of IL-8 was not due to contamination with lipopolysaccharide (LPS) because preincubation of IL-8 with a specific antibody or boiling the IL-8 for 30 min abolished its activity but not that of LPS; also, IL-8 but not LPS induced fever in LPS-tolerant rats. Indomethacin significantly reduced the pyrogenic effects of intracerebroventricular injections of LPS and IL-1 beta but not responses to IL-8, suggesting that pyrogenic responses to IL-8 were mediated independently of prostaglandins. In contrast, dexamethasone markedly attenuated pyrogenic responses to IL-8 and IL-1 beta. These data suggest that inhibition of IL-8 by glucocorticoids contributes to the antipyretic effects of these drugs in fevers resistant to cyclooxygenase inhibitors.

Indomethacin blocks the febrile response induced by interleukin-8 in rabbits.

Interleukin (IL)-8 induces fever in rats by a mechanism independent of the release of cyclooxygenase products. The purpose of this study was to investigate whether a similar mechanism is responsible for the pyrogenic effect of IL-8 in rabbits. Intravenous (0.31-5.0 ng/kg) or intracerebroventricular (15.6-250 pg) injections of IL-8 induced a dose-dependent increase in body temperature. The correlations between the doses of recombinant human IL-8 and the fever index were 0.98 and 0.99 for the intravenous and intracerebroventricular injections, respectively. The pyrogenic activity of IL-8 was not due to contamination with lipopolysaccharide (LPS), inasmuch as the Limulus amoebocyte lysate test showed < 10 pg endotoxin/micrograms IL-8, and boiled IL-8 lost its pyrogenic activity. Indomethacin (2 and 5 mg/kg i.p.) abolished the febrile response induced by the intravenous injection of LPS (5.0 ng/kg), IL-1 beta (5 ng/kg), and IL-8 (5 ng/kg). Indomethacin also abolished the fever induced by the intracerebroventricular injection of IL-8 (62.5 pg) but only partially reduced the response induced by the injection of IL-1 beta (25 pg icv). These results show that, different from rats, indomethacin blocks the febrile response induced by the central or peripheral administration of IL-8 in rabbits.

Importance of the vagus nerve for fever and neutrophil migration induced by intraperitoneal LPS injection.

OBJECTIVE: We investigated the importance of the vagus nerve in fever, neutrophil migration and neutrophilia simultaneously induced by intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) and in terms of the production of pre-formed pyrogenic factor (PFPF) and of the fever induced by this factor. METHODS: Naïve, sham-operated or subdiaphragmatically vagotomized male Wistar rats received either LPS (i.p. or i.pl.) or PFPF (i.v., i.c.v., i.p.). The number of neutrophils was evaluated in peritoneal or pleural fluid and in blood. Fever was monitored using a rectal probe. RESULTS: In naïve animals, LPS (0.02-200 microg kg(-1), i.p.) induced dose-related neutrophilia and fever while on neutrophil migration it resulted in a bell-shaped curve. Vagotomy reduced the peritoneal resident cell population (56%), fever (71%) and neutrophil migration (43%) but not the neutrophilia or neutrophil migration to the pleural cavity. Vagotomy did not affect the PFPF production or PFPF-induced fever. CONCLUSIONS: Vagus nerve integrity is important not only for fever but also for the neutrophil influx to the peritoneal cavity by controlling the number of resident cells in this cavity.

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats.

OBJECTIVE: Compare the antipyretic effects of dipyrone and indomethacin. MATERIALS AND METHODS: Fever was induced in rats by i. v. LPS or i. c. v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-alpha (TNF-alpha) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA. RESULTS: Indomethacin or dipyrone reduced LPS, IL-1beta, IL-6 or TNF-alpha induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2alpha fever. Only indomethacin inhibited fever induced by AA or IL-1beta, plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 microM) blocked COX-1 and COX-2 in COS-7 cells. CONCLUSION: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.