Prostaglandins are the most widely known central mediators of the febrile response. Prostaglandin E2 (PGE2) is synthesized through the metabolism of arachidonic acid by cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES1) , , . The production of this eicosanoid occurs mainly in the brain, specifically in the endothelial cells , although some evidence indicate that the peripheral synthesis of prostaglandins also contributes to the initial phase of fever . Once released, these prostanoids can easily reach the medial preoptic nucleus of the hypothalamus to produce fever by acting on prostaglandin EP3 receptors , , . Most of the studies above, which reported the involvement of prostaglandins in fever, used lipopolysaccharide (LPS) to induce fever to simulate Gram-negative bacterial infections , , , , , .
However, LPS-induced fever also relies on other central mediators beyond prostaglandins. The first of the mediators identified was the corticotropin-releasing factor (CRF). In 1989, Rothwell showed that the CRF receptor antagonist α-helical CRF9-41 attenuated the increases in body temperature, oxygen consumption, and brown adipose tissue activity, which were observed after the central administration of interleukin-1β (IL-1β), one of the main pyrogenic cytokines . Subsequent studies have shown that the CRF receptor antagonists reduced the febrile response induced by several pyrogenic mediators , . The blockade of the actions of substance P in the brain using the neurokinin-1 (NK1) receptor antagonists reduced LPS-induced fever , , , suggesting that substance P is also a central mediator of LPS-induced fever. However, substance P does not participate in the febrile response induced by some endogenous pyrogens, such as IL-1β and the chemokine CCL3 ; however, it is involved in the febrile response induced by tumor necrosis factor-α (TNF-α), IL-6, and PGE2 .
In 1998, Fabrício et al.  showed that endothelin-1 (ET-1) induced fever when it was injected directly in the brain, and that the ETB receptor antagonist BQ788 reduced LPS-induced fever. These results and those of the following studies ,  confirmed that ET-1 is a central mediator of fever. The first study that suggested that endogenous opioids (eOPs) also participated in the LPS-induced febrile response was published by Blatteis et al. . Fraga et al.  further confirmed these results by proving that μ-opioid receptors (MORs) are involved in modulating fever. Some redundancy in the pathways that lead to the release of these central mediators (i.e. eOPs and ET-1) induces fever independently of the prostaglandin synthesis, whereas substance P appears to act in concert with prostaglandins in the autocrine circuitry , . The CRF appears to be involved in both prostaglandin-dependent and -independent mechanisms (for review, see ).
Zymosan is an insoluble protein-carbohydrate complex from the yeast cell and has been used to induce fever, thus mimicking the febrile response that is induced by fungi . Past studies that used this pyrogen have intraperitoneally (i.p.) or intra-articularly (i.a.) injected zymosan, but the mediators that are involved in this febrile response are scarcely known. Kanashiro et al.  showed that an i.a. injection of zymosan produced a febrile response in rats that depended on the prostaglandin synthesis. In contrast to the LPS, the zymosan-induced febrile response did not depend on CRF or ET-1 synthesis .
We and others recently showed that cytokines, such as IL-1β, IL-6, and TNF-α, which are produced peripherally or in the central nervous system are involved in the febrile response induced by both i.p. and i.a. zymosan administration , . However, it remains unknown whether substance P and eOPs participate in the febrile response induced by zymosan.
The aim of the present study was to evaluate the central mediators, specifically substance P and eOPs, which are involved in the febrile response induced by the i.p. injected zymosan. We also evaluated whether, similar to what was observed with the i.a. injection of zymosan, the febrile response induced by the i.p. injected zymosan is mediated by prostaglandins but not by ET-1.
Materials and methods
The experiments were conducted with 132 male Wistar rats (180–220 g), which were housed 4 per cage at 22°C±1°C under a 12 h/12 h light–dark cycle (lights on at 7:00 a.m.) with free access to food chow and tap water. All of the experiments were approved by the institution’s Ethical Committee on Animal Use, in accordance with the Brazilian and International Guidelines for Animal Care.
Abdominal temperature measurement
Abdominal body temperature (Tb) was measured in conscious unrestrained rats using data loggers (Subcue, Calgary, Canada). Briefly, data loggers were implanted in the peritoneal cavity under ketamine (90 mg/kg) and xylazine (7.5 mg/kg) anesthesia under aseptic conditions at least 5 days prior to the experiment. The animals were treated with oxytetracycline hydrochloride (400 mg/kg, intramuscular) and ketoprofen (10 mg/kg, orally) immediately after surgery. On the day of the experiment, body temperature was continuously monitored at 15-min intervals beginning 2 h before any injection until 6 h after the injection of the pyrogenic stimulus. During the experiment, the room temperature was maintained at 28°C±1°C (i.e. the thermoneutral zone for rats) .
Intracerebral cannula implantation and microinjection
For the intracerebroventricular (i.c.v.) administration of BQ788, SR140333B, and CTAP, a 22-gauge stainless-steel guide cannula (0.8 mm outer diameter, 12 mm length) was sterotaxically implanted above the right lateral ventricle under the same anesthesia that was used for data logger implantation according to the following coordinates: 0.8 mm lateral to the midline, 1.5 mm posterior to the bregma, and 2.5 mm below the brain surface, with the incisor bar lowered 3.3 mm below the horizontal zero . The cannulas were fixed to the skull with jeweler’s screws and embedded in dental acrylic cement. Microinjections were made aseptically using a 30-gauge needle that was connected to a polyethylene-10 tubing. The needle protruded 2 mm beyond the cannula tip to reach the right lateral ventricle, and a 2 μL volume was slowly injected over 1 min using a 25 μL Hamilton syringe. After the injection, the needle was left in place for 30 s before it was withdrawn to prevent the backflow of the injection fluid through the cannula. After the experiment, each rat received a microinjection of Evans blue (2.5% in saline) in the lateral ventricle. Finally, the brains were removed, and animals that had cannula misplacements, cannula blockage, or abnormal body weight gain after surgery were excluded from the study.
All the pyrogenic stimuli were injected between 9:00 a.m. and 11:00 a.m. to avoid the circadian differences in body temperature. In the first set of experiments, we evaluated the participation of prostaglandins in the febrile response induced by the i.p. injected zymosan. The animals received zymosan (3 mg/kg, i.p.) that was suspended in sterile saline or the same volume of vehicle and after 2.5 h, they received the nonselective COX inhibitor indomethacin (2 mg/kg, i.p.) or the same volume of vehicle (Tris-HCl buffer, 0.2 M). In another experiment, the animals received the nonselective COX inhibitor indomethacin (2 mg/kg, i.p.), the selective COX-2 inhibitor celecoxib (5 mg/kg, v.o.), or the same volume of vehicle (Tris-HCl buffer, 0.2 M). After 30 min, the animals received zymosan (3 mg/kg, i.p.) suspended in sterile saline or the same volume of vehicle. The doses of indomethacin, celecoxib, and zymosan were based on previous works , , . The core body temperature was measured for 6 h. In a different group of animals, the same protocol described above for the pre-treatment with indomethacin was used, but the experiment was interrupted 3 h after the zymosan injection to collect the cerebrospinal fluid (CSF) for PGE2 measurements (described below).
Next, we evaluated the participation of substance P, ET-1, and eOPs in the zymosan-induced febrile response. The animals intracerebroventricularly (i.c.v.) received the NK1 receptor antagonist SR140333B (3 μg/site), ETB receptor antagonist BQ788 (0.7 μg/site), or MOR antagonist CTAP (1 μg/site) in a volume of 2 μL or the same volume of sterile saline. The effective doses of antagonists were based on previous studies using LPS and polyinosinic: polycytidylic acid . After 30 min, the animals received zymosan (3 mg/kg, i.p.).
Previous studies have shown that the i.a. injected zymosan induces a febrile response that is not blocked by a ETB receptor antagonist . To clarify the specific pathways that involve ET-1 and eOPs, a separate group of animals received the same dose of i.c.v. CTAP. After 30 min, the animals received an i.a. injection of zymosan (4 mg/site, 50 μL) in the right knee joint as described previously .
Determination of PGE2 concentrations in the cerebrospinal fluid
A single sample of CSF was collected from each animal according to Consiglio and Lucion . Briefly, 3 h after the zymosan or saline injection, animals that were pre-treated with vehicle or indomethacin were anesthetized with ketamine/xylazine at the same doses described above and fixed in a stereotaxic apparatus. The cisterna magna could be easily visualized by shaving the top and back of the animal and moistening this region with a cotton swab embedded in ethanol. CSF was directly aspirated from the cisterna magna through a syringe in a volume of 50–100 μL. The CSF samples were transferred to small plastic tubes that contained 2 μL of indomethacin (2.5 mg/mL) to stop further the prostaglandin synthesis and stored in the dark under ice. The samples were then centrifuged at 1000×g at 4°C for 10 min and stored at –80°C for further analysis. The PGE2 levels in the CSF were analyzed by an enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI, USA). All the samples were assayed according to the manufacturer’s instructions.
Zymosan, indomethacin, celecoxib, BQ788, and CTAP were purchased from Sigma Aldrich (St. Louis, MO, USA). SR140333B was a kind donation from Sanovis-Aventis Laboratories. Ketamine hydrochloride was purchased from Vetnil Veterinary Products (Louveira, SP, Brazil). Xylazine hydrochloride was purchased from Syntec Laboratory (Cotia, SP, Brazil). Oxytetracycline hydrochloride was purchased from Pfizer Laboratories (São Paulo, SP, Brazil). Ketoprofen was purchased from Medley Laboratories (São Paulo, SP, Brazil).
The body temperature data were analyzed by two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s test. PGE2 levels were analyzed by one-way ANOVA followed by Bonferroni’s test. All the results were expressed as mean±SEM and were analyzed using Prism 6 software (GraphPad, San Diego, CA, USA). Values of p<0.05 were considered statistically significant.
The i.p. injected zymosan induced a febrile response that began 2 h after the injection and lasted until 5 h (Figures 1 and 2). The treatment of the animals with indomethacin 2.5 h after the injection of zymosan (at the time that the animals were already febrile) significantly reduced the duration of the fever which lasted until 3.5 h. The significative differences between the zymosan plus indomethacin-treated group and the zymosan plus vehicle-treated group were observed from 255 and 355 min, respectively (Figure 1). Additionally, the pre-treatment of the animals with indomethacin completely blocked this febrile response (Figure 2A). The zymosan-induced febrile response was followed by an increase in the levels of PGE2 in CSF that was also completely blocked by the pretreatment with indomethacin (Figure 2B). Treatment with the COX-2 selective inhibitor celecoxib also completely inhibited the febrile response by zymosan (Figure 2C). Figures 3A and B show that both the NK1 receptor antagonist SR140333B and ETB receptor antagonist BQ788 significantly inhibited the febrile response induced by zymosan. Furthermore, the zymosan-induced febrile response was significantly reduced by the MOR antagonist CTAP (Figure 4A). Intra-articular zymosan administration induced a more prolonged and intense febrile response that began approximately 1.5 h after administration and lasted until the end of the experiment (Figure 4B). Treatment with CTAP significantly reduced this response.
The present study showed that the central mediators involved in the i.p. zymosan-induced febrile response are similar to those of the LPS-induced febrile response. Zymosan is a protein-carbohydrate complex that is extracted from the cell wall of the yeast Saccharomyces cerevisae. Zymosan is commonly used to induce experimental inflammation, and can induce signs of local inflammation, such as neutrophil migration , hyperalgesia , and edema . It also induces systemic effects, such as sickness behavior  and fever , . Zymosan was originally described as a Toll-like receptor 2 agonist , but it also binds to the pattern-recognition receptor dectin-1, a C-like lectin receptor . We previously showed that the increase in body temperature induced by the i.p. injected zymosan, similar to the i.a. injected zymosan, involves an integrated febrile response, in which the increase in the core body temperature is preceded by a decrease in tail skin temperature .
Bastos-Pereira found that IL-1β, IL-6, and TNF-α participate in the febrile response induced by the i.p. injected zymosan . These cytokines are known to induce fever through the induction of COX-2 in the brain microvasculature , , . The participation of eicosanoids in the febrile response induced by zymosan was also investigated. The zymosan administration significantly increased PGE2 levels in the CSF. Moreover, the nonselective COX inhibitor indomethacin, before or after the induction of fever, significantly reduced the febrile response and abolished the increase in PGE2 levels in the brain. A similar reduction of the febrile response was observed with the treatment using the selective COX-2 inhibitor celecoxib. These results suggest that zymosan-induced fever depends on prostaglandins that are produced via COX-2, thus corroborating previous findings reported by Kanashiro et al.  using an i.a. injection of zymosan.
In a recent study, we demonstrated that TNF-α, IL-6, and prostaglandins in autocrine circuitry, together with substance P, participate in fever induction . The generation of PGE2 in the central nervous system during fever can initiate the release of substance P, which in turn generates more prostaglandins. Therefore, we evaluated the participation of substance P in the zymosan-induced febrile response. Treatment with the NK1 antagonist SR140333B reduced the febrile response induced by zymosan, suggesting that this neuropeptide participates in this response. In models of acute visceral inflammation and nociception induced by zymosan, the internalization of NK1 receptors has been observed in neurons in lamina I and sympathetic preganglionic neurons, indicating that the release of substance P and activation of NK1 receptors are induced by this stimulus , . These results support the hypothesis that zymosan induces substance P release from neurons.
We also analyzed ET-1, which has been shown to promote fever through ETB receptor activation . An effective treatment with the ETB receptor antagonist BQ788 significantly reduced the febrile induced by the i.p. injected zymosan. In contrast, Kanashiro et al.  reported that BQ788 did not reduce the febrile response induced by i.a. zymosan. The inflammatory process is different in the joints and other sites of the body. For example, Griffiths et al. compared the effectiveness of 5-lipooxygenase inhibitors against zymosan-induced inflammation in the rat knee joint and peritoneal cavity . An i.p. injection of zymosan induced a single initial peak of eicosanoid production, whereas the i.a. injection of this polysaccharide led to two different peaks: one initial peak and a later peak associated with leukotriene and prostaglandin production. The initial phase of higher permeability in the joint and peritoneal cavity was also differentially affected by leukotriene inhibitors. These results demonstrate that different mediators are involved in the inflammatory reaction that is induced by zymosan in the peritoneum and knee joint. Previous studies have also shown that the antibodies against adhesion molecules do not affect zymosan-induced inflammation in joints but reduced zymosan-induced peritonitis, suggesting that the characteristics of inflammation are site-specific . It has been demonstrated that, specifically for the febrile response, the route of administration may have an important impact on the kind of mediators generated and their access to the central nervous system . This may also be caused by the access of pyrogens to the cells bearing the specific Toll-like receptor involved. Voss et al.  demonstrated that the administration of synthetic RNA into the artificial subcutaneously implanted Teflon chambers, in contrast to LPS, had no pyrogenic activity or cytokine-inducing effects, whereas the i.p. or intra-arterial, and intramuscular injection caused a pronounced or moderated fever and cytokine induction, respectively. In the model of joint inflammation (localized inflammatory response) this might not be the case. The induction of the ET-1 synthesis by zymosan has been previously reported. Zymosan increased the levels of prepro-ET1 mRNA in rat knee joints . The ETA and ETB receptor antagonist administration reduced edema formation, neutrophil influx, and TNF-α production in rat knee joints . These results suggest that zymosan can induce the production of ET-1, at least locally. However, the central, rather than peripheral, production of ET-1 seems to be important for the febrile response . In contrast with the i.a injection, the i.p. injection of zymosan might result in sufficient amounts of this pyrogen in the circulation; hence, the fever-related brain sites with an incomplete blood-brain barrier can be directly activated by this pyrogen  to produce ET-1 in the central nervous system. These studies and the present results suggest that different inflammatory sites can produce different mediators of inflammation, which may lead to varying inflammatory and febrile responses.
Notably, ET-1 also participates in the febrile response induced by the i.p. injected zymosan (present study) and LPS  but not polyinosinic:polycytidylic acid , suggesting that ET-1 participates in the febrile response induced by bacteria and fungus but not viruses.
Endogenous opioids also participate in the zymosan-induced febrile response. An i.c.v. injection of the MOR antagonist CTAP significantly reduced the febrile response induced by the i.p. injected zymosan. We previously found that eOPs participate in the febrile response that is induced by ET-1 and CRF but not prostaglandins . Kanashiro et al.  reported that ET-1 and CRF do not participate in the febrile response induced by i.a. zymosan. In the present study, we evaluated whether eOPs are involved in the febrile response induced by i.a. zymosan. Surprisingly, the febrile response induced by i.a. zymosan, similar to i.p. administration, was reduced by the MOR antagonist CTAP. The CRF/ET-1 pathway is not involved in i.a. zymosan-induced fever , indicating that the eOPs are not released through the CRF or ET-1 during the febrile response; instead, they appear to be released through cytokines, such as TNF-α and IL-6 , .
The present results indicate that centrally produced prostaglandins, substance P, ET-1, and eOPs participate in the febrile response induced by an i.p. injection of zymosan. Although ET-1 does not appear to participate in the febrile response observed after the zymosan-induced knee joint inflammation, it appears to participate in the zymosan-induced fever after peritonitis.
This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (Grant No. 473873/2011-7). D. Fraga had a postdoctoral scholarship from REUNI, UFPR.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
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