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Involvement of Cytokine-Induced Neutrophil Chemoattractant in Hypothalamic Thermoregulation of Luteinizing Hormone-Releasing Hormone

Tsumura Research Laboratory (M.N., M.Y., Y.K.), Ibaraki 300-1192, Japan; and Department of Obstetrics and Gynecology (T.Y., M.I.), Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan

Address all correspondence and requests for reprints to: Masamichi Noguchi, Tsumura Research Laboratory, 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan. E-mail: noguchi_masamichi{at}mail.tsumura.co.jp.

	Abstract

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We demonstrated in a previous study that serum IL-8 concentrations were significantly higher in women with hot flashes than without hot flashes. To clarify the role of IL-8 in the pathoetiology of menopausal hot flashes, we examined the effect of rat cytokine-induced neutrophil chemoattractant (CINC), a member of the IL-8 family, on thermoregulation using ovariectomized (OVX) rats treated with intracerebroventricular (i.c.v.) injection of LHRH agonist (LHRHa) as a model of hot flashes. We found that: 1) expression of CINC mRNA was increased around the periventricular area in the hypothalamus at 1 h, and the serum CINC concentration was increased at 2 h after i.c.v. injection of LHRHa; 2) the increase in serum CINC concentration in hypophysectomized rats was significantly lower than that in sham-operated rats; 3) i.c.v. but not iv injection of CINC elevated the rectal temperature of OVX rats; 4) i.c.v. injection of LHRHa into OVX rats produced a rapid rise (maximal increase: 10–25 min) in tail skin temperature, and the elevation was augmented by injection of an anti-CINC antibody; and 5) changes in serum CINC concentration and skin temperature after i.c.v. injection of LHRHa were reversed by replacement of estradiol. In conclusion, the production of CINC in the hypothalamus due to LHRHa injection in OVX rats was increased after elevation of skin temperature, suggesting that CINC plays a key role in the homeostasis of body temperature. Disturbance of the thermoregulatory mechanism involving LHRH and CINC may be related to the pathoetiology of hot flashes.

	Introduction

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A HOT FLASH, which often occurs in menopausal women due to estrogen withdrawal, generally begins with a sudden outpouring of sweat and an increase in heart rate and peripheral blood flow, causing a drastic increase in skin temperature (1). Because hot flashes clearly accompany estrogen withdrawal, estrogen replacement therapy is suggested to be effective for menopausal hot flashes (2). However, the mechanism underlying hot flashes has not been elucidated. It has been reported that analysis of serial blood samples in patients with frequent hot flashes revealed that the subjective onset of the hot flash corresponded to the nadir of the LH pulses and the surge in LHRH release from the hypothalamus (2, 3). We also found that intracerebroventricular (i.c.v.) but not iv injection of an LHRH agonist (LHRHa) elevated the tail skin temperature of conscious rats (4). These reports suggest that LHRH acts as a central temperature modulator and plays a key role in the occurrence of hot flashes.

Recently we demonstrated that serum IL-8 concentrations in pre-, peri-, and postmenopausal women and bilaterally oophorectomized women with hot flashes were significantly higher than in women without hot flashes (5). It is well known that IL-8 plays an important role in inflammatory events such as neutrophil activation and neutrophil chemotaxis (6, 7). The rat orthologs of the human IL-8 receptor has been identified (8), and the functions of IL-8 appear to be performed by cytokine-induced neutrophil chemoattractant (CINC) in rodents (9). There is considerable evidence that CINC is produced in the central nervous system (10, 11), although CINC was originally identified as an essential mediator in the inflammatory system. In addition, it has been shown that CINC stimulates the secretion of prolactin, GH, and ACTH but suppresses the secretion of LH and FSH from rat anterior pituitary cells (12). These reports suggest that CINC may be involved in regulating the hypothalamus-pituitary-adrenal (HPA) axis, contributing to the neuroendocrine interface. Therefore, there are important interactions between the immune and neuroendocrine systems that may explain the pathophysiology of menopausal hot flashes. Although it has been shown that i.c.v. injection of IL-8 induced fever by a mechanism independent of prostaglandins (13), the role of IL-8 on the central nervous system in menopausal hot flashes has not been completely elucidated.

To clarify the role of IL-8 (CINC in rats) in the pathoetiology of menopausal hot flashes, we examined the effects of changes in the serum level, source, and role of CINC on hypothalamic thermoregulation of LHRH after i.c.v. injection of LHRHa in bilaterally ovariectomized (OVX) rats as a model of hot flashes. In addition, the effect of estrogen replacement was examined in this model.

	Materials and Methods

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Animals
Ten-week-old female Sprague Dawley rats weighing 200–250 g were purchased from Charles River Laboratories (Yokohama, Japan). The animals were allowed free access to water and standard laboratory food and housed in stainless steel cages at a temperature of 23 ± 2 C, relative humidity of 55 ± 10%, and a 12-h light, 12-h dark cycle, with lights on from 0700 to 1900 h daily.

All experimental procedures were performed according to the guidelines for the care and use of laboratory animals approved by the Laboratory Animal Committee of Tsumura & Co.

Drugs and reagents
An LHRH agonist [des-gly¹⁰-(im-BZl-D-His⁶)LHRH ethylamide], urethane, and -chloralose were purchased from Sigma Chemical (St. Louis, MO). CINC-1 was purchased from Peptide Institute (Osaka, Japan). Antirat CINC-1 antibody was purchased from R & D Systems (AF-515-NA; Minneapolis, MN). 17β-Estradiol was purchased from Wako Pure Chemical Industries (Osaka, Japan). Other reagents used for analysis were the highest purity commercially available.

Surgical procedures
Ovariectomy.
All rats were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg) and bilaterally ovariectomized. All ovariectomized rats were used 3 wk after surgery. We used a surgical technique for ovariectomy that was established for monitoring the decrease in plasma estradiol concentration (14).

Adrenalectomy.
Adrenalectomies were performed under sodium pentobarbital anesthesia via a dorsolateral approach. Adrenalectomized rats were given immediate access to 0.9% saline and were allowed to recover for more than 7 d before testing.

Hypophysectomy.
Hypophysectomies were performed by Charles River Laboratories under sodium pentobarbital anesthesia via a transsphenoidal approach. Hypophysectomized rats were allowed to recover for more than 10 d before testing. Hypophysectomized rats were decapitated after completion of the experiments, and the complete removal of the pituitary gland was verified visually.

Implantation of i.c.v. cannula.
Rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) and placed on a stereotaxic frame. A stainless steel guide cannula (AG-8; Eicom, Kyoto, Japan) was implanted into the right lateral ventricle (coordinates: 0.8 mm posterior and 1.4 mm right lateral from the bregma and 3.4 mm ventral from the skull surface) according to a rat brain atlas (15). The rats were allowed to recover for more than 5 d before the experiments.

Measurements of serum CINC concentration
Rats were anesthetized with diethyl ether, and then blood (approximately 6 ml) was collected from the abdominal aorta in a polypropylene tube. The blood was centrifuged at 1500 x g and 4 C for 15 min, and the serum was stored at –80 C until CINC assay. Serum CINC concentration were measured using a commercial CINC-1 ELISA kit (Panapharm Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. The lowest level of detection for CINC was 12.5 pg/ml. The intraassay coefficient of variation was 4.3%, and the interassay coefficient of variation was 5.2%.

CINC mRNA expression assay using real-time RT-PCR
Rats were decapitated, and the hypothalamus was removed. Total RNA was extracted from isolated hypothalamus using an RNeasy lipid tissue minikit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. In brief, tissue samples were homogenized in QIAzol lysis reagent. After addition of chloroform, the homogenate was separated into aqueous and organic phases by centrifugation (12000 x g, 2 min, 4 C). The upper, aqueous phase was extracted, and ethanol was added to provide appropriate binding conditions. The sample was then applied to the RNeasy minispin column, in which the total RNA binds to the membrane, and phenol and other contaminants were efficiently washed away. Total RNA was eluted in 50 µl of RNase-free water. The quantity and purity of the RNA were determined by measuring absorbance at 260 and 280 nm by UV-Vis spectroscopy. The integrity of the RNA was analyzed using an Agilent 2100 bioanalyzer and a Eukaryote total RNA nano kit (Agilent Technologies, Palo Alto, CA). Quantitative gene expression analysis was performed by real-time RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Real-time RT-PCR was performed in a two-step manner. cDNA synthesis and real-time detection were carried out in a PCR Thermal Cycler SP (Takara Bio, Shiga, Japan) and an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA), respectively. TaqMan reverse transcription reagent (Applied Biosystems) was used to generate cDNA from 1 µg of total RNA, as described in Applied Biosystems user bulletin no. 2. A TaqMan PCR core reagent kit (Applied Biosystems) and Assays-on-Demand gene expression probes [CINC (CXCL1): Rn00578225_m1; GAPDH: Rn99999916_s1; Applied Biosystems] were used in subsequent PCRs according to the manufacturer’s protocols. Relative quantitation of gene expression was performed using the relative standard curve method. All real-time RT-PCRs were performed in triplicate.

In situ hybridization
Paraffin-embedded tissue blocks and sections of rat brain for in situ hybridization were obtained from Genostaff Co., Ltd. (Tokyo, Japan). The rat brains were dissected after perfusion, fixed with Tissue Fixative (Genostaff), and then embedded in paraffin by their proprietary procedures and sectioned at 6 µm.

For in situ hybridization, tissue sections were dewaxed with xylene and rehydrated through an ethanol series and PBS. The sections were fixed in 4% paraformaldehyde in PBS for 15 min and then washed with PBS. The sections were treated with 3 µg/ml proteinase K in PBS for 30 min at 37 C, washed with PBS, refixed with 4% paraformaldehyde in PBS, again washed with PBS, and placed in 0.2 N HCl for 10 min. After washing with PBS, the sections were acetylated by incubation in 0.1 M triethanolamine-HCl (pH 8.0) in 0.25% acetic anhydride for 10 min. After washing with PBS, the sections were dehydrated through a series of ethanols.

The cDNA template for CINC was a 387-bp fragment corresponding to bases 20–406 of the rat chemokine (C-X-C motif) ligand 1 cDNA (GenBank accession no. NM_030845). Sense and antisense cRNA probes for CINC mRNA were synthesized using a digoxigenin RNA labeling kit (Roche, Stockholm, Switzerland) according to the manufacturer’s protocol. Hybridization was performed with probes at concentrations of 300 ng/ml in the probe diluent (Genostaff) at 60 C for 16 h. After hybridization, the sections were washed in 5x HybriWash (Genostaff), equal to 5x saline sodium citrate, at 60 C for 20 min and then in 50% formamide, 2x HybriWash at 60 C for 20 min, followed by RNase treatment in 50 µg/ml RNase A in 10 mM Tris-HCl (pH 8.0), 1 M NaCl, and 1 mM EDTA for 30 min at 37 C. Then the sections were washed twice with 2x HybriWash at 60 C for 20 min, twice with 0.2x HybriWash at 60 C for 20 min, and once with 0.1% Tween 20 in Tris-buffered saline (TBST). After treatment with 0.5% blocking reagent (Roche) in TBST for 30 min, the sections were incubated with antidigoxigenin AP conjugate (Roche) diluted 1:1000 with TBST for 2 h at room temperature. The sections were washed twice with TBST and then incubated in 100 mM NaCl, 50 mM MgCl₂, 0.1% Tween 20, and 100 mM Tris-HCl (pH 9.5). Coloring reactions were performed with 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt solution (Sigma) overnight, and then sections were washed with PBS. The sections were counterstained with Kernechtrot red solution (Mutoh, Hokkaido, Japan), dehydrated, and then mounted with Malinol (Mutoh).

Measurement of changes in body temperature
Measurements of skin temperature and rectal temperature were carried out according to a procedure described previously (4). In brief, each rat was restrained in a Ballman’s cage. Two thermistor probes (SXN-54; Technol Seven Co., Yokohama, Japan) were used: one thermistor probe was taped to the dorsal surface of the tail about 3 cm from its base, and the other thermistor probe was inserted 4 cm into the rectum. Forty minutes later, the mean temperature was automatically measured at 10-min intervals throughout the experiment. Data were recorded by a K932 recording device (Technol Seven). The area under the curve (AUC) of body temperature was calculated using Pharmacokinetic Analysis and Graphics for Clinical Pharmacology (Medical Research AS Medica, Osaka, Japan).

In studies of the effects of CINC on body temperature changes, the dosage of CINC was based on previous studies (13, 16). CINC dissolved in saline was centrally (50, 250 ng/10 µl/head, i.c.v.) or peripherally (0.4, 2 µg/kg, iv) injected after the basal temperature was stable.

In another set of experiments, the effects of antirat CINC antibody on LHRHa-induced body temperature changes were examined by comparing the LHRHa and LHRHa + antirat CINC groups. The LHRHa group and the antirat CINC antibody group were injected with LHRHa (10 µg/10 µl/head, i.c.v.) and anti-CINC antibody (1 µg/10 µl/head, i.c.v.), respectively, dissolved in saline after the basal temperature was stable. For the LHRHa + antirat CINC group, after the anti-CINC antibody and LHRHa were dissolved in saline, the anti-CINC antibody (1 µg/5 µl) and LHRHa (10 µg/5 µl) were mixed. The mixture (10 µl) was injected into i.c.v. of rats simultaneously.

Body temperatures were measured between 1100 and 1530 h in all experiments to limit fluctuation of the basal levels of body temperature.

Effects of estradiol replacement on skin temperature and serum CINC concentration after i.c.v. injection of LHRHa
Olive oil (1 ml/kg, sc) or 17β-estradiol (10 µg/ml/kg, sc) dissolved in olive oil was administered for 7 d (once a day) from the second week after ovariectomy. Measurement of the tail skin temperature was carried out on the day after the final administration according to a procedure described in Measurement of changes in body temperature. The tail skin temperature was measured for 90 min after i.c.v. injection of saline or LHRHa. The AUC of tail skin temperature was calculated using Pharmacokinetic Analysis and Graphics for Clinical Pharmacology (Medical Research AS Medica).

In another set of experiments, olive oil (1 ml/kg, sc) or 17β-estradiol (10 µg/ml/kg, sc) was administered to ovariectomized rats by the same schedule. On the day after the final administration, blood was collected at 2 h after i.c.v. injection of saline or LHRHa. The serum CINC concentration was measured according to the procedure described in Measurements of serum CINC concentration.

Statistical analysis
All values were represented as the mean ± SEM. The statistical significance was evaluated by one-way ANOVA followed by Dunnett’s test or Student’s t test. For all tests, the significance level was accepted at P < 0.05.

	Results

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Serum CINC concentration for 24 h after i.c.v. injection of LHRHa
We assessed serum levels of CINC at 0, 2, 4, 8, and 24 h after i.c.v. injection of LHRHa (10 µg per 10 µl/head) in OVX rats. As can be seen in Fig. 1, the serum CINC concentration was significantly (P < 0.01) increased at 2 h after i.c.v. injection of LHRHa in OVX rats. The elevated CINC level was recovered to the basal level by 8 h.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Serum CINC concentration for 24 h after i.c.v. injection of LHRHa (10 µg/head) in OVX rat. Each value is expressed as the mean ± SEM (n = 8). Significance with post hoc Dunnett’s t test after a one-way ANOVA is indicated: **, P < 0.01 vs. before LHRHa injection (0 h).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Effects of adrenalectomy and hypophysectomy on serum CINC concentration after i.c.v. injection of LHRHa (10 µg/head). Each value is expressed as the mean ± SEM. Significance by Student’s t test is indicated: **, P < 0.001 vs. hypophysectomized rats. ADX, Adrenalectomized rats; HPX, hypophysectomized rats; NS, not significant.

View larger version (35K): [in this window] [in a new window]   FIG. 3. CINC mRNA expression in the hypothalamus at 1 h after i.c.v. injection of LHRHa (10 µg/head) by in situ hybridization histochemistry. A, Coronal diagram of the rat brain (posterior 0.8 mm from the bregma). The section of hypothalamus was hybridized with antisense (B) and sense (C) probes. AH, Anterior hypothalamus; 3V, third ventricle.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effects of i.c.v. (A) or iv (B) injection of CINC on tail skin temperature and rectal temperature in OVX rats. Each value is expressed as the mean ± SEM (n = 6–7). Time-dependent changes in the skin temperature and rectal temperatures after i.c.v. or iv injection of CINC were evaluated by post hoc Dunnett’s t test after a one-way ANOVA. The significances are indicated: #, P < 0.05; ##, P < 0.01 vs. before CINC (50 ng/head) injection (0 min); *, P < 0.05; **, P < 0.01 vs. before CINC (250 ng/head) injection (0 min).

View larger version (21K): [in this window] [in a new window]   FIG. 5. Effects of anti-CINC antibody (1 µg/head) on tail skin temperature (A) and rectal temperature (B) after i.c.v. injection of LHRHa (10 µg/head) in OVX rats. C, AUC data after i.c.v. injection of LHRHa in anti-CINC antibody treatment. Each value is expressed as the mean ± SEM (n = 11–12). Time-dependent changes in the skin and rectal temperatures after i.c.v. injection of LHRHa were evaluated by post hoc Dunnett’s t test after a one-way ANOVA. The significances are indicated: **, P < 0.01 vs. before LHRHa injection (0 h); #, P < 0.05; ##, P < 0.01 vs. before LHRHa and anti-CINC antibody injection (0 h). In the AUC data, significance with Student’s t test is indicated: *, P < 0.05 vs. LHRHa-alone group. NS, Not significant.

View larger version (11K): [in this window] [in a new window]   FIG. 6. Effects of estradiol replacement on the skin temperature (A) and serum CINC concentration (B) after i.c.v. injection of LHRHa (10 µg/head) in OVX rats. Olive oil (1 ml/kg) or 17β-estradiol (10 µg/ml·kg) dissolved in olive oil was administered sc once a day for 7 d from the second week after ovariectomy. Measurement of tail skin temperature was carried out on the day after the final administration. The tail skin temperature was measured for 90 min after i.c.v. injection of saline or LHRHa. The AUC of tail skin temperature was calculated, and the AUC of the olive oil/LHRHa group is shown as 100%. In A, significance according to Student’s t test is indicated: ***, P < 0.001 vs. olive oil/saline group; #, P < 0.05 vs. olive oil/LHRHa group. In another set of experiments, olive oil (1 ml/kg) or 17β-estradiol (10 µg/ml·kg) was administered sc to OVX rats by the same schedule. On the day after the final administration, the serum CINC concentration was measured at 2 h after i.c.v. injection of saline or LHRHa. In B, significance with Student’s t test is indicated: **, P < 0.01 vs. olive oil/saline group; ##, P < 0.01 vs. olive oil/LHRHa group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The immune system is regulated in part by the central nervous system, acting principally via the HPA axis (17). To clarify the source of the increase in serum CINC, we examined serum CINC concentrations in OVX rats after adrenalectomy or hypophysectomy. The increase in serum CINC concentration induced by administering LHRHa was reduced about 66% by hypophysectomy but not adrenalectomy, suggesting that CINC was partly released into the circulation from the pituitary after central LHRHa injection. However, part of the increase in the serum CINC concentration was not reduced by hypophysectomy. It has been suggested that two major pathway systems are involved in the cross talk between the brain and the immune system: the HPA axis and the autonomic nervous system (18). Because i.c.v. injection of LHRHa induced elevation of skin temperature in addition to hypothalamic CINC production, LHRH might stimulate the autonomic nervous system, directly or indirectly. Therefore, part of the increase in serum CINC level may be due to a release of CINC from peripheral sources through the autonomic nervous system. On the other hand, although serum CINC concentrations before LHRHa injection were increased by adrenalectomy, the percentage increase in serum CINC concentration induced by LHRHa did not differ between sham-operated and adrenalectomized rats. This suggests that the increase in the serum CINC concentration induced by administering LHRHa is not produced by the adrenal gland. We speculate that the increase in the serum CINC concentration before LHRHa injection induced by adrenalectomy may be due to a lack of glucocorticoids because it has been reported that glucocorticoids inhibited the production of CINC in vitro (19).

To assess the contribution of CINC in the hypothalamo-hypophysial system, we examined the expression of CINC mRNA in the hypothalamus using quantitative RT-PCR and in situ hybridization. We showed that the CINC mRNA level was increased approximately 130-fold at 1 h after LHRHa injection, compared with the CINC mRNA level before LHRHa injection. This result suggests that CINC synthesis is activated by LHRH in the hypothalamus and that an LHRH-induced chemokinergic pathway exists in the hypothalamo-hypophysial system. In addition, we observed that the expression of CINC mRNA was highest around the periventricular area, which contains the paraventricular nucleus (PVN) of the hypothalamus. It has been suggested that the PVN of the hypothalamus is a site of integration of autonomic and endocrine cardiovascular responses (20). Indeed, the expression of CINC mRNA in the PVN has been shown to be increased by immobilization stress (11) and osmotic stimulation (21). It has been reported that the PVN and preoptic area (POA) communicate bidirectionally in the hypothalamus (22, 23). The POA is well known as the thermoregulatory center (24), and it regulates the synthesis of LHRH (25). In addition, an immunoreactive study has revealed the existence of lamprey GnRH-like neurons and mammalian LHRH neurons in the POA of the rat (26). Therefore, CINC produced in the PVN may be associated with hypothalamic thermoregulation of LHRH.

To clarify the significance of the increase in CINC production, we focused on the effects of CINC on thermoregulation. Zampronio et al. (13) reported that i.c.v. but not iv injection of IL-8, which exhibits a homology to rat CINC, evoked a dose-dependent increase in rectal temperature in normal male rats. In the present study, we confirmed that i.c.v. but not iv injection of CINC resulted in an increase of rectal temperature in OVX rats. Therefore, CINC or IL-8 may act on the hypothalamic thermoregulatory center. In mammals, body temperature is regulated by a neuronal system, the major controller of which is located in the hypothalamus. The regulatory mechanism has been described by Hammel’s model in which a simple synaptic network of hypothalamic neurons regulates body temperature around a set-point temperature (27). An elevation in the core temperature is an upward setting of the central hypothalamic thermostats (28). The present study demonstrated that i.c.v. injection of CINC elevated the core temperature, suggesting that CINC induces a higher setting of the central hypothalamic thermostat. In contrast, it has been suggested that LHRH induces elevation of skin temperature by lowering the setting of the central hypothalamic thermostat because microinjection of LHRHa (29) or GnRH (30) into the POA resulted in an increase in tail skin temperature. Therefore, hypothalamic thermoregulation by CINC and LHRH may be a reciprocal relationship. Our result that the elevated skin temperature induced by LHRHa was augmented by treatment with anti-CINC antibody suggests that the CINC produced in the hypothalamus may play a role in restoring the excessive skin temperature change induced by LHRH. This hypothesis is supported by the changes in skin temperature and CINC synthesis: maximal increases in skin temperature (approximately 10–25 min) induced by LHRHa preceded the maximal increase in hypothalamic CINC synthesis (1 h) and serum CINC concentration (2 h). In the present study, the anti-CINC antibody did not significantly affect rectal temperature changes induced by LHRHa. This may be attributable to the fact that LHRHa cannot decrease rectal temperature below a certain threshold under this condition.

It is thought that the thermoregulatory response to LHRH is controlled mainly by CINC produced in the hypothalamus rather than by circulating CINC because the body temperature in OVX rats was not changed by iv injection of CINC. However, it has been reported that circulating CINC may cross the blood-brain barrier (31). Therefore, there may be a feedback regulation mechanism of CINC through the hypothalamus-pituitary-gonadal and HPA axis. Although i.c.v. injection of LHRHa increased the hypothalamic CINC production and serum CINC concentration, it is uncertain whether it was caused by a direct effect of LHRHa or a secondary effect, such as a change in body temperature due to LHRHa. Further study is needed to clarify action of relationship between LHRHa and LHRH antagonist on CINC production in an in vitro study.

We showed that the skin temperature and serum CINC level after i.c.v. injection of LHRHa were reversed by estradiol replacement. Therefore, the elevated skin temperature and increase in serum CINC level are related to estrogen decline. Because the increase in serum CINC concentration followed elevation of the skin temperature, we speculate that estradiol may decrease the serum CINC concentration by attenuating skin temperature elevation in response to LHRHa.

In conclusion, the production of CINC in the hypothalamus due to LHRHa injection in OVX rats increased after the elevation of skin temperature, suggesting that CINC plays a key role in the homeostasis of body temperature. Disturbance of the thermoregulatory mechanism involving LHRH and CINC may be related to the pathoetiology of hot flashes.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2008

Abbreviations: AUC, Area under the curve; CINC, cytokine-induced neutrophil chemoattractant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPA, hypothalamus-pituitary-adrenal; i.c.v., intracerebroventricular; LHRHa, LHRH agonist; OVX, ovariectomized; POA, preoptic nucleus; PVN, paraventricular nucleus; TBST, Tween 20 in Tris-buffered saline.

Received November 6, 2007.

Accepted for publication February 27, 2008.

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