Expression of hypothalamic feeding-related peptide genes and neuroendocrine responses in an experimental allergic encephalomyelitis rat model.

Experimental allergic encephalomyelitis (EAE) is considered to be a useful animal model of human multiple sclerosis (MS). However, among the various symptoms of MS, the mechanisms contributing to inflammatory anorexia remain unclear. In the present study, we used an EAE rat model to examine changes in expression levels of hypothalamic feeding-related peptide genes and neuroendocrine responses such as the hypothalamo-neurohypophysial system and the hypothalamo-pituitary-adrenal (HPA) axis. The weight gain and cumulative food intake in EAE rats in the early days after immunization was significantly lower than that of the control group. The expression of orexigenic peptide genes Npy and Agrp were significantly increased, whereas the levels of anorectic peptide genes (Pomc and Cart) were significantly decreased in the hypothalamus of EAE rats. There was also a significant increase in the mRNA and plasma oxytocin (OXT) but not of arginine vasopressin (AVP) in the supraoptic and paraventricular nuclei (PVN) of EAE rats at days 12 and 18 after immunization. The expression of corticotropin-releasing hormone (Crh) and Avp was downregulated and upregulated, respectively, in the parvocellular division of the PVN at day 12 after immunization. The expression level of Pomc in the anterior pituitary significantly increased, accompanied by increased plasma corticosterone levels, at days 6, 12, and 18 after immunization. These results suggest that inflammatory anorexia in rat EAE may be caused by activation of the OXT-ergic pathway and HPA axis via changes in the expression of hypothalamic feeding-related peptides, including Avp but not Crh.

Effects of acute kidney dysfunction on hypothalamic arginine vasopressin synthesis in transgenic rats.

Acute loss of kidney function is a critical internal stressor. Arginine vasopressin (AVP) present in the parvocellular division of the paraventricular nucleus (PVN) plays a key role in the regulation of stress responses. However, hypothalamic AVP dynamics during acute kidney dysfunction remain unclear. In this study, we investigated the effects of bilateral nephrectomy on AVP, using a transgenic rat line that expressed the AVP-enhanced green fluorescent protein (eGFP). The eGFP fluorescent intensities in the PVN were dramatically increased after bilateral nephrectomy. The mRNA levels of eGFP, AVP, and corticotrophin-releasing hormone in the PVN were dramatically increased after bilateral nephrectomy. Bilateral nephrectomy also increased the levels of Fos-like immunoreactive cells in brainstem neurons. These results indicate that bilateral nephrectomy upregulates the AVP-eGFP synthesis. Further studies are needed to identify the neural and/or humoral factors that activate AVP synthesis and regulate neuronal circuits during acute kidney dysfunction.

Centrally administered kisspeptin suppresses feeding via nesfatin-1 and oxytocin in male rats.

Kisspeptin (KP), known as a hypothalamic neuropeptide, plays a critical role in the regulation of not only reproduction but also food intake. The anorectic neuropeptides, nesfatin-1 and oxytocin (OXT), are expressed in central nervous system, particulaly in various hypothalamic nuclei, and peripheral tissue. We examined the effects of the intracerebroventricular (icv) administration of KP-10 on feeding and nesfatin-1-immunoreactive (ir) or OXT-ir neurons in the rat hypothalamus, using Fos double immunohistochemistry in male rats. Cumulative food intake was remarkably decreased 0.5-3 h after icv administration of KP-10 (6.0 µg) compared to the vehicle treated and the KP-10 (3.8 µg) treated group. The icv administration of KP-10 significantly increased the number of nesfatin-1-ir neurons expressing Fos in the supraoptic nucleus (SON), paraventricular nucleus (PVN), arcuate nucleus (ARC), dorsal raphe nucleus, locus coeruleus, and nucleus tractus solitarius. The decreased food intake induced by KP-10 was significantly attenuated by pretreatment with the icv administration of antisense RNA against nucleobindin-2. After icv administration of KP-10, the percentages of OXT-ir neurons expressing FOS were remarkably higher in the SON and PVN than for vehicle treatment. The KP-10-induced anorexia was partially abolished by pretreatment with OXT receptor antagonist (OXTR-A). The percentage of nesfatin-1-ir neurons expressing Fos-ir in the ARC was also decreased by OXTR-A pretreatment. These results indicate that central administration of KP-10 activates nesfatin-1- and OXT neurons, and may play an important role in the suppression of feeding in male rats.

Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs.

The effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in sham-operated (Sham) and vestibular-lesioned (VL) mice were examined by in situ hybridization histochemistry. Corticotrophin-releasing hormone (CRH) in the paraventricular nucleus was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2 g environment compared with a 1 g environment. Significant decreases in pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript and significant increases in neuropeptide Y, agouti-related protein in the arcuate nucleus and orexin in the lateral hypothalamic area were observed in both Sham and VL mice. After 2 weeks of exposure, CRH and POMC were increased significantly in Sham but not in VL mice. After 8 weeks of exposure, the hypothalamic feeding-related neuropeptides were comparable between Sham and VL mice. These results suggest that the hypothalamic feeding-related neuropeptides may be affected during the exposed duration of hypergravity via vestibular inputs.

Involvement of central nesfatin-1 neurons on oxytocin-induced feeding suppression in rats.

Peripheral anorectic hormones, such as peptide YY (PYY) and oxytocin (OXT), suppress food intake. A newly identified anorectic neuropeptide, nesfatin-1, is synthesized in both peripheral tissue and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. Here, we examined the effects of intraperitoneal (ip) administration of PYY3-36, OXT, and OXT analog, on nesfatin-1-immunoreactive (ir) neurons in the rat hypothalamus and brainstem, using Fos double fluorescence-immunohistochemistry. The ip administration of OXT and OXT analog significantly increased the number of nesfatin-1-ir neurons expressing Fos-ir in the paraventricular nucleus, the arcuate nucleus, and the nucleus tractus solitarius, but not in the supraoptic nucleus, the lateral hypothalamic area, and the area postrema. No differences in the percentage of nesfatin-1-ir neurons expressing Fos in the nuclei of the hypothalamus and brainstem were observed, between rats treated with vehicle or those treated with PYY3-36. The decreased food intake, induced by OXT and OXT analog, was attenuated significantly by pretreatment with intracerebroventricular administration of antisense nesfatin-1. These results suggested that nesfatin-1-expressing neurons in the hypothalamus and brainstem may play a role in sensing the peripheral level of OXT and its suppression of feeding in rats.

Comparison of the induction of c-fos-eGFP and Fos protein in the rat spinal cord and hypothalamus resulting from subcutaneous capsaicin or formalin injection.

We evaluated whether a c-fos-enhanced green fluorescent protein (eGFP) transgenic rat line, which expresses the c-fos and eGFP fusion gene, can be useful for the study of nociceptive pathways and processing. Capsaicin solution (15%) or formalin (5%) was subcutaneously injected bilaterally into the hind paws (100µL per each paw) of adult male c-fos-eGFP transgenic or wild-type rats. Control rats were injected with ethanol or physiological saline respectively. Transgenic and wild-type rats were perfused at 1.5, 3 and 6h post injection, with some transgenic rats being perfused 24h post injection. A comparison of eGFP in transgenic rats and Fos-like immunoreactivity (LI) in wild-type rats was made in the dorsal spinal cord, paraventricular nucleus (PVN) and supraoptic nucleus (SON). Oxytocin-LI (OXT-LI) was carried out to examine the activation of OXT neurons in the PVN and SON. Following capsaicin or formalin treatment, eGFP was maximally expressed at 6h in the spinal cord and 3h in the PVN and SON, whereas, Fos-LI was maximally expressed at 1.5h in all the regions we analyzed. Induction of eGFP in the OXT neurons was observed after capsaicin or formalin treatment, while Fos-LI in the OXT neurons was observed only after formalin treatment. These results demonstrate that the peak induction of c-fos-eGFP following exposure to acute nociceptive stimuli was delayed by around 1.5-4.5h, but more sensitive than endogenous Fos, suggesting that the c-fos-eGFP rat line can be useful for the study of nociceptive pathways and processing.

Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50µg/kg) and secretin (100µg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

Reduced effectiveness of oseltamivir in children infected with oseltamivir-resistant influenza A (H1N1) viruses with His275Tyr mutation.

BACKGROUND: Little is known about whether neuraminidase inhibitors are effective for children infected with oseltamivir-resistant influenza A(H1N1) viruses. METHODS: Children aged 15 years and younger having influenza-like illness and who visited outpatient clinics within 48 hours of fever onset were enrolled from 2006-2007 to 2008-2009 influenza seasons in Japan. Patients received oseltamivir, zanamivir, or no treatment after screening by a rapid antigen test. Nasopharyngeal swabs were collected before antiviral therapy and were used for virus isolation. Oseltamivir resistance was determined by detection of the H275Y mutation in neuraminidase, and susceptibility test using neuraminidase inhibition assay. Daily body temperature was evaluated according to drug type and susceptibility by univariate and multivariate analyses. RESULTS: Of 1647 patients screened, 238 oseltamivir-resistant H1N1 cases (87 oseltamivir-treated, 64 zanamivir-treated, and 87 nontreated) and 110 oseltamivir-susceptible cases (60 oseltamivir-treated and 50 nontreated) were evaluated. In oseltamivir-resistant cases, fever on days 4 to 5 after the start of treatment was significantly higher in oseltamivir-treated and nontreated than in zanamivir-treated patients (P < 0.05). In oseltamivir-susceptible cases, fever was significantly lower in oseltamivir-treated than nontreated on days 3 to 6 (P < 0.01). Similar findings were obtained for duration of the fever and proportion of recurrent fever. Reduced effectiveness of oseltamivir was more prominent in children 0 to 6 years old than in those 7 to 15 years old. Multiple logistic regression analysis showed that lower age, nontreatment, and oseltamivir treatment of oseltamivir-resistant patients were factors associated with the duration of the longer fever. CONCLUSIONS: Infection with oseltamivir-resistant viruses significantly reduced the effectiveness of oseltamivir, and this tendency was more apparent in younger children.