Gut microbiota composition modulates inflammation and structure of the vagal afferent pathway.

Vagal afferent neurons (VAN), located in the nodose ganglion (NG) innervate the gut and terminate in the nucleus of solitary tract (NTS) in the brainstem. They are the primary sensory neurons integrating gut-derived signals to regulate meal size. Chronic high-fat diet (HFD) consumption impairs vagally mediated satiety, resulting in overfeeding. There is evidence that HFD consumption leads to alterations in both vagal nerve function and structural integrity. HFD also leads to marked gut microbiota dysbiosis; in rodent models, dysbiosis is sufficient to induce weight gain. In this study, we investigated the effect of microbiota dysbiosis on gut-brain vagal innervation independently of diet. To do so, we recolonized microbiota-depleted rats with gastrointestinal (GI) contents isolated from donor animals fed either a HFD (45 or 60% fat) or a low fat diet (LFD, 13% fat). We used two different depletion models while maintaining the animals on LFD: 1) conventionally raised Fischer and Wistar rats that underwent a depletion paradigm using an antibiotic cocktail and 2) germ free (GF) raised Fischer rats. Following recolonization, receiver animals were designated as ConvLF and ConvHF. Fecal samples were collected throughout these studies and analyzed via 16S Illumina sequencing. In both models, bacteria that were identified as characteristic of HFD were successfully transferred to recipient animals. Three weeks post-colonization, ConvHF rats showed significant increases in ionized calcium-binding adapter molecule-1 (Iba1) positive immune cells in the NG compared to ConvLF animals. Additionally, using isolectin B4 (IB4) staining to identify c-fibers, we found that, compared to ConvLF animals, ConvHF rats displayed decreased innervation at the level of the medial NTS; c-fibers at this level are believed to be primarily of vagal origin. This alteration in vagal structure was associated with a loss in satiety induced by the gut peptide cholecystokinin (CCK). Increased presence of immunocompetent Iba1+ cells along the gut-brain axis and alterations in NTS innervation were still evident in ConvHF rats compared to ConvLF animals 12 weeks post-colonization and were associated with increases in food intake and body weight (BW). We conclude from these data that microbiota dysbiosis can alter gut-brain vagal innervation, potentially via recruitment and/or activation of immune cells.

Cell-specific cytotoxic effect of pyrazole derivatives on breast cancer cell lines MCF7 and MDA-MB-231.

Pyrazoles and their derivatives belong to a class of compounds that demonstrate a great potential in design of anticancer, antiangiogenic, and antimetastatic drugs. Our earlier studies showed that pyrazole derivatives TOSPYRQUIN and TOSIND diminished viability of colorectal adenocarcinoma cells HT-29. Here we demonstrated for the first time in human mammary gland adenocarcinoma cell lines MCF7 and MDA-MB-231 cells the cytotoxic effects of four pyrazole derivatives: TOSIND, PYRIND, METPYRIND, and DIPYR. Three pyrazoles: PYRIND, METPYRIND, and one novel unpublished derivative DIPYR were tested for the first time in living cells. Viability of MCF7 did not significantly change in the presence of TOSIND but it decreased after 72 hours of treatment with PYRIND (IC-50 39.7 ± 5.8 µM). In the presence of METPYRIND the viability was also diminished, while DIPYR increased MCF7 viability after 24 hours of incubation. The viability of MDA-MB-231 cells was strongly decreased by TOSIND (IC-50 17.7 ± 2.7 µM 72 h), and was not influenced by PYRIND and METPYRIND, while DIPYR increased the viability and stimulated the growth of MDA-MB-231 cells. PYRIND, METPYRIND and DIPYR caused a gradual decrease of caspase-3 and caspase-7 activities in MDA-MB-231 cells and there was no influence of TOSIND on the activity of both caspases. Our results open the way to search for other compounds with pendant pyrazole residues in order to increase their cytotoxic activity; especially with regard to its anti-breast cancer activity. It appears that the pyrazoles synthesized by us diminish cell viability in a cell-specific manner. This observation might be useful in designing 'off-DNA' anticancer drugs, compounds which are not harmful to the healthy cells.

Molecular effects of fractional ablative erbium:YAG laser treatment with multiple stacked pulses on standardized human three-dimensional organotypic skin models.

The molecular changes in gene expression following ablative laser treatment of skin lesions, such as atrophic scars and UV-damaged skin, are not completely understood. A standardized in vitro model of human skin, to study the effects of laser treatment on human skin, has been recently developed. Therefore, the aim of the investigation was to examine morphological and molecular changes caused by fractional ablative erbium:YAG laser treatment on an in vitro full-thickness 3D standardized organotypic model of human skin. A fractional ablative erbium:YAG laser was used to irradiate organotypic human 3D models. Laser treatments were performed at four different settings using a variety of stacked pulses with similar cumulative total energy fluence (60 J/cm2). Specimens were harvested at specified time points and real-time PCR (qRT-PCR) and microarray studies were performed. Frozen sections were examined histologically. Three days after erbium:YAG laser treatment, a significantly increased mRNA expression of matrix metalloproteinases and their inhibitors (MMP1, MMP2, MMP3, TIMP1, and TIMP2), chemokines (CXCL1, CXCL2, CXCL5, and CXCL6), and cytokines such as IL6, IL8, and IL24 could be detected. qRT-PCR studies confirmed the enhanced mRNA expression of IL6, IL8, IL24, CXCLs, and MMPs. In contrast, the mRNA expression of epidermal differentiation markers, such as keratin-associated protein 4, filaggrin, filaggrin 2, and loricrin, and antimicrobial peptides (S100A7A, S100A9, and S100A12) as well as CASP14, DSG2, IL18, and IL36ß was reduced. Four different settings with similar cumulative doses have been tested (N10%, C10%, E10%, and W25%). These laser treatments resulted in different morphological changes and effects on gene regulations. Longer pulse durations (1000 µs) especially had the strongest impact on gene expression and resulted in an upregulation of genes, such as collagen-1A2, collagen-5A2, and collagen-6A2, as well as FGF2. Histologically, all treatment settings resulted in a complete regeneration of the epidermis 3 days after irradiation. Fractional ablative erbium:YAG laser treatment with a pulse stacking technique resulted in histological alterations and shifts in the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling depending on the treatment setting applied. A standardized in vitro 3D model of human skin proved to be a useful tool for exploring the effects of various laser settings both on skin morphology and gene expression during wound healing. It provides novel data on the gene expression and microscopic architecture of the exposed skin. This may enhance our understanding of laser treatment at a molecular level.

Levels of polybrominated diphenyl ethers in house dust in Central Poland.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in numerous products, from which they are emitted to the environment, including house dust. House dust is a source of human exposure to these compounds by ingestion. The aim of this article was to determine the levels of selected PBDEs in the house dust and indicate their potential sources of origin. PBDE congeners: BDE-47, BDE-99, BDE-153 and BDE-209, were analyzed in 129 samples. The geometric mean levels (and 95% CIs) of the aforementioned congeners amounted to 3.8 (3.1-4.7) ng/g, 4.5 (3.5-5.6) ng/g, 2.2 (2.1-2.4) ng/g and 345 (269-442) ng/g respectively. BDE-209 was the dominant congener in the majority of tested samples. We found a statistically significant correlation between the concentrations of BDE-47 and the computer operating time per day (rs - 0.18) and the living area (rs - 0.20). Statistically significant higher levels of BDE-99 were found in homes where the floor was not replaced during the last 2 years.

No association between body mass index and sperm DNA integrity.

STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including mainly subfertile men, have shown conflicting results regarding the influence of overweight and obesity on sperm DNA integrity. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was based on semen samples from 1503 men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included two cohorts (cohort A and B) of military recruits (n = 275, n = 304, respectively), one group (cohort C) of fertile men and men without known fertility problems (n = 724), and one group (cohort D) of men between 19 and 40 years without known fertility problems (n = 200). In all cohorts, data were available on BMI, DFI as measured by the sperm chromatin structure assay (SCSA), standard semen characteristics, and potential confounders (age, abstinence time, smoking habits). The subjects were categorized according to BMI into four groups: underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)), overweight (25.0-29.9 kg/m(2)) and obese (≥30.0 kg/m(2)). Using a linear regression model, the inter-group differences in DFI were calculated. Furthermore with the normal-weight group as the reference, the odds ratios (ORs) for DFI > 20% and DFI > 30%, were calculated for the other groups. Calculations were made for the material as a whole and after exclusion of cohort C which included proven fertile men. MAIN RESULTS AND THE ROLE OF CHANCE: We found that normal-weight men had significantly higher DFI than overweight men, with a mean difference of 1.13% (95% CI: 1.05-1.22%); P = 0.001). Overweight men had a reduced risk of having DFI ≥ 20% and DFI ≥ 30%, compared with normal-weight men; adjusted odds ratio (OR) = 0.61 (95% CI: 0.42-0.88; P < 0.01) and adjusted OR = 0.48 (95% CI: 0.28-0.84; P < 0.01), respectively. When excluding cohort C, the statistical significance was lost. Regarding standard semen parameters, we found that obese men had a higher percentage of progressive motile spermatozoa than normal-weight men; mean difference 1.15% (95% CI: 1.02-1.30%, P < 0.05) but the significance was lost when excluding cohort C. All other standard semen parameters were unaffected by BMI. LIMITATIONS, REASONS FOR CAUTION: A main limitation might be the cross-sectional nature of the data. Furthermore our study included a significant proportion of men with proven fertility (75% of cohort C, n = 550), and could therefore be biased toward fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our study indicates that overweight per se is not associated with a higher level of sperm DNA damage. STUDY FUNDING/COMPETING INTERESTS: This research has been given grants from the following: EU 5th and 7th framework program (Inuendo and Clear projects, [Contracts no. QLK4-CT-2001-00202 and FP7-ENV-2008-1-226217)]), the Swedish Research Council (Grants No. 2007-2590, 521-2004-6072 and 521-2002-3907); the Swedish Governmental Funding for Clinical Research, Skåne county council's research and development foundation, MAS Funds, University Hospital MAS Foundation in Malmö, Crafoordska Fund, Ove Tulefjords Fund, Foundation for Urological Research, Fundacion Federico SA, and Gunnar Nilssons Cancer Fund. The authors declare that there are no conflicts of interest.

Sleeve gastrectomy and Roux-en-Y gastric bypass alter the gut-brain communication.

This study investigated the anatomical integrity of vagal innervation of the gastrointestinal tract following vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) operations. The retrograde tracer fast blue (FB) was injected into the stomach to label vagal neurons originating from nodose ganglion (NG) and dorsal motor nucleus of the vagus (DMV). Microglia activation was determined by quantifying changes in the fluorescent staining of hindbrain sections against an ionizing calcium adapter binding molecule 1 (Iba1). Reorganization of vagal afferents in the hindbrain was studied by fluorescent staining against isolectin 4 (IB4). The density of Iba1- and IB4-immunoreactivity was analyzed using Nikon Elements software. There was no difference in the number of FB-labeled neurons located in NG and DMV between VSG and VSG-sham rats. RYGB, but not RYGB-sham rats, showed a dramatic reduction in number of FB-labeled neurons located in the NG and DMV. VSG increased, while the RYGB operation decreased, the density of vagal afferents in the nucleus tractus solitarius (NTS). The RYGB operation, but not the VSG procedure, significantly activated microglia in the NTS and DMV. Results of this study show that the RYGB, but not the VSG procedure, triggers microglia activation in vagal structures and remodels gut-brain communication.

Characterising the individual health risk in infants exposed to organochlorine pesticides via breast milk by applying appropriate margins of safety derived from estimated daily intakes.

Milk secretion being an important way of elimination of organochlorine pesticides (OCPs) poses a concern due to potential risk for breastfed infants. This study aims to provide a tool for assessing such risks to infants exposed to OCPs (through accumulation in the mother's body), using calculated individual margins of safety (MoS). Selected OCPs included; p,p'-DDT, p,p'-DDD, p,p'-DDE, ß-HCH, γ-HCH and HCB which were analysed in 28 samples of maternal milk. The highest intakes were recorded for p,p'-DDE (at 2.90 µg kg(-1)bw d(-1)) whilst the lowest was for γ-HCH, (at 0.019 µg kg(-1)bwd(-1)). For the risk characterisation purposes MoSs were calculated for the compounds for which toxicological reference values (e.g. ADI, TDI) were adopted. The MoS for average ∑DDT concentrations was found to be relatively low (2.82) somewhat similar to that for HCB at 7.08, and for γ-HCH, the MoS was substantially higher at 263.1. This, however does not take into account the extremely high individual concentrations. Thus, it was decided to calculate estimated daily intake (EDI) values based on OCP levels in individual milk samples. MoS levels of <1 (meaning unacceptable risk) were noted both for HCB in one sample as well as for ∑DDT in 3 samples indicating likely threats to infant's health. The lowest MoS noted for γ-HCH equalled to 60.6, indicating that this compound was not a threat to the health of any of the breastfed infants from the study group.

A comprehensive analysis of the binding of anti-KIR antibodies to activating KIRs.

Analysis of killer cell immunoglobulin-like receptor (KIR) expression has been notoriously difficult because of the cross-reactivity of available antibodies, in particular between activating and inhibitory isoforms. We undertook a comprehensive study of available anti-KIR antibodies binding to activating KIRs (a-KIRs). Using cell lines stably transfected with a-KIRs (KIR2DS1-S5 and KIR3DS1), we confirmed documented binding specificities. In addition, we show that clones HPMA4 and 143211-previously assumed to be specific for KIR2DS1/L1 and KIR2DL1, respectively-bind KIR2DS5 and KIR2DS3 (HPMA4), and KIR2DS5 (143211). Other antibodies with previously undocumented binding were JJC11.6 (recognizing KIR2DS3) and 5.133 (recognizing all a-KIRs except KIR2DS1 and KIR2DS3). The novel KIR2DS5 reactivities were confirmed by blocking with soluble KIR-Fc fusion proteins, and by reverse transcriptase-PCR analysis of sorted primary natural killer cells. In conclusion, we show formerly undocumented binding properties of anti-KIR antibodies. These cross-reactivities should be taken into account when analyzing KIR expression.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1ß, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

HLA-Bw4 identifies a population of HIV-infected patients with an increased capacity to control viral replication after structured treatment interruption.

OBJECTIVES: After structured treatment interruption (STI) of treatment for HIV-1, a fraction of patients maintain suppressed viral loads. Prospective identification of such patients might improve HIV-1 treatment, if selected patients are offered STI. METHODS: We analysed the effect of previously identified genetic modulators of HIV-1 disease progression on patients' ability to suppress viral replication after STI. Polymorphisms in the genes killer cell immunoglobulin-like receptor 3DLI (KIR3DL1)/KIR3DS1, human leucocyte antigen B (HLA-B) and HLA Complex P5 (HCP5), and a polymorphism affecting HLA-C surface expression were analysed in 130 Swiss HIV Cohort Study patients undergoing STI. Genotypes were correlated with viral load levels after STI. RESULTS: We observed a statistically significant reduction in viral load after STI in carriers of HLA-B alleles containing either the Bw480Thr or the Bw480Ile epitope (mean adjusted effect on post-STI viral load: -0.82 log HIV-1 RNA copies/ml, P < 0.001; and -1.12 log copies/ml, P < 0.001, respectively). No significant effects were detected for the other polymorphisms analysed. The likelihood of being able to control HIV-1 replication using a prespecified cut-off (viral load increase < 1000 copies/ml) increased from 39% in Bw4-negative patients to 53% in patients carrying Bw4-80Thr, and to 65% in patients carrying Bw4-80Ile (P = 0.02). CONCLUSIONS: These data establish a significant impact of HLA-Bw4 on the control of viral replication after STI.

Changes in microglial activation within the hindbrain, nodose ganglia, and the spinal cord following subdiaphragmatic vagotomy.

Damage to peripheral nerve branches triggers activation of microglia in CNS areas containing motor neuron soma and primary afferent terminals of the damaged fibers. Furthermore, microglial activation occurs in areas containing the soma and terminals of spared nerve branches of a damaged nerve. Because the abdominal viscera are innervated by spinal afferents as well as vagal afferents and efferents, we speculated that spinal nerves might respond like spared nerve branches following damage to vagal fibers. Therefore, we tested the hypothesis that damage to the abdominal vagus would result in microglial activation in vagal structures-the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMV), and nodose ganglia (NG)-as well as spinal cord (SC) segments that innervate the abdominal viscera. To test this hypothesis, rats underwent subdiaphragmatic vagotomy or sham surgery and were treated with saline or the microglial inhibitor, minocycline. Microglial activation was determined by quantifying changes in the intensity of fluorescent staining with a primary antibody against ionizing calcium adapter binding molecule 1 (Iba1). We found that subdiaphragmatic vagotomy significantly activated microglia in the NTS, DMV, and NG two weeks post-vagotomy. Microglial activation remained significantly increased in the NG and DMV for at least 42 days. Surprisingly, vagotomy significantly decreased microglial activation in the SC. Minocycline treatment attenuated microglial activation in all studied areas. Our results indicate that microglial activation in vagal structures following abdominal vagal damage is accompanied by suppression of microglial activation in associated areas of the spinal cord.

Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.

BACKGROUND: Interleukin (IL)-31 is a novel Th2 T-cell cytokine that induces pruritus and dermatitis in transgenic mice. While enhanced mRNA expression of this cytokine is detected in skin samples of inflammatory skin diseases, the regulation of IL-31 expression is poorly understood. OBJECTIVES: To assess the effects of ultraviolet (UV) B radiation and H2O2 on IL-31 mRNA and protein expression in skin and different peripheral blood mononuclear cells (PBMCs). METHODS: The effects of UVB radiation and H2O2, as a prototypic reactive oxygen species, on IL-31 mRNA and protein expression were analysed in various inflammation-related cells and murine skin tissue. RESULTSTreatment of cells with UVB radiation and H2 O2 strongly induced IL-31 mRNA and protein expression in human PBMCs and in the skin of SKH-1 mice. Following exposure to UVB or H2O2, we observed increased expression of IL-31 mRNA in T cells, monocytes, macrophages, and immature and especially mature dendritic cells. H2O2 treatment but not UVB radiation led to a moderate upregulation of IL-31 mRNA expression in epidermal keratinocytes and dermal fibroblasts. Pretreatment of T lymphocytes with the MAPK p38 inhibitor SB203580 or the MEK1 inhibitor U0126 reduced the stimulatory effect of H2O2. These experiments suggest that p38 is involved in the regulation of IL-31 expression in human skin. CONCLUSIONS: Our studies reveal that UVB and reactive oxygen species stimulate the expression of IL-31 in PBMCs and skin, especially in T cells, monocytes and monocyte-derived dendritic cells.

High-resolution transcriptional profiling of chemical-stimulated dendritic cells identifies immunogenic contact allergens, but not prohaptens.

Allergic contact dermatitis is a complex syndrome and knowledge about the in vitro detection of small-molecular-weight compounds, particularly prohaptens, is limited. Therefore, we investigated chemical-induced gene expression changes in human antigen-presenting cells upon stimulation with immunogenic contact allergens, prohaptens and irritants. Monocyte-derived dendritic cells (moDCs) and THP-1 cells were stimulated with the prohapten cinnamic alcohol (CAlc), the hapten cinnamic aldehyde (CAld), an irritant and an obligatory sensitizer in vitro. Whole-genome screening and consecutive PCR analysis of differential gene expression in moDCs stimulated with either CAld or the obligatory sensitizer revealed coregulation of 11 marker genes which were related to immunological reactions (IL-8, CD1e, CD200R1, PLA2G5, TNFRSF11A), oxidative or metabolic stress responses (AKR1C3, SLC7A11, GCLM) or other processes (DPYLS3, TFPI, TRIM16). In contrast, the prohapten CAlc and the irritant did not change marker gene expression. In THP-1 cells, CAld and the positive control elicited similar expression changes in only 4 of the previously identified genes (IL-8, TRIM16, CD200R1, GCLM). In conclusion, we provide important insights into the pathophysiological basis of allergic contact dermatitis, identify marker genes suitable for skin hazard assessment and demonstrate that contact-allergenic prohaptens escape in in vitro detection if their skin metabolism is not taken into account.

Plasticity of nodose ganglion neurons after capsaicin- and vagotomy-induced nerve damage in adult rats.

Previous reports show that vagal afferent innervation of the stomach eventually regenerates from surviving nodose ganglion (NG) neurons after subdiaphragmatic vagotomy. Systemic capsaicin treatment destroys gastric vagal afferent neurons expressing vanilloid receptor 1 (VR1). However, it is not known whether gastric innervation lost after neuronal destruction can be restored. Here, we report that capsaicin-induced damage of NG neurons innervating the stomach in adult rats is followed by restoration of vagal afferent projections. Specifically, we compared measures of neuronal plasticity in NG and vagi after subdiaphragmatic vagotomy or capsaicin treatment. The numbers of VR1-immunoreactive neurons projecting to the stomach were significantly reduced 10 days after either capsaicin treatment or vagotomy. However, the VR1-immunoreactive afferent innervation of the stomach was restored to levels exceeding those of vagotomized rats by 37 days after capsaicin, whereas neither total afferent innervation nor VR1-immunoreactive innervation reached control levels, even by 67 days after vagotomy. Capsaicin treatment significantly increased NG neuronal nitric oxide synthase (nNOS) immunoreactivity at 10 days after capsaicin, and this increase was sustained for the duration of the study, indicating higher nNOS demand in restoration of vagal projections. Vagotomy was associated with a much smaller increase in the number of nNOS-immunoreactive NG neurons, detectable only at 10 days after surgery. The number of nNOS-immunopositive gastric-projecting neurons was dramatically reduced 10 days after either capsaicin treatment or vagotomy but returned to the control level in both groups at 67 days. We found a significantly higher number of growth cones in capsaicin-treated animals compared with controls. Capsaicin significantly increased the number of nNOS-immunopositive and nNOS-immunonegative growth cones in NG at all time points. Vagotomy did not increase the number of nNOS(-) growth cones in NG. We conclude that capsaicin treatment may result in more significant restorative capacities than vagotomy, mainly because of sprouting of capsaicin-insensitive nerve fibers.

Capsaicin-induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats.

To evaluate the potential for neuronal replacement following destruction of vagal afferent neurons, we examined nodose ganglia following i.p. capsaicin treatment of adult rats. Rats received capsaicin or vehicle followed by a regimen of 5'-bromo-2'-deoxyuridine injections (BrdU) to reveal DNA replication. Nodose ganglia were harvested at various times post-treatment and processed for 4',6-diamidino-2-phenylindole (DAPI) nuclear staining and immunofluorescence to estimate neuronal numbers and to determine vanilloid receptor, cleaved caspase 3, TUNEL, BrdU, the neuron-selective marker protein gene product (PGP) -9.5 and neurofilament-M-immunoreactivity. Twenty-four hours after capsaicin approximately 40% of nodose ganglion neurons expressed cleaved caspase 3-immunoreactivity and 16% revealed TUNEL staining, indicating that primary sensory neurons are killed by the capsaicin treatment of adult rats. The occurrence of neuronal death was confirmed by counts of DAPI-stained neuronal nuclei, which revealed >or=50% reduction of nodose neuron number by 30 days post-capsaicin. However, by 60 days post-capsaicin, the total numbers of neuronal nuclei in nodose ganglia from capsaicin-treated rats were not different from controls, suggesting that new neurons had been added to the nodose ganglia. Neuronal proliferation was confirmed by significant BrdU incorporation in nuclei of nodose ganglion cells immunoreactive for the neuron-specific antigen PGP-9.5 revealed 30 and 60 days post-capsaicin. Collectively, these observations suggest that in adult rats massive scale neurogenesis occurs in nodose ganglia following capsaicin-induced neuronal destruction. The adult nodose ganglion, therefore, provides a novel system for studying neural plasticity and adult neurogenesis after peripheral injury of primary sensory neurons.

Hypothalamic neurons innervating fat tissue in the pig express leptin receptor immunoreactivity.

While leptin receptors have been found in both the autonomic ganglion neurons and the hypothalamic nuclei, studies dealing with the projections from the central nervous system to the adipose tissue have been conducted mainly in laboratory animals. Therefore, the purpose of our study was to establish whether hypothalamic neurons are transsynaptically connected to adipose tissue depots in the pig, and if these neurons express leptin receptor immunoreactivity. Pseudorabies virus (PRV; Bartha's K strain) was introduced in perirenal or subcutaneous adipose tissue depots in domestic pigs. On day 9, animals were euthanized and hypothalami were collected and processed immunohistochemically with primary antisera against PRV and leptin receptor (OBR). PRV-labeled neurons were localized in paraventricular nucleus, supraoptic nucleus and arcuate nucleus following injections in both the perirenal and the subcutaneous adipose tissue depots. Ventromedial nucleus, dorsomedial nucleus and preoptic area-labeled neurons were observed after injection of the PRV into the perirenal adipose tissue, while in the lateral hypothalamic area-labeled neurons projected only to the subcutaneous adipose tissue. The majority of the PRV-labeled neurons simultaneously expressed OBR-immunoreactivity. Our results provide the morphological data on multisynaptic projections from hypothalamus to the fat tissue in the pig and demonstrate that these neurons, located in areas involved in reproductive processes and feeding behavior, may regulate fat tissue metabolism.

Transsynaptic connections between the hypothalamus and adipose tissue: relationship to reproduction.

Neurophysiological mechanisms that control energy balance are reciprocally linked to those that control reproduction. Neuromorphological studies using retrograde tracing methods revealed that nerve cells within the central (CNS) and autonomic (ANS) nervous systems in different species, including the pig, are transsynaptically connected to different fat tissue depots. In the pig, neurons localised in the paraventricular nucleus, supraoptic nucleus and arcuate nucleus were infected with pseudorabies virus (PRV) 9 days after injections into both the perirenal and subcutaneous adipose tissue depots. Infected neurons were in the ventromedial nucleus, dorsomedial nucleus and preoptic area after injection of PRV into perirenal adipose tissue, while infected cells in the lateral hypothalamic area projected only to the subcutaneous adipose tissue depot. Additionally, numerous centres of the ANS innervate adipose tissue depots in the pig. Fast blue stained (FB+) neurons, which projected to the subcutaneous adipose tissue overlaying the thoracolumbar area were located in the thoraco-lumbar region of the sympathetic chain ganglia (SChG). However, neurons supplying perirenal and mesentery adipose tissue depots were found in both the SChG and prevertebral ganglia. The vast majority of labelled neurons, in both the CNS and ANS, which innervated adipose tissue depots, expressed leptin receptor (OBR) immunoreactivity. The purpose of this brief review is to establish evidence for a multisynaptic circuit of neurons, which innervate adipose tissue in the pig and demonstrate that hypothalamic nuclei and sympathetic ganglion neurons involved in reproductive processes are transsynaptically connected to different adipose tissue depots.

The influence of experimental ileitis on the neuropeptide coding of enteric neurons in the pig.

In the present study, both the ELISA test and immunohistochemical staining were used to investigate the influence of artificially induced ileitis on the chemical coding of enteric neurons in the pig. The ileum wall in experimental (E) pigs was injected in multiple sites with 4% paraformaldehyde to induce inflammation, while in the control (C) animals, the organ was injected with 0.1M phosphate buffer (pH 7.4). Three days after ileitis induction, samples of ileum wall from all the animals were evaluated for VIP, SP, CGRP, NPY, GAL and SOM concentration (ELISA test) and the expression of these biologically active substances by the enteric neurons (immunohistochemical staining). Quantitative results showed that ileitis decreased tissue concentration of VIP, CGRP and SOM but increased tissue concentration of SP, NPY and GAL. Immunochemistry revealed that in both the experimental and control pigs, VIP-positive (VIP+) nerve fibers supplied mainly ileal blood vessels, and the labeled pericarya were located in the inner (ISP) and outer submucous plexus (OSP). SP+ and CGRP+ nerve terminals were found in both the mucous and muscular membrane, while the labeled pericarya were found in ISP, OSP and myenteric plexus (MP). In both C and E pigs, the very few nerve terminals containing NPY and SOM were located mainly in the mucous membrane. NPY- or/and SOM-immunopositive nerve cell bodies were found in ISP, OSP and MP. GAL+ nerve fibers supplied all layers of the ileum and were most numerous in the muscular membrane, while the labeled pericarya were present in all the enteric plexuses. The present results suggest that enteric neurons are highly plastic in their response to inflammation.

Leptin: a metabolic signal affecting central regulation of reproduction in the pig.

The discovery of the obesity gene and its product, leptin, it is now possible to examine the relationship between body fat and the neuroendocrine axis. A minimum percentage of body fat may be linked to onset of puberty and weaning-to-estrus interval in the pig. Adipose tissue is no longer considered as only a depot to store excess energy in the form of fat. Recent findings demonstrate that numerous genes, i.e., relaxin, interleukins and other cytokines and biologically active substances such as leptin, insulin-like growth factor-I (IGF-I), IGF-II and Agouti protein are produced by porcine adipose tissue, which could have a profound effect on appetite and the reproductive axis. Hypothalamic neurons are transsynaptically connected to porcine adipose tissue and may regulate adipose tissue function. In the pig nutritional signals such as leptin are detected by the central nervous system (CNS) and translated by the neuroendocrine system into signals, which regulate appetite, hypothalamic gonadotropin-releasing hormone (GnRH) release and subsequent luteinizing hormone (LH) secretion. Furthermore, leptin directly affects LH secretion from the pituitary gland independent of CNS input. Changes in body weight or nutritional status are characterized by altered adipocyte function a reduction in adipose tissue leptin expression, serum leptin concentrations and a concurrent decrease in LH secretion. During pubertal development serum leptin levels, hypothalamic leptin receptor mRNA and estrogen-induced leptin gene expression in fat increased with age and adiposity in the pig and this occurred at the time of expected puberty. In the lactating sow serum and milk leptin concentrations were positively correlated with backfat thickness and level of dietary energy fed during gestation as well as feed consumption. Although, these results identify leptin as a putative signal that links metabolic status and neuroendocrine control of reproduction, other adipocyte protein products may play an important role in regulating the reproductive axis in the pig.