Perinatal exposure to tributyltin affects feeding behavior and expression of hypothalamic neuropeptide Y in the paraventricular nucleus of adult mice.

Organotins such as tributyltin chloride (TBT), are highly diffused environmental pollutants, which act as metabolism disrupting chemicals, i.e. may interfere with fat tissue differentiation, as well as with neuroendocrine circuits, thus impairing the control of energetic balance. We have previously demonstrated that adult exposure to TBT altered the expression of neuropeptides in the hypothalamus. In this study, we orally administered daily a solution containing oil, or TBT (0.25, 2.5, or 25 µg/kg body weight/day) to pregnant females from gestational day 8 until birth, and to their pups from day 0 until post-natal day 21. Our results showed that TBT exposure of female mice during gestation and of pups during lactation permanently altered the feeding efficiency of pups of both sexes and subcutaneous fat distribution in adult males. In addition, the neuropeptide Y system was affected at the level of the paraventricular nucleus, with a decrease in immunoreactivity in both sexes (significant in females for all TBT doses and in males only for intermediate TBT doses), while no effect was observed in other hypothalamic areas (arcuate, ventromedial and dorsomedial nuclei). Metabolic syndrome, as well as obesity and diabetes, which are significant health issues, are considered multifactorial diseases and may be caused by exposure to metabolic disruptors, both in adults and during perinatal life. In addition, our work indicates that TBT doses defined as the tolerably daily intake had a profound and sex-specific long-term effect.

Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro.

BACKGROUND: Mesenchymal stem cells (MSCs) have been used therapeutically in equine medicine. MSCs release extracellular vesicles (EVs), which affect cell processes by inhibiting cell apoptosis and regulating inflammation. To date, little is known about equine EVs and their regenerative properties. OBJECTIVES: To characterise equine MSC-derived extracellular vesicles (EVs) and evaluate their effect on equine chondrocytes treated with pro-inflammatory cytokines in vitro. STUDY DESIGN: In vitro experiments with randomised complete block design. METHODS: Mesenchymal stem cells from bone marrow, adipose tissue, and synovial fluid were cultured in vitro. The MSC culture medium was centrifuged and filtered. Isolated particles were analysed for size and concentration (total number of particles per mL). Transmission electron microscopy analysis was performed to evaluate the morphology and CD9 expression of the particles. Chondrocytes from healthy equines were treated with the inflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor-alpha. MSC-derived EVs from bone marrow and synovial fluid cells were added as co-treatments in vitro. Gene expression analysis by real-time PCR was performed to evaluate the effects of EVs. RESULTS: The particles isolated from MSCs derived from different tissues did not differ significantly in size and concentration. The particles had a round-like shape and positively expressed CD9. EVs from bone marrow cells displayed reduced expression of metalloproteinase-13. MAIN LIMITATIONS: Sample size and characterisation of the content of EVs. CONCLUSIONS: EVs isolated from equine bone marrow MSCs reduced metalloproteinase 13 gene expression; this gene encodes an enzyme related to cartilage degradation in inflamed chondrocytes in vitro. EVs derived from MSCs can reduce inflammation and could potentially be used as an adjuvant treatment to improve tissue and cartilage repair in the articular pathologies.

Early Postnatal Genistein Administration Affects Mice Metabolism and Reproduction in a Sexually Dimorphic Way.

The phytoestrogen genistein (GEN) may interfere with permanent morphological changes in the brain circuits sensitive to estrogen. Due to the frequent use of soy milk in the neonatal diet, we aimed to study the effects of early GEN exposure on some physiological and reproductive parameters. Mice of both sexes from PND1 to PND8 were treated with GEN (50 mg/kg body weight, comparable to the exposure level in babies fed with soy-based formulas). When adult, we observed, in GEN-treated females, an advanced pubertal onset and an altered estrous cycle, and, in males, a decrease of testicle weight and fecal testosterone concentration. Furthermore, we observed an increase in body weight and altered plasma concentrations of metabolic hormones (leptin, ghrelin, triiodothyronine) limited to adult females. Exposure to GEN significantly altered kisspeptin and POMC immunoreactivity only in females and orexin immunoreactivity in both sexes. In conclusion, early postnatal exposure of mice to GEN determines long-term sex-specific organizational effects. It impairs the reproductive system and has an obesogenic effect only in females, which is probably due to the alterations of neuroendocrine circuits controlling metabolism; thus GEN, should be classified as a metabolism disrupting chemical.

Hypothalamic Expression of Neuropeptide Y (NPY) and Pro-OpioMelanoCortin (POMC) in Adult Male Mice Is Affected by Chronic Exposure to Endocrine Disruptors.

In the arcuate nucleus, neuropeptide Y (NPY) neurons, increase food intake and decrease energy expenditure, and control the activity of pro-opiomelanocortin (POMC) neurons, that decrease food intake and increase energy expenditure. Both systems project to other hypothalamic nuclei such as the paraventricular and dorsomedial hypothalamic nuclei. Endocrine disrupting chemicals (EDCs) are environmental contaminants that alter the endocrine system causing adverse health effects in an intact organism or its progeny. We investigated the effects of long-term exposure to some EDCs on the hypothalamic NPY and POMC systems of adult male mice that had been previously demonstrated to be a target of some of these EDCs after short-term exposure. Animals were chronically fed for four months with a phytoestrogen-free diet containing two different concentrations of bisphenol A, diethylstilbestrol, tributyltin, or E2. At the end, brains were processed for NPY and POMC immunohistochemistry and quantitatively analyzed. In the arcuate and dorsomedial nuclei, both NPY and POMC immunoreactivity showed a statistically significant decrease. In the paraventricular nucleus, only the NPY system was affected, while the POMC system was not affected. Finally, in the VMH the NPY system was affected whereas no POMC immunoreactive material was observed. These results indicate that adult exposure to different EDCs may alter the hypothalamic circuits that control food intake and energy metabolism.

Postnatal genistein administration selectively abolishes sexual dimorphism in specific hypothalamic dopaminergic system in mice.

As demonstrated in previous studies, early postnatal genistein (GEN) administration to mice pups of both sexes, at doses similar to that of infant soy-based formulas, may affect the development of some steroid-sensitive neuronal circuits (i.e. nitrergic and vasopressinergic systems), causing irreversible alterations in adults. Here, we investigated the hypothalamic and mesencephalic dopaminergic system (identified with tyrosine hydroxylase immunohistochemistry). GEN administration (50 mg/kg) to mice of both sexes during the first week of postnatal life specifically affected tyrosine hydroxylase immunohistochemistry in the hypothalamic subpopulation of neurons, abolishing their sexual dimorphism. On the contrary, we did not observe any effects in the mesencephalic groups. Due to the large involvement of dopamine in circuits controlling rodent sexual behavior and food intake, these results clearly indicate that the early postnatal administration of GEN may irreversibly alter the control of reproduction, of energetic metabolism, and other behaviors. These results suggest the need for a careful evaluation of the use of soy products in both human and animal newborns.

Chronic treatment with tributyltin induces sexually dimorphic alterations in the hypothalamic POMC system of adult mice.

Tributyltin (TBT), an antifouling agent found in boat paints, is a common contaminant of marine and freshwater ecosystems. It is rapidly absorbed by organic materials and accumulated in many aquatic animals. Human exposure may depend on ingestion of contaminated food or by indirect exposure from household items containing organotin compounds. TBT is defined as an endocrine disruptor compound (EDC) because it binds to androgen receptors. Moreover, it is also included on the list of metabolic disruptors. The brain is a known target of TBT and this compound interferes with the orexigenic system, inducing a strong decrease in NPY expression in the hypothalamus. In the present experiment, we investigated the effect of a chronic treatment with TBT on the mouse anorexigenic system in both sexes, to look at the pro-opiomelanocortin (POMC) expression in the paraventricular (PVN), dorsomedial (DMN), ventromedial (VMN), and arcuate (ARC) hypothalamic nuclei. The results show a sexually dimorphic effect of TBT on both systems. TBT induced a significant decrease of POMC-positive structures only in female mice in DMN, ARC, and in PVN for both sexes. Apparently, these results show that TBT may interfere with the anorexigenic system in hypothalamic areas involved in the control of food intake, by inhibiting POMC in a sexually dimorphic way. In conclusion, in addition to having a direct effect on fat tissue, the effects of TBT as metabolic disruptor, may be due to gender-specific actions on both orexigenic and anorexigenic hypothalamic systems.

Sex Steroids and Adult Neurogenesis in the Ventricular-Subventricular Zone.

The forebrain ventricular-subventricular zone (V-SVZ) continuously generates new neurons throughout life. Neural stem cells (type B1 cells) along the lateral ventricle become activated, self-renew, and give rise to proliferating precursors which progress along the neurogenic lineage from intermediate progenitors (type C cells) to neuroblasts (type A cells). Neuroblasts proliferate and migrate into the olfactory bulb and differentiate into different interneuronal types. Multiple factors regulate each step of this process. Newly generated olfactory bulb interneurons are an important relay station in the olfactory circuits, controlling social recognition, reproductive behavior, and parental care. Those behaviors are strongly sexually dimorphic and changes throughout life from puberty through aging and in the reproductive age during estrous cycle and gestation. Despite the key role of sex hormones in regulating those behaviors, their contribution in modulating adult neurogenesis in V-SVZ is underestimated. Here, we compare the literature highlighting the sexual dimorphism and the differences across the physiological phases of the animal for the different cell types and steps through the neurogenic lineage.

Kisspeptin system in ovariectomized mice: Estradiol and progesterone regulation.

The kisspeptin system is clustered in two main groups of cell bodies (the periventricular region, RP3V and the arcuate nucleus, ARC) that send fibers mainly to the GnRH neurons and in a few other locations, including the paraventricular nucleus, PVN. In physiological conditions, gonadal hormones modulate the kisspeptin system with expression changes according to different phases of the estrous cycle: the highest being in estrus phase in RP3V and PVN (positive feedback), and in ARC during the diestrus phase (negative feedback). In this work we wanted to study these hormonal fluctuations during the estrous cycle, investigating the role played by progesterone (P) or estradiol (E2), alone or together, on the kisspeptin system. Gonadectomized CD1 female mice were treated with P, E2 or both (E2 + P), following a timing of administration that emulates the different phases of estrous cycle, for two cycles of 4 days. As expected, the two cell groups were differentially affected by E2; the RP3V group was positively influenced by E2 (alone or with the P), whereas in the ARC the administration of E2 did not affect the system. However P (alone) induced a rise in the kisspeptin immunoreactivity. All the treatments significantly affected the kisspeptin innervation of the PVN, with regional differences, suggesting that these fibers arrive from both RP3V and ARC nuclei.

Metabolism Disrupting Chemicals and Alteration of Neuroendocrine Circuits Controlling Food Intake and Energy Metabolism.

The metabolism-disrupting chemicals (MDCs) are molecules (largely belonging to the category of endocrine disrupting chemicals, EDCs) that can cause important diseases as the metabolic syndrome, obesity, Type 2 Diabetes Mellitus or fatty liver. MDCs act on fat tissue and liver, may regulate gut functions (influencing absorption), but they may also alter the hypothalamic peptidergic circuits that control food intake and energy metabolism. These circuits are normally regulated by several factors, including estrogens, therefore those EDCs that are able to bind estrogen receptors may promote metabolic changes through their action on the same hypothalamic circuits. Here, we discuss data showing how the exposure to some MDCs can alter the expression of neuropeptides within the hypothalamic circuits involved in food intake and energy metabolism. In particular, in this review we have described the effects at hypothalamic level of three known EDCs: Genistein, an isoflavone (phytoestrogen) abundant in soy-based food (a possible new not-synthetic MDC), Bisphenol A (compound involved in the manufacturing of many consumer plastic products), and Tributyltin chloride (one of the most dangerous and toxic endocrine disruptor, used in antifouling paint for boats).

Kisspeptin innervation of the hypothalamic paraventricular nucleus: sexual dimorphism and effect of estrous cycle in female mice.

The hypothalamic paraventricular nucleus (PVN) is the major autonomic output area of the hypothalamus and a critical regulatory center for energy homeostasis. The organism's energetic balance is very important for both the regular onset of puberty and regulation of fertility. Several studies have suggested a relationship among neural circuits controlling food intake, energy homeostasis and the kisspeptin peptide. The kisspeptin system is clustered in two main groups of cell bodies [the anterior ventral periventricular region (AVPV) and the arcuate nucleus (ARC)] projecting mainly to gonadotropin-releasing hormone (GnRH) neurons and to a few other locations, including the PVN. In the present study, we investigated the distribution of the kisspeptin fibers within the PVN of adult CD1 mice. We observed a significant sexual dimorphism for AVPV and ARC, as well as for the PVN innervation. Kisspeptin fibers showed a different density within the PVN, being denser in the medial part than in the lateral one; moreover, in female, the density changed, according to different phases of the estrous cycle (the highest density being in estrus phase). The presence of a profound effect of estrous cycle on the kisspeptin immunoreactivity in AVPV (with a higher signal in estrus) and ARC, and the strong co-localization between kisspeptin and NkB only in ARC and not in PVN suggested that the majority of the kisspeptin fibers found in the PVN might arise directly from AVPV.

Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice.

Proliferating neural stem cells and intermediate progenitors persist in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. This extensive germinal layer in the walls of the lateral ventricles is the site of birth of different types of interneurons destined for the olfactory bulb. The cell cycle dynamics of stem cells (B1 cells), intermediate progenitors (C cells), and neuroblasts (A cells) in the V-SVZ and the number of times these cells divide remain unknown. Using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs, we determined the proliferation dynamics of B1, C, and A cells in vivo. Achaete-scute complex homolog (Ascl)1(+) C cells were heterogeneous with a cell cycle length (T(C)) of 18-25 h and a long S phase length (T(S)) of 14-17 h. After C cells, Doublecortin(+) A cells were the second-most common dividing cell type in the V-SVZ and had a T(C) of 18 h and T(S) of 9 h. Human glial fibrillary acidic protein (hGFAP)::GFP(+) B1 cells had a surprisingly short Tc of 17-18 h and a T(S) of 4 h. Progenitor population analysis suggests that following the initial division of B1 cells, C cells divide three times and A cells once, possibly twice. These data provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ and how large numbers of new neurons continue to be produced in the adult mammalian brain.

Genesis of neuronal and glial progenitors in the cerebellar cortex of peripuberal and adult rabbits.

Adult neurogenesis in mammals is restricted to some brain regions, in contrast with other vertebrates in which the genesis of new neurons is more widespread in different areas of the nervous system. In the mammalian cerebellum, neurogenesis is thought to be limited to the early postnatal period, coinciding with end of the granule cell genesis and disappearance of the external granule cell layer (EGL). We recently showed that in the rabbit cerebellum the EGL is replaced by a proliferative layer called 'subpial layer' (SPL) which persists beyond puberty on the cerebellar surface. Here we investigated what happens in the cerebellar cortex of peripuberal rabbits by using endogenous and exogenously-administered cell proliferation antigens in association with a cohort of typical markers for neurogenesis. We show that cortical cell progenitors extensively continue to be generated herein. Surprisingly, this neurogenic process continues to a lesser extent in the adult, even in the absence of a proliferative SPL. We describe two populations of newly generated cells, involving neuronal cells and multipolar, glia-like cells. The genesis of neuronal precursors is restricted to the molecular layer, giving rise to cells immunoreactive for GABA, and for the transcription factor Pax2, a marker for GABAergic cerebellar interneuronal precursors of neuroepithelial origin that ascend through the white matter during early postnatal development. The multipolar cells are Map5+, contain Olig2 and Sox2 transcription factors, and are detectable in all cerebellar layers. Some dividing Sox2+ cells are Bergmann glia cells. All the cortical newly generated cells are independent from the SPL and from granule cell genesis, the latter ending before puberty. This study reveals that adult cerebellar neurogenesis can exist in some mammals. Since rabbits have a longer lifespan than rodents, the protracted neurogenesis within its cerebellar parenchyma could be a suitable model for studying adult nervous tissue permissiveness in mammals.

Adult mammalian neurogenesis and the New Zealand white rabbit.

Although the central nervous system is unable to undergo spontaneous repair and is hostile to the integration of exogenously delivered cells, various examples of adult structural plasticity have been shown to occur. It is now widely accepted that endogenous proliferative activity leading to the production of new neurons exists, at least within two restricted brain sites: the hippocampal dentate gyrus and the forebrain subventricular zone. A substantial insight into spontaneous neurogenesis within these allocortical regions in rodents has been obtained, but less is known regarding its occurrence in other mammalian brain regions. In this review, differences in the structural and temporal characteristics of protracted neurogenesis in mammals will be considered. Attention will be focused on the rabbit cerebrum and cerebellum, where unexpected features of structural plasticity have been found to occur despite the relative closeness of the Orders Lagomorpha and Rodentia.

Cellular composition and cytoarchitecture of the rabbit subventricular zone and its extensions in the forebrain.

Persistent neurogenic sites, harboring neurogenic progenitor cells, which give rise to neuronal precursors throughout life, occur in different mammals, including humans. The telencephalic subventricular zone (SVZ) is the most active adult neurogenic site. Despite remarkable knowledge of its anatomical and cellular composition in rodents, detailed arrangement of SVZ in other mammals is poorly understood, yet comparative studies suggest that differences might exist. Here, by analyzing the cellular composition/arrangement in the SVZ of postnatal, young, and adult rabbits, we found a remarkably heterogeneous distribution of its chain and glia compartments. Starting from postnatal stages, this heterogeneity leads to a distinction between a ventricular SVZ and an abventricular SVZ, whereby the former contains small chains and isolated neuroblasts and the latter is characterized by large chains and a loose astrocytic meshwork. In addition to analysis of the SVZ proper, attention has been focused on its extensions, called parenchymal chains. Anterior parenchymal chains are compact chains surrounded by axon bundles and frequently establish direct contact with blood vessels. Posterior parenchymal chains are less compact, being squeezed between gray and white matter. In the shift from neonatal to adult rabbit SVZ, chains occur very early, both in the SVZ and within the brain parenchyma. Comparison of these results with the pattern in rodents reveals different types of chains, displaying a variety of relationships with glia or other substrates in vivo, an issue that might be important in understanding differences in the adaptation of persistent germinative layers to different mammalian brain anatomies.

A subpial, transitory germinal zone forms chains of neuronal precursors in the rabbit cerebellum.

Protracted neurogenesis occurs at different postnatal stages in different brain locations, whereby leading to site-specific adult neurogenesis in some cases. No spontaneous genesis of neurons occurs in the cerebellum after the postnatal genesis of granule cells from the external germinal layer (EGL), a transitory actively proliferating zone which is thought to be exhausted before puberty. Here, we show the protracted genesis of newly generated neuronal precursors in the cerebellar cortex of young rabbits, persisting beyond puberty. Neuroblasts generated within an actively proliferating subpial layer thus extending the postnatal EGL are arranged to form thousands of tangential chains reminiscent of those responsible for cell migration in the forebrain subventricular zone. These subpial chains cover the whole cerebellar surface from the 2nd to the 5th month of life, then disappearing after puberty. In addition, we describe the appearance of similar groups of cells at the end of granule cell genesis in the mouse cerebellum, here limited to the short period of EGL exhaustion (4-5 days). These results show common features do exist in the postnatal reorganization of secondary germinal layers of brain and cerebellum at specific stages, parallel to differences in the slowing down of cerebellar neurogenesis among mammalian species.