The regulatory effect of sex steroids on the RhoA/ROCK pathway in the rat distal vagina.

BACKGROUND: Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. AIM: This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. METHODS: Ovariectomized (OVX) Sprague-Dawley rats were treated with 17ß-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. OUTCOMES: Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. RESULTS: ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. CLINICAL IMPLICATIONS: Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. STRENGTHS AND LIMITATIONS: This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.

Consequences of Anabolic-Androgenic Steroid Abuse in Males; Sexual and Reproductive Perspective.

The real epidemiology and the possible consequences of anabolic-androgenic steroids (AAS) use still represent a very tricky task due to the difficulties in the quantification and detection of these drugs. Chronic use of AAS, frequently combined with other illicit substances, can induce tremendous negative effects on the reproductive system, but it is also associated with an increased overall and cardiovascular mortality risk. In the present review we summarize and discuss the available evidence regarding the negative impact of AAS on the male reproductive system, providing practical suggestions to manage these problems. For this purpose a meta-analysis evaluating the effects of AAS abusers vs. controls on several hormonal, reproductive and metabolic parameters was performed. In addition, in order to overcome possible limitations related to the combined use of different AAS preparations, we also retrospectively re-analyzed data on animal models treated with supraphysiological dosage of testosterone (T), performed in our laboratory. Available data clearly indicated that AAS negatively affect endogenous T production. In addition, increased T and estradiol circulating levels were also observed according to the type of preparations used. The latter leads to an impairment of sperm production and to the development of side effects such as acne, hair loss and gynecomastia. Furthermore, a worse metabolic profile, characterized by reduced high density lipoprotein and increased low density lipoprotein cholesterol levels along with an increased risk of hypertension has been also detected. Finally sexual dysfunctions, often observed upon doping, represent one the most probable unfavorable effects of AAS abuse.

Insight on the Intracrinology of Menopause: Androgen Production within the Human Vagina.

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers. hvSMCs were isolated from vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated mRNA expression of several steroidogenic enzymes and sex steroid receptors using real time reverse transcription-polymerase chain reaction. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LC-MS/MS). In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α, whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of proandrogenic steroidogenic enzymes (HSD3ß1/ß2, HSD17ß3/ß5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short-term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone and DHT secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of genitourinary syndrome in menopause.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

The G protein-coupled oestrogen receptor, GPER1, mediates direct anti-inflammatory effects of oestrogens in human cholinergic neurones from the nucleus basalis of Meynert.

It has been well established, particularly in animal models, that oestrogens exert neuroprotective effects in brain areas linked to cognitive processes. A key protective role could reside in the capacity of oestrogen to modulate the inflammatory response. However, the direct neuroprotective actions of oestrogens on neurones are complex and remain to be fully clarified. In the present study, we took advantage of a previously characterised primary culture of human cholinergic neurones (hfNBM) from the foetal nucleus basalis of Meynert, which is known to regulate hippocampal and neocortical learning and memory circuits, aiming to investigate the direct effects of oestrogens under inflammatory conditions. Exposure of cells to tumour necrosis factor (TNF)α (10 ng mL-1 ) determined the activation of an inflammatory response, as demonstrated by nuclear factor-kappa B p65 nuclear translocation and cyclooxygenase-2 mRNA expression. These effects were inhibited by treatment with either 17ß-oestradiol (E2 ) (10 nmol L-1 ) or G1 (100 nmol L-1 ), the selective agonist of the G protein-coupled oestrogen receptor (GPER1). Interestingly, the GPER1 antagonist G15 abolished the effects of E2 in TNFα-treated cells, whereas the ERα/ERß inhibitor tamoxifen did not. Electrophysiological measurements in hfNBMs revealed a depolarising effect caused by E2 that was specifically blocked by tamoxifen and not by G15. Conversely, G1 specifically hyperpolarised the cell membrane and also increased both inward and outward currents elicited by a depolarising stimulus, suggesting a modulatory action on hfNBM excitability by GPER1 activation. Interestingly, pretreating cells with TNFα completely blocked the effects of G1 on membrane properties and also significantly reduced GPER1 mRNA expression. In addition, we found a peculiar subcellular localisation of GPER1 to focal adhesion sites that implicates new possible mechanisms of action of GPER1 in the neuronal perception of mechanical stimuli. The results obtained in the present study indicate a modulatory functional role of GPER1 with respect to mediating the oestrogen neuroprotective effect against inflammation in brain cholinergic neurones and, accordingly, may help to identify protective strategies for preventing cognitive impairments.

Physical activity counteracts metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction in the rabbit.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.

Neuroprotective effects of quercetin 4'-O-ß-d-diglucoside on human striatal precursor cells in nutrient deprivation condition.

Several investigations have demonstrated neuroprotective effects of quercetin, a polyphenol widely present in nature, against neurotoxic chemicals, as well as in neuronal injury/neurodegenerative disease models. Most of these studies have been performed with quercetin aglycone and its metabolites, while scanty data are available on its glycosides. This study is aimed at investigating the neuroprotective effects of quercetin 3,4'-O-ß-d-diglucoside (Q3,4'dG), isolated from the bulbs of the white cultivar (Allium cepa L.), using an in vitro model of human striatal precursor cells (HSPs), a primary culture isolated from the striatal primordium and previously characterized. To study the effect of Q3,4'dG on cell survival, HSPs were exposed to nutrient deprivation created by replacing culture medium with phosphate buffer saline (PBS). Our findings showed that Q3,4'dG treatment significantly promoted cell survival and strongly decreased apoptosis induced by nutrient deprivation, as evaluated by cell proliferation/death analyses. In addition, since the adhesive capacities of cells are essential for cell survival, the expression of some adhesion molecules, such as pancadherin and focal adhesion kinase, was evaluated. Interestingly, PBS exposure significantly decreased the expression of both molecules, while in the presence of Q3,4'dG this effect was prevented. This study provides evidence of a neuroprotective role exerted by Q3,4'dG and suggests its possible implication in sustaining neuronal survival for prevention and treatment of neurodegenerative disorders.

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer's Disease.

The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer's disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-ß-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.

Sialic acid expression in human fetal skeletal muscle during limb early myogenesis.

Investigations on animal models demonstrated that changes of sialic acid (SA) expression, particularly the polymeric form, in the skeletal muscle during embryonic and post-natal development seem to be related to muscle differentiation and functionality onset. The aim of this study was to evaluate the monomeric and polymeric SA expression in human skeletal muscle during early stages of fetal development, when important morphofunctional events occur. Specimens of fetal skeletal muscle from limb, between 9 and 12 weeks of gestation (wg), were obtained from 19 pregnant women. To investigate some morphofunctional features occurring during this development period, haematoxylin-eosin staining, tunel assay and immunohistochemistry for connexin-43 (Cx43) and parvalbumin were performed. SA expression and characterization was evaluated using lectin histochemistry (MAA, SNA, PNA, SBA, DBA), associated with enzymatic and chemical treatments. Polysialic acid (PSA) expression was also evaluated using immunohistochemistry. The results showed apoptotic myotubes between 9 and 10.5 wg, disappearing from 11 wg; Cx43 was more abundant in myotubes/myoblasts between 9 and 9.5 wg, decreasing and/or disappearing from 10 wg and parvalbumin was present in myotubes between 10 and 10.5 wg. PSA was revealed in myotubes/myoblasts from 9 to 10.5 wg; from 11 wg it was reduced or disappeared. Monomeric SA appeared in myotubes/myoblasts from 10 wg, increasing successively; acetylated SA was present from 11 wg. These findings demonstrated that changes in expression of various types of SA, occurring in human fetal skeletal muscle during early development, seem to be related to some morphofunctional aspects distinctive of this organogenesis crucial period.

Tumor Necrosis Factor-α Impairs Kisspeptin Signaling in Human Gonadotropin-Releasing Hormone Primary Neurons.

Context: Inflammatory pathways may impair central regulatory networks involving gonadotropin-releasing hormone (GnRH) neuron activity. Studies in humans are limited by the lack of human GnRH neuron cell lines. Objective: To establish an in vitro model of human GnRH neurons and analyze the effects of proinflammatory cytokines. Design: The primary human fetal hypothalamic cells (hfHypo) were isolated from 12-week-old fetuses. Responsiveness to kisspeptin, the main GnRH neurons' physiological regulator, was evaluated for biological characterization. Tumor necrosis factor alpha (TNF-α) was used as a proinflammatory stimulus. Main Outcome Measures: Expression of specific GnRH neuron markers by quantitative reverse transcription-polymerase chain reaction, flow cytometry, and immunocytochemistry analyses; and GnRH-releasing ability and electrophysiological changes in response to kisspeptin. Results: The primary hfHypo showed a high percentage of GnRH-positive cells (80%), expressing a functional kisspeptin receptor (KISS1R) and able to release GnRH in response to kisspeptin. TNF-α exposure determined a specific inflammatory intracellular signaling and reduced GnRH secretion, KISS1R expression, and kisspeptin-induced depolarizing effect. Moreover, hfHypo possessed a primary cilium, whose assembly was inhibited by TNF-α. Conclusion: The hfHypo cells represent a novel tool for investigations on human GnRH neuron biology. TNF-α directly affects GnRH neuron function by interfering with KISS1R expression and ciliogenesis, thereby impairing kisspeptin signaling.

Cardiopulmonary protective effects of the selective FXR agonist obeticholic acid in the rat model of monocrotaline-induced pulmonary hypertension.

Farnesoid X receptor (FXR) activation by obeticholic acid (OCA) has been demonstrated to inhibit inflammation and fibrosis development and even induce fibrosis regression in liver, kidney and intestine in multiple disease models. OCA also inhibits liver fibrosis in nonalcoholic steatohepatitis patients. FXR activation has also been demonstrated to suppress the inflammatory response and to promote lung repair after lung injury. This study investigated the effects of OCA treatment (3, 10 or 30mg/kg, daily for 5days a week, for 7 and/or 28 days) on inflammation, tissue remodeling and fibrosis in the monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rat model. Treatment with OCA attenuated MCT-induced increased pulmonary arterial wall thickness and right ventricular hypertrophy, by i) blunting pathogenic inflammatory mechanisms (downregulation of interleukin 6, IL-6, and monocyte chemoattractant protein-1, MCP-1) and ii) enhancing protective mechanisms counteracting fibrosis and endothelial/mesenchymal transition. MCT-injected rats also showed a marked decrease of pulmonary artery responsiveness to both endothelium-dependent and independent relaxant stimuli, such as acetylcholine and a nitric oxide donor, sodium nitroprusside. Administration of OCA (30mg/kg) normalized this decreased responsiveness. Accordingly, OCA treatment induced profound beneficial effects on lung histology. In particular, both OCA doses markedly reduced the MCT-induced medial wall thickness increase in small pulmonary arteries. To evaluate the objective functional improvement by OCA treatment of MCT-induced PAH, we performed a treadmill test and measured duration of exercise. MCT significantly reduced, and OCA normalized treadmill endurance. Results with OCA were similar, or even superior, to those obtained with tadalafil, a well-established treatment of PAH. In conclusion, OCA treatment demonstrates cardiopulmonary protective effects, modulating lung vascular remodeling, reducing right ventricular hypertrophy and significantly improving exercise capacity. Thus, OCA can restore the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions.

Differential expression of vascular endothelial growth factor in human fetal skeletal site-specific tissues: Mandible versus femur.

Vascular endothelial growth factor (VEGF) is a well-known mediator that signals through pathways in angiogenesis and osteogenesis. Angiogenesis and bone formation are coupled during either skeletal development or bone remodeling and repair occurring in postnatal life. In this study, we examined for the first time the expression of VEGF in human fetal mandibular and femoral bone in comparison with the respective adult tissues. Similarly to other craniofacial bones, but at variance with the axial and appendicular skeleton, during development mandible does not arise from mesoderm but neural crest cells of the neuroectoderm germ layer, and undergoes intramembranous instead of endochondral ossification. By quantitative real-time PCR technique, we could show that VEGF gene expression levels were significantly higher in fetal than in adult samples, especially in femoral tissue. Western blotting analysis confirmed higher protein expression of VEGF in the fetal femur respect to the mandible. Moreover, immunohistochemistry revealed that in both fetal tissues VEGF expression was mainly localized in pre- and osteoblasts. Differential expression of VEGF in femoral and mandibular bone tissues could be related to their different structure, function and development during organogenesis.

Human striatum remodelling after neurotransplantation in Huntington's disease.

BACKGROUND: Restoration of functions in Huntington's disease (HD) by neurotransplantation stems from the formation of a striatum-like structure capable of establishing host connections as a result of grafted striatal neuroblast maturation. For the first time, we demonstrated some developmental steps accomplished by progenitor cells in the brain of an HD patient and analysed the molecular asset of the human primordium. CASE REPORT: Surgery involved bilateral (two sessions) stereotactic, caudate-putaminal transplantation of whole ganglionic eminence fragments from single legally aborted fetuses. MRI showed that the tissue deposits of the left hemisphere grew and joined to constitute a single tissue mass that remodelled basal ganglia anatomy and remained stable in size over time. No evidence of graft growth was observed contralaterally. PET demonstrated increased striatal and stable cortical metabolism. Unified Huntington's Disease Rating Scale assessments demonstrated improvement of motor performances, which faded over the 36-month follow-up. Cognitive performance tended to decrease at a lower rate than before transplantation. CONCLUSION: The striatal primordium grew into the host brain and this process was associated with metabolic change and some clinical benefit. The study suggests the plasticity and reparative potential of un-manipulated primordium in an era where promising cell-based therapies are still in their infancy.

Multifaceted roles of BDNF and FGF2 in human striatal primordium development. An in vitro study.

Grafting fetal striatal cells into the brain of Huntington's disease (HD) patients has raised certain expectations in the past decade as an effective cell-based-therapy for this devastating condition. We argue that the first requirement for successful transplantation is defining the window of plasticity for the striatum during development when the progenitor cells, isolated from their environment, are able to maintain regional-specific-identity and to respond appropriately to cues. The primary cell culture from human fetal striatal primordium described here consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons. These cells express trophic factors, such as BDNF and FGF2. We show that these neurotrophins maintain cell plasticity, inducing the expression of neuronal precursor markers and cell adhesion molecules, as well as promoting neurogenesis, migration and survival. We propose that BDNF and FGF2 play an important autocrine-paracrine role during early striatum development in vivo and that their release by fetal striatal grafts may be relevant in the setting of HD cell therapy.

Nonalcoholic steatohepatitis as a novel player in metabolic syndrome-induced erectile dysfunction: an experimental study in the rabbit.

A pathogenic link between erectile dysfunction (ED) and metabolic syndrome (MetS) is now well established. Nonalcoholic steatohepatitis (NASH), the hepatic hallmark of MetS, is regarded as an active player in the pathogenesis of MetS-associated cardiovascular disease (CVD). This study was aimed at evaluating the relationship between MetS-induced NASH and penile dysfunction. We used a non-genomic, high fat diet (HFD)-induced, rabbit model of MetS, and treated HFD rabbits with testosterone (T), with the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA), or with the anti-TNFα mAb infliximab. Rabbits fed a regular diet were used as controls. Liver histomorphological and gene expression analysis demonstrated NASH in HFD rabbits. Several genes related to inflammation (including TNFα), activation of stellate cells, fibrosis, and lipid metabolism parameters were negatively associated to maximal acetylcholine (Ach)-induced relaxation in penis. When all these putative liver determinants of penile Ach responsiveness were tested as covariates in a multivariate model, only the association between hepatic TNFα expression and Ach response was confirmed. Accordingly, circulating levels of TNFα were increased 15-fold in HFD rabbits. T and OCA dosing in HFD rabbits both reduced TNFα liver expression and plasma levels, with a parallel increase of penile eNOS expression and responsiveness to Ach. Also neutralization of TNFα with infliximab treatment fully normalized HFD-induced hypo-responsiveness to Ach, as well as responsiveness to vardenafil, a phosphodiesterase type 5 inhibitor. Thus, MetS-induced NASH in HFD rabbits plays an active role in the pathogenesis of ED, likely through TNFα, as indicated by treatments reducing liver and circulating TNFα levels (T or OCA), or neutralizing TNFα action (infliximab), which significantly improve penile responsiveness to Ach in HFD rabbits.

Metabolic syndrome induces inflammation and impairs gonadotropin-releasing hormone neurons in the preoptic area of the hypothalamus in rabbits.

Rabbits with high fat diet (HFD)-induced metabolic syndrome (MetS) developed hypogonadotropic hypogonadism (HH) and showed a reduced gonadotropin-releasing hormone (GnRH) immunopositivity in the hypothalamus. This study investigated the relationship between MetS and hypothalamic alterations in HFD-rabbits. Gonadotropin levels decreased as a function of MetS severity, hypothalamic gene expression of glucose transporter 4 (GLUT4) and interleukin-6 (IL-6). HFD determined a low-grade inflammation in the hypothalamus, significantly inducing microglial activation, expression and immunopositivity of IL-6, as well as GLUT4 and reduced immunopositivity for KISS1 receptor, whose mRNA expression was negatively correlated to glucose intolerance. Correcting glucose metabolism with obetcholic acid improved hypothalamic alterations, reducing GLUT4 and IL-6 immunopositivity and significantly increasing GnRH mRNA, without, however, preventing HFD-related HH. No significant effects at the hypothalamic level were observed after systemic anti-inflammatory treatment (infliximab). Our results suggest that HFD-induced metabolic derangements negatively affect GnRH neuron function through an inflammatory injury at the hypothalamic level.

Transepithelial riboflavin/ultraviolet. a corneal cross-linking in keratoconus: morphologic studies on human corneas.

PURPOSE: To evaluate histologic and molecular changes in human keratoconic corneas after the procedure of transepithelial collagen cross-linking (CXL), without the removal of corneal epithelium. DESIGN: Experimental laboratory investigation. METHODS: Thirty corneal buttons were examined, 18 of which were from patients affected by severe keratoconus and submitted to penetrating keratoplasty (PK). Among these, 8 were analyzed without any treatment, 4 were treated with transepithelial CXL 2 hours before PK, and 6 were treated with transepithelial CXL 3 months before PK. Twelve normal corneal buttons from healthy donors were used as controls. The corneal buttons were then evaluated by hematoxylin-eosin staining and by immunostaining with markers of epithelial junction proteins (ß-catenin and connexin 43), of stromal keratocytes (CD34), of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] assay), and of collagen type I fibers. RESULTS: The analysis of epithelial markers showed a clear defective expression in keratoconic corneas before and soon after the transepithelial CXL treatment, returning to normal in corneas analyzed 3 months after transepithelial CXL. The analysis of stroma components indicated a loss of keratocytes in the upper stroma of keratoconic corneas and a trend toward a normal situation 3 months after transepithelial CXL; similarly, collagen fibers appeared disorganized in keratoconus, while their pattern appears to be close to normal 3 months after treatment. CONCLUSIONS: Histologic and immunohistochemical findings on human keratoconic corneas showed the presence of biochemical and morphologic alterations in the epithelium and the upper stroma that are significantly improved 3 months after transepithelial CXL. However, further studies are necessary to assess to what extent these results correlate with measurable biomechanical effects.

Mechanism of action of phosphodiesterase type 5 inhibition in metabolic syndrome-associated prostate alterations: an experimental study in the rabbit.

BACKGROUND: Phosphodiesterase type 5 (PDE5) inhibitors improve benign prostatic hyperplasia (BPH)-related lower urinary tract symptoms (LUTS), often associated with metabolic syndrome (MetS). This study investigated the effects of PDE5 inhibition in the prostate of rabbits fed a high fat diet (HFD) for 12 weeks. HFD-rabbits develop the most important features of human MetS (glucose intolerance, dyslipidemia, increased abdominal adiposity, and hypertension), along with hypogonadism and LUT abnormalities (prostate and bladder inflammation/tissue remodeling). METHODS: Gene expression was evaluated by quantitative RT-PCR. Prostate morphological changes and oxygenation were evaluated by immunohistochemistry. RESULTS: HFD prostates showed increased PDE5 expression, suggesting a peculiar sensitivity of prostate to the action of PDE5 inhibitors during MetS. Accordingly, prostate PDE5 mRNA was negatively associated to plasma testosterone/estradiol ratio, whose reduction characterizes MetS, and positively with the expression in prostate of several genes exploring pathogenetic processes for BPH/LUTS, such as inflammation, leukocyte infiltration, and fibrosis/myofibroblast activation. Most of these genes was up-regulated by HFD, and significantly reduced by PDE5 inhibition, through either chronic (12 weeks) or, at a lower extent, acute (1-week) tadalafil dosing. Tadalafil was also able to reduce blood pressure and visceral fat in HFD rabbits, without changing any other MetS parameter. Interestingly, 1-week tadalafil administration to HFD rabbits, significantly blunted prostate inflammation (increased CD45 immunopositivity), fibrosis (reduced muscle/fiber ratio) and hypo-oxygenation, thus suggesting a potential curative effect of PDE5 inhibition on MetS-related prostate alterations. CONCLUSIONS: Our data provide the experimental evidences to support the multiple potentiality of PDE5 inhibitors as a useful therapeutic tool in LUTS.