Silodosin improves functional consequences of lower urinary tract obstruction secondary to benign prostate hypertrophy, a proof of concept study in the spontaneously hypertensive rat supplemented with testosterone.

BACKGROUND: The main purpose of this study is to investigate the effect of silodosin on the urodynamic consequences in a previously established model of lower urinary tract symptoms associated with benign prostate hyperplasia, the spontaneously hypertensive rats (SHR) supplemented with testosterone. METHODS: Three groups of animals (8-week-old; n = 10/group) were considered: Wistar Kyoto (control) rats (WKY), SHR supplemented with testosterone at 3 mg/kg/day and treated with either vehicle (SHR-T, n = 10) or silodosin at 0.1 mg/kg/day (SHR-T + silodosin, n = 10) by oral gavage for 6 weeks. Cystometry experiments were performed. The bladder was harvested, weighed and paraffin-embedded for morphometric analysis. The prostate was also harvested and weighed. RESULTS: The number of animals included in the analysis were n = 10/10 for WKY and n = 7-8/10 for each SHR rats supplemented with testosterone group. SHR-T displayed a significant decrease in the intercontraction interval, infused volume and mean flow rate whereas the frequency of non-voiding contractions was increased. Silodosin improved the voiding behavior of SHR-T by significantly increasing the intercontraction interval, the infused volume and the mean flow rate and decreasing the number of non-voiding contractions. SHR-T displayed a significant increase in prostate and bladder weights and a 15% increase in the detrusor wall area compared to WKY. CONCLUSIONS: Chronic silodosin treatment relieved storage symptoms in SHR supplemented with testosterone and decreased the frequency of non-voiding detrusor contractions during the filling phase.

Extracorporeal Shock Waves Therapy Delivered by Aries Improves Erectile Dysfunction in Spontaneously Hypertensive Rats Through Penile Tissue Remodeling and Neovascularization.

BACKGROUND: Low-intensity extracorporeal shock wave therapy (Li-ESWT) has been reported to improve erectile function in patients with moderate-to-severe erectile dysfunction (ED) or even convert phosphodiesterase type 5 inhibitors nonresponders to responders. ED is highly prevalent in hypertensive patients. The effect of Li-ESWT on an animal model of hypertension-associated ED has not been reported. AIM: To investigate the effect of Li-ESWT on hypertension-associated ED and provide plausible mechanisms of action of Li-ESWT on local mechanisms of penile erection. METHODS: Spontaneously hypertensive rats (SHRs) in the active group (n = 13) received Li-ESWT at energy flux density 0.06 mJ/mm2 (Aries; Dornier MedTech, Wessling, Germany) twice weekly for 6 weeks. The emitter was set to zero for SHRs in the sham group (n = 12). Erectile function was assessed 4 weeks post-treatment by monitoring intracavernosal pressure (ICP) in response to electrical stimulation of cavernous nerve before and after single dose of 0.3 mg/kg intravenous sildenafil. Cavernosal tissue was then evaluated for collagen/smooth muscle content, neuronal nitric oxide synthase (nNOS), and vascular endothelial factor (CD31) expression. OUTCOMES: Erectile function was assessed with ICP, erectile tissue remodeling was studied by smooth muscle/collagen ratio, nNOS and CD31 were semiquantitatively evaluated on cavernosal sections. RESULTS: The improvement of ICP parameters was greater in Li-ESWT-treated rats compared with controls with and without sildenafil. Sildenafil led to 20% increase in area under the intracavernosal pressure curve measured during the entire response/mean arterial pressure at 10 Hz in ESWT_SHR + sildenafil compared with ESWT_SHR. The smooth muscle/collagen ratio increased 2.5-fold in Li-ESWT compared with sham. Expression of CD31 tended to be increased whereas nNOS was unchanged. CONCLUSIONS: Li-ESWT by Aries may represent an effective noninvasive therapeutic alternative and a relevant add-on therapy to phosphodiesterase type 5 inhibitors for ED in hypertensive patients, and it is suggested that it acts via remodeling of the penile tissue and promoting cavernosal vascularization. Assaly R, Giuliano F, Clement P, et al. Extracorporeal Shock Waves Therapy Delivered by Aries Improves Erectile Dysfunction in Spontaneously Hypertensive Rats Through Penile Tissue Remodeling and Neovascularization. Sex Med 2019;7:441-450.

The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes.

INTRODUCTION: Following the results of the EMPA-REG Outcome trial, we hypothesized that empagliflozin, a highly potent and specific sodium/glucose cotransporteur 2 inhibitor, could improve type 2 diabetes mellitus (T2DM)-associated erectile dysfunction (ED), a highly prevalent complication of T2DM, very often coexisting with cardiovascular complications and considered as a prognostic factor of cardiovascular disease in men with diabetes. AIM: To investigate the effects of chronic treatment with empagliflozin on ED in a T2DM rat model in the presence or absence of sildenafil. METHODS: Male Goto-Kakizaki (GK), a model of T2DM, and age-matched Wistar rats received placebo or empagliflozin treatment at 25.3 ± 0.9 mg/kg/d for 4 weeks. Then, the in vivo effect of empagliflozin on erectile function was assessed by electrical stimulation of the cavernous nerve at different frequencies under anesthesia in the presence or absence of acute intravenous injection of sildenafil. Endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from the rats were studied. MAIN OUTCOME MEASURES: Body weight, food consumption, metabolic parameters, plasma inflammation biomarkers, and in vivo erectile responses elicited by electrical stimulation of the cavernous nerve in empagliflozin-treated and untreated GK rats and control Wistar rats were assessed and followed by concentration or frequency response curves to endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from these rats. RESULTS: Chronic empagliflozin followed by acute sildenafil significantly improved erectile responses in adult GK rats (n = 12-15/group). Ratios of intracavernous pressure and area under the curve/mean arterial pressure during the electrical stimulation were significantly increased in empagliflozin-treated vs untreated GK rats. Nitrergic relaxations of cavernosal strips from GK rats were significantly increased with empagliflozin compared with placebo. Moreover, the effect of sildenafil on erectile function was not altered by empagliflozin treatment. CLINICAL IMPLICATIONS: Empagliflozin may benefit T2DM patient with ED. STRENGTHS & LIMITATIONS: The mechanism(s) by which empagliflozin shows favorable effect on erectile function in GK rats needs to be further elucidated. CONCLUSION: Empagliflozin shows favorable effect on erectile function in diabetic GK rats mediated by an improvement of nitrergic relaxation of erectile tissue. Whether this favorable effect on ED in the experimental context of T2DM is due to better glycemic control or to another effect of empagliflozin deserves further investigation. Assaly R, Gorny D, Compagnie S, et al. The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes. J Sex Med 2018;15:1224-1234.

Low Intensity Extracorporeal Shock Wave Therapy Improves Erectile Function in a Model of Type II Diabetes Independently of NO/cGMP Pathway.

PURPOSE: Erectile dysfunction is highly prevalent in type II diabetes mellitus. Low intensity extracorporeal shock wave therapy improves erectile function in patients with erectile dysfunction of vasculogenic origin, including diabetes. However, its mode of action remains unknown. We investigated the effects of low intensity extracorporeal shock wave therapy compared to or combined with sildenafil on erectile dysfunction in a type II diabetes mellitus model. Our purpose was to test our hypothesis of a mode of action targeting the cavernous nitric oxide/cyclic guanosine monophosphate pathway. MATERIALS AND METHODS: GK rats, a validated model of type II diabetes mellitus, and age matched Wistar rats were treated with low intensity extracorporeal shock wave therapy twice weekly for 3 weeks. Treatment was repeated after a 3-week no-treatment interval. The penis was stretched and dipped in a specifically designed water-filled cage. Shock waves were delivered by a calibrated probe yielding a controlled energy flux density (0.09 mJ/mm(2)). The probe was attached to an electrohydraulic unit with a focused shock wave source, allowing for accurate extrapolation to humans. Following a 4-week washout period erectile function was assessed as well as endothelium dependent and independent, and nitrergic relaxations of the corpus cavernosum of GK rats. RESULTS: Low intensity extracorporeal shock wave therapy significantly improved erectile function in GK rats to the same extent as sildenafil. Treatment effects were potentiated when combined with sildenafil. Shock wave effects were not associated with improved cavernous endothelium dependent or independent, or nitrergic reactivity. CONCLUSIONS: Low intensity extracorporeal shock wave therapy improved erectile function in GK rats. Unexpectedly, this was not mediated by a nitric oxide/cyclic guanosine monophosphate dependent mechanism. Sildenafil increased shock wave efficacy. This preclinical paradigm to deliver low intensity extracorporeal shock wave therapy to the rat penis should help further exploration of the mode of action of this therapy on erectile tissue.

Bladder and erectile dysfunctions in the Type 2 diabetic Goto-Kakizaki rat.

Despite the fact that urogenito-sexual complications significantly impact the quality of life of diabetic patients, a robust in vivo experimental model is lacking. Bladder and erectile function in the Type 2 diabetic Goto-Kakizaki (GK) rat and responses to standard-of-care treatments for each disorder have been assessed. GK rats (n = 25, 18-wk-old, GK/Par colony) and age-matched Wistar rats (n = 23), characterized for their metabolic parameters, were used. Bladder function was assessed by cystometry in conscious rats treated by intravenous solifenacin (1 mg/kg). Subsequently, erectile function was assessed under anesthesia following electrical stimulation of the cavernous nerve in presence of intravenous sildenafil (0.3 mg/kg). GK rats displayed detrusor overactivity with a significant increase in frequency/amplitude of nonvoiding contractions during the filling phase, together with an increase in bladder capacity, intercontraction interval, voided volume, and maximal pressure of voiding contraction. Solifenacin significantly decreased parameters characterizing voiding contractions without modifying voiding efficiency. Erectile function in GK rats was markedly impaired and remained so after sildenafil treatment despite a significant improvement. GK rats display both bladder and erectile dysfunctions and respond at least partially to standard-of-care treatments for each disorder, thus representing a suitable model to investigate the pathophysiology and assess the efficacy of new therapeutic agents for Type 2 diabetes-associated bladder and erectile complications.

Inhibition of ejaculation by the non-peptide oxytocin receptor antagonist GSK557296: a multi-level site of action.

BACKGROUND AND PURPOSE: Oxytocin (OT) plays a major role in the control of male sexual responses. Notably, blockade of OT receptors has been reported to inhibit ejaculation in animals. The study aimed to investigate the action of a highly selective, non-peptide OT antagonist GSK557296 in a model of pharmacologically induced ejaculation in anaesthetized rats. The site of action was assessed by investigating different delivery routes for this compound. EXPERIMENTAL APPROACH: Urethane-anaesthetized Wistar rats were implanted with a cerebral ventricle cannula for i.c.v. injections or with a subdural catheter for intrathecal (i.t.) GSK557296 injections. Occurrence of ejaculation was assessed following i.v. 7-hydroxy-2-(di-N-propylamino)tetralin (7-OH-DPAT), a dopamine D3 receptor agonist. In addition, seminal vesicle pressures (SVP) and bulbospongiosus muscle (BS) EMG were recorded as physiological markers of emission and expulsion phases of ejaculation respectively. KEY RESULTS: Highest i.v. GSK557296 dose reduced occurrence of ejaculation and increases in SVP but had no effect on BS-EMG. I.c.v. GSK557296 dose dependently inhibited ejaculation, increases in SVP and BS contractions. At spinal thoracic level, GSK557296 dose dependently inhibited ejaculation and increases in SVP but BS-EMG was impaired only with the highest dose. When delivered at lumbar level, GSK557296 dose dependently inhibited ejaculation, increases in SVP and BS contractions. CONCLUSIONS AND IMPLICATIONS: In the 7-OH-DPAT-induced ejaculation model, GSK557296 acts peripherally and centrally to inhibit ejaculation with different modalities. Blockade of brain OT receptors seems to be the most effective mechanism of action. Targeting central OT receptors with highly selective antagonist seems a promising approach for the treatment of premature ejaculation.

Effect of dapoxetine on ejaculatory performance and related brain neuronal activity in rapid ejaculator rats.

INTRODUCTION: A brain network specifically activated when ejaculation occurs has been described in rats. Increasing serotonin (5-hydroxytryptamine [5-HT]) tone impairs ejaculation and chronic 5-HT selective serotonin reuptake inhibitors (SSRIs) are known to inhibit ejaculation. However, efficacy of acute treatment with SSRI varies from one compound to another. The SSRI dapoxetine has been reported to delay ejaculation when given on demand to men with premature ejaculation (PE), although the mechanism of action is unclear. Effects of acute SSRIs on activity of the brain ejaculation circuit in relation with ejaculation have never been examined. AIM: To test the effects of acute administration of the short half-life SSRI dapoxetine on ejaculatory performance and activity in brain ejaculation circuit in rapid ejaculator rats taken as PE model. METHODS: Standard copulatory test was used to attribute one sexual category (sluggish, middle, or rapid) to male rats on the basis of their ejaculatory performance. Parameters of sexual, including ejaculatory, behavior, and Fos level of expression in discrete brain areas were assessed in the three sexual categories and in rapid category following acute oral treatment with dapoxetine. MAIN OUTCOME MEASURES: Ejaculation frequency (EF) and latency (EL) were measured as primary end points of ejaculatory behavior. Density of Fos-immunopositive cells in specific brain areas of brain stem, hypothalamus, and thalamus was determined as marker of neuronal activity. RESULTS: EL and Fos level of expression in hypothalamic and thalamic structures were found related. Dapoxetine acute oral administration (300 mg/kg) to rapid ejaculator rats resulted in (i) diminution of ejaculatory performance (lengthened EL and decreased EF); and (ii) modulation of Fos level of expression in hypothalamic and thalamic nuclei of the brain ejaculatory circuit. CONCLUSION: Acute treatment with dapoxetine, which reduced ejaculatory performance in rapid ejaculator rats, was also accompanied with changes in neuronal activity in components of the brain ejaculatory network.

A new experimental rat model of erectile dysfunction and lower urinary tract symptoms associated with benign prostatic hyperplasia: the testosterone-supplemented spontaneously hypertensive rat.

UNLABELLED: What's known on the subject? and What does the study add? Lower urinary tract symptoms (LUTS) resulting from benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) are common problems in the aging male population. Moreover, several recent studies have shown that ED is closely associated with the presence and severity of LUTS independently of co-morbidities. However, the pathophysiological mechanisms linking LUTS/BPH and ED remain largely unexplored. The major difficulty in studying such relationships between ED and LUTS/BPH, and of exploring the impact of new therapeutic approaches for both LUTS/BPH and ED, is the lack of experimental model combining ED, prostate enlargement and bladder dysfunction all at once. The present study describes a new model of BPH, the SHR supplemented with testosterone which is the first animal model which displays all at once the key features of BPH: prostate enlargement and an increased sympathetic tone of bladder outlet mimicking the static and the dynamic components of voiding symptoms of BPH, a significant impairment of bladder function which reflects the storage symptoms of BPH and finally, ED. This model could be very relevant to better characterize the close relationship that exists between BPH/LUTS and ED, and to evaluate new therapeutic strategies for BPH together with their side effect profile on sexual function on the same animal, thus allowing a reduction of the number of animals to be used in such studies. Study Type - Aetiology (case control) Level of Evidence 3b. OBJECTIVE: • To design a new experimental model combining erectile dysfunction, prostate enlargement and urodynamic impairment characteristic of lower urinary tract symptoms (LUTS) associated with benign prostate hyperplasia (BPH). MATERIALS AND METHODS: • Three groups of animals (12-week-old; n= 7/group) were considered: Wistar Kyoto (control) rats (WKY), untreated spontaneously hypertensive rats (SHR) and SHR treated with testosterone (SHR-T, 3 mg/kg/day) for 3 weeks. • Cystometry experiments and evaluation of erectile function were performed. Prostate enlargement was evaluated. RESULTS: • SHR displayed a significant decrease in the intercontraction interval (ICI) and in the voided volume (VV) whereas non-voiding contractions (NVC) were increased. SHR-T exhibited a further decreased ICI and VV and an increased frequency of NVC. • Erectile responses to electrical stimulation of the cavernous nerve were significantly impaired in both SHR (-66%) and SHR-T (-58%). • The prostate weight was similar in WKY and SHR, but significantly increased in SHR-T. CONCLUSIONS: • The testosterone-supplemented SHR represents an experimental model for urodynamic impairment combining both static and dynamic components of voiding symptoms with erectile dysfunction and prostate enlargement. • This model is suitable for the assessment of sexual side effects of LUTS/BPH treatments and efficacy of new therapeutic agents in LUTS/BPH and associated erectile dysfunction.

Minimal effective dose of dysport and botox in a rat model of neurogenic detrusor overactivity.

BACKGROUND: Two botulinum toxins A have been evaluated for the treatment of refractory neurogenic detrusor overactivity (NDO) in humans: Dysport (abobotulinumtoxinA) and Botox (onabotulinumtoxinA). However, these two distinct commercialized products have different potency units and are not interchangeable. OBJECTIVE: Assessment of the dose response and determination of minimal effective dose (MED) for Dysport and Botox in spinal cord-injured (SCI) rats with NDO. DESIGN, SETTING, AND PARTICIPANTS: Female, adult, Sprague-Dawley rats (n=98) underwent T8-T9 spinal cord transection. Nineteen days after spinal cord injury, rats received intradetrusor injections (25µl injected, eight sites) of vehicle (V); Dysport 2, 5, 7.5, 10, and 12.5 U; and Botox 0.8, 2, 5, 7.5, and 10 U. Two days after injection, continuous cystometry was performed in conscious rats. MEASUREMENTS: Voiding contractions (VC) were assessed by duration of VC, intercontraction interval, voided volume, maximal pressure, pressure threshold change, and intravesical baseline pressure (BP), while nonvoiding contractions (NVC) were evaluated by amplitude, frequency, and volume threshold to elicit NVC. MEDs for Dysport and Botox were determined by analysis of variance step-down trend test. RESULTS AND LIMITATIONS: MEDs for Dysport and Botox were 10 U and 7.5 U, respectively. Regarding VC, only BP significantly decreased after 10 U Dysport and 7.5 U Botox compared to V (from 3.7±0.6 to 1.5±0.1 and 1.4±0.3mm Hg, respectively; p<0.01 and p<0.001, respectively). Dysport (10 U) and Botox (7.5 U) significantly inhibited NVC by decreasing their amplitude (from 7.4±1.1 to 5.8±0.5 and 5.4±0.6mm Hg, respectively; p<0.05); frequency (from 2.2±0.4 to 1.5±0.2 and 1.3±0.3 NVC per minute, respectively; p<0.01); and increasing volume threshold to elicit NVC (from 29.8±3.7 to 47.6±6.9 and 47.7±6.3%, respectively; p<0.05 and p<0.001, respectively). CONCLUSIONS: This is the first preclinical dose-ranging study with Dysport and Botox under standardized conditions showing similar inhibiting effects on NDO, albeit at different MEDs. It highlights the importance of distinguishing each preparation for predicted outcomes and doses to be used. Further studies in patients with NDO are warranted to confirm these experimental results.

Combination of BAY 60-4552 and vardenafil exerts proerectile facilitator effects in rats with cavernous nerve injury: a proof of concept study for the treatment of phosphodiesterase type 5 inhibitor failure.

BACKGROUND: Radical prostatectomy (RP) is frequently responsible for erectile dysfunction (ED). Post-RP patients often show a failure to respond to phosphodiesterase type 5 (PDE5) inhibitors. OBJECTIVE: The acute effect of BAY 60-4552, the soluble guanylate cyclase (sGC) stimulator, and vardenafil were evaluated alone or in combination on erectile responses to electrical stimulation of the cavernous nerve (ES CN) in rats with cavernous nerve (CN) crush injury-induced ED. DESIGN, SETTING, AND PARTICIPANTS: Male adult Sprague-Dawley rats underwent laparotomy (sham, n=10) or bilateral CN crush injury (n=56). After 3 wk of recovery, erectile function was evaluated under urethane anaesthesia following ES CN at different frequencies. MEASUREMENTS: The acute effects of intravenous (IV) injection of vehicle, vardenafil 0.03 mg/kg, BAY 60-4552 0.03 mg/kg or 0.3 mg/kg, or a BAY 60-4552 0.03 mg/kg plus vardenafil 0.03 mg/kg combination were evaluated in CN-crushed rats. RESULTS AND LIMITATIONS: Bilateral CN crush injury followed by a 3-wk recovery period decreased erectile responses to ES CN by about 50%. In CN-crushed rats, IV vardenafil 0.03 mg/kg and BAY 60-4552 (0.03 or 0.3 mg/kg) increased erectile responses to ES CN to the same extent: Δ intracavernosal pressure/mean arterial pressure (ICP/MAP) at 10 Hz ES CN was 21±1% after vehicle, 25±3% (p<0.001) after vardenafil, and 26±5% and 27±5% after BAY 60-4552 0.03 mg/kg (p<0.01) and 0.3 mg/kg (p<0.001), respectively. The combination of vardenafil with BAY 60-4552 in CN-crushed rats totally restored erectile responses to ES CN equivalent to sham rats (ΔICP/MAP at 10 Hz ES CN: 34±4% after BAY 60-4552/vardenafil combination vs 39±4% in sham rats; not significant). CONCLUSIONS: The present study supports the concept that the combined administration of a sGC stimulator, BAY 60-4552, and vardenafil provides synergistic beneficial effects and might therefore salvage patients who experience treatment failures with PDE5 inhibitors after RP.

Vardenafil decreases bladder afferent nerve activity in unanesthetized, decerebrate, spinal cord-injured rats.

BACKGROUND: Phosphodiesterase type 5 inhibitors (PDE5-Is) improve storage symptoms in benign prostatic hyperplasia patients, despite a lack of effect on peak urinary flow rate. Moreover, vardenafil improves urodynamic parameters in spinal cord-injured (SCI) patients with neurogenic detrusor overactivity (NDO). SCI rats also display NDO characterized by nonvoiding contractions (NVCs) during bladder filling, resulting in an increased bladder afferent nerve firing (BANF). OBJECTIVE: We postulated that vardenafil could improve urodynamic parameters by reducing BANF. The effect of vardenafil has been investigated on intravesical pressure by cystometry experiments while recording BANF in response to bladder filling. DESIGN, SETTING, AND PARTICIPANTS: Complete T7-T8 spinalization was performed in 15 female adult Sprague-Dawley rats (250-275 g). MEASUREMENTS: At 21-29 d postspinalization, fine filaments were dissected from the L6 dorsal roots and placed across a bipolar electrode. Bladder afferent nerve fibers were identified by electrical stimulation of the pelvic nerve and bladder distension. SCI rats were decerebrated before cystometry experiments. Bladders were filled to determine the maximal bladder filling volume (BFV) for each rat. Then, after bladder stabilization at 75% of maximal BFV, saline (n=7) or vardenafil 1 mg/kg (n=8) was delivered intravenously. NVCs and BANF were recorded for 45 min. RESULTS AND LIMITATIONS: In all SCI rats, BANF was already present and regular at resting conditions (26.2±4.1 spikes per second). During bladder filling, intravesical pressure (IVP) slowly increased with transient NVCs superimposed. Concomitantly, BANF progressively increased up to 2.4-fold at maximal BFV (2.08±0.24 ml). After stabilization at submaximal BFV, BANF was increased by 186±37%. Vardenafil injection induced an immediate decrease in NVCs compared to saline (p<0.001) and BANF (52% decrease vs 28% in saline after 45 min; p<0.001). CONCLUSIONS: Systemic vardenafil reduced both NVCs and BANF in unanesthetized, decerebrate, SCI rats. These findings provide new insights into the mechanism of action by which PDE5-Is improve storage symptoms in SCI patients.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain.

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina.

INTRODUCTION: The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. AIM: To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). METHODS: Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. MAIN OUTCOME MEASURE: Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. RESULTS: The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. CONCLUSION: These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function.

Combination of alfuzosin and tadalafil exerts an additive relaxant effect on human detrusor and prostatic tissues in vitro.

BACKGROUND: Lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) are highly prevalent in aging men and are strongly linked. Alpha1-blockers such as alfuzosin are effective monotherapies for LUTS. Phosphodiesterase type 5 (PDE5) inhibitors such as tadalafil are the first-line treatment for ED. Both drugs act by two different mechanisms of action on common urogenital target organs and, thus, may have additive effects. OBJECTIVES: We evaluated in vitro the effects of alfuzosin, tadalafil, and the combination of both on human detrusor and prostatic smooth muscle. DESIGN, SETTING, AND PARTICIPANTS: Prostatic and bladder tissue were obtained from patients (n=20 and n=17, respectively) undergoing cystoprostatectomy for bladder cancer. MEASUREMENTS: In organ baths, isolated prostatic strips and isolated bladder strips were incubated with vehicle, tadalafil (10⁻6 M and 10⁻5 M), alfuzosin (3×10⁻8 M or 10⁻6 M and 10⁻5 M) or a combination. Concentration-response curves (CRCs) to norepinephrine were generated on prostatic strips and detrusor strips precontracted with carbachol. Strips were also submitted to electrical field stimulation (EFS). RESULTS AND LIMITATIONS: When alfuzosin and tadalafil were combined, the maximal relaxation to norepinephrine on carbachol-precontracted detrusor strips was significantly increased compared with tadalafil alone, and EFS-induced detrusor contractions were better inhibited compared with each compound alone. Tadalafil significantly inhibited norepinephrine-induced prostatic strip contractions by reducing the maximal effect, whereas alfuzosin shifted the CRC of norepinephrine to the right. Combining both tadalafil and alfuzosin resulted in a greater relaxant effect. Likewise, the combination was more effective at reducing EFS-induced contractions compared with each compound alone. CONCLUSIONS: The combination of alfuzosin and tadalafil exerts an additive effect of inhibiting adrenergic smooth muscle tone of prostatic tissue and EFS-induced detrusor contractions and conversely, of enhancing adrenergic relaxation of detrusor precontracted with carbachol. These experiments provide experimental support for the clinical investigation of the combination of α1-blockers and PDE5 inhibitors in the treatment of LUTS.

How does chronic sildenafil prevent vascular oxidative stress in insulin-resistant rats?

INTRODUCTION: Insulin resistance features both endothelial dysfunction and increased oxidative stress. Both disorders are targeted by a chronic treatment with sildenafil. However, the mechanism of action by which chronic sildenafil exerts its effects on reactive oxygen species sources is still largely unknown. AIM: We therefore investigated how chronic sildenafil administration could impact vascular endothelial NO and superoxide release in a rat model of insulin resistance induced by fructose overload. METHODS: Adult male Wistar rats were fed a fructose-enriched diet (fructose-fed rats [FFR]) for 9 weeks. From weeks 6-8, sildenafil was administered subcutaneously twice daily (20 mg/kg), followed by a 1-week washout. MAIN OUTCOME MEASURES: Vascular endothelial NO and superoxide release were monitored in vitro in thoracic aortic segments using oxidative fluorescence. Specific inhibitors were used to distinguish the respective role of the main superoxide-producing systems within the vascular wall (i.e., mitochondrial respiratory chain and NADPH oxidases). The levels of expression of eNOS, Akt, and NADPH oxidase subunits were determined in the abdominal aorta. RESULTS: Chronic sildenafil administration corrected hyperglycemia, hyperinsulinemia, and hypertriglyceridemia in FFR. Moreover, after 9 weeks of diet, while global unstimulated aortic endothelial NO and superoxide release were unchanged in FFR, the relative contribution of the mitochondrial respiratory chain and NADPH oxidases was modified. Chronic sildenafil treatment, even after the 1-week washout period, was able to increase endothelial NO release independently of Akt-dependent phosphorylation by up-regulating eNOS expression, and restored the relative contribution of each superoxide-producing system examined, yielding endothelial superoxide release. Finally, in vitro incubation of aortic segments with sildenafil markedly decreased the endothelial aortic superoxide release. CONCLUSIONS: The present study showed that chronic sildenafil produced sustained vascular antioxidant effects in insulin-resistant rats by increasing NO release and regulating vascular superoxide release, supporting therefore further investigations using chronic sildenafil administration in preventing cardiovascular alterations associated with oxidative stress.

Differences in the spinal command of ejaculation in rapid ejaculating rats.

INTRODUCTION: It has been hypothesized that lifelong premature ejaculation is part of a biological variation in the intravaginal ejaculation latency, but what causes this variation remains poorly understood. AIM: The aim of this study is to elucidate whether variations in ejaculation latencies in an experimental rat model for premature ejaculation are linked to differences in the spinal command of ejaculation. MAIN OUTCOME MEASURES: Electrical microstimulation of the spinal generator for ejaculation revealed an accelerated expulsion phase in rapid ejaculating rats. METHODS: Adult male Wistar rats were categorized as "sluggish,""normal," or "rapid" ejaculators on the basis of their ejaculation frequency in sexual mating tests. One to three weeks after selection, males were urethane anesthetized and electrically microstimulated in the spinal generator for ejaculation, evoking ejaculation. Bulbospongiosus muscle electromyographic and intraluminal vas deferens pressure were measured simultaneously, representing, respectively, the expulsion and emission phase in ejaculation. RESULTS: Electrical microstimulation of the spinal generator for ejaculation evoked ejaculation in "sluggish" (N = 9), "normal" (N = 13), and "rapid" (N = 11) ejaculating rats. Vas deferens contraction (emission phase) was evoked at different stimulation strengths, but response properties were not statistically different between "sluggish,""normal," and "rapid" ejaculator rats. Bulbospongiosus muscle contractions (expulsion phase) following microstimulation was significantly accelerated in "rapid" rats as compared with "sluggish" and "normal" rats. The total duration of bulbospongiosus muscle contractions remained unchanged between the three ejaculator groups. CONCLUSIONS: Our results provide the first scientific evidence supporting a neurophysiological difference between "rapid,""normal," and "sluggish" ejaculators, expressed as an accelerated expulsion phase in "rapid" ejaculator rats. This bridges the gap between a sexual behavior trait and the spinal command of ejaculation.

Delay of ejaculation induced by SB-277011, a selective dopamine D3 receptor antagonist, in the rat.

INTRODUCTION: Dopamine (DA) plays a key role in different aspects of the male sexual response, including sexual motivation and arousal, penile erection, and ejaculation. The modalities of action of DA are however unclear, although the various DA receptors may differentially mediate the activity of DA in different aspects of the male sexual response. AIM: To clarify the role of DA D(3) receptors in the control of the male sexual response. METHODS: The effects of a highly selective DA D(3) receptors antagonist (SB-277011; intraperitoneal) were tested in experimental paradigms exploring several aspects of the male sexual response in (i) anesthetized rats using 7-hydroxy-N,N-di-n-propylaminotetralin to induce ejaculation and (ii) conscious rats using sexual incentive motivation and mating tests. MAIN OUTCOME MEASURES: Physiological markers of erection and emission and expulsion phases of ejaculation were measured in anesthetized rats. Behavioral parameters of sexual incentive motivation and mating tests were quantified. RESULTS: In anesthetized rats, we found that SB-277011 specifically and dose-dependently inhibited the expulsion phase of ejaculation without impairing either emission phase or erection, and this resulted in delayed ejaculation. Administration of SB-277011 had no effect on the spontaneous preference that males displayed for sexually receptive females as shown in sexual incentive motivation test. Delayed ejaculation was confirmed when male rats were administered with the highest dose of SB-277011 (10 mg/kg) in mating test, where males were free to copulate with estrous females. In addition, the refractory period following ejaculation was lengthened in rats treated with SB-277011. CONCLUSION: As a whole, the present data demonstrate the specific and primary role of D(3) receptors in the expulsion phase of ejaculation and provide preclinical evidence for the investigation of the therapeutic potential of D(3) antagonism for treating premature ejaculation.

Combination of doxazosin and sildenafil exerts an additive relaxing effect compared with each compound alone on human cavernosal and prostatic tissue.

INTRODUCTION: Phosphodiesterase 5 inhibitors (PDE5) such as sildenafil are first-line treatment for erectile dysfunction (ED). Alpha1 (alpha1)-adrenoceptor antagonists such as doxazosin are indicated for the treatment of patients with lower urinary tract symptoms (LUTS)/benign prostatic hyperplasia (BPH). ED and LUTS/BPH are conditions that are often associated. Accordingly, alpha1-adrenoceptor antagonists and PDE5 inhibitors will be often prescribed in real life setting together. AIM: To evaluate the effects of the combination of sildenafil and doxazosin on human cavernosal and prostatic tissue. METHODS: Prostatic and erectile tissues were obtained from nine to 12 patients, respectively. Patients underwent cystoprostatectomy for infiltrating bladder cancer or penile surgery for penile implant, congenital curvature or Peyronie's disease. MAIN OUTCOME MEASURES: In organ baths, prostatic and cavernosal strips were submitted to either concentration-response curves (CRC) to phenylephrine (Phe) or norepinephrine (NE), respectively, in presence of vehicle, sildenafil (10(-6) M, 10(-5) M), doxazosin (10(-8) M, 3.10(-8) M, or 10(-7) M), or a combination of both. Continuous electrical field stimulation (EFS; 32 Hz, 5 ms, 5 seconds, 300 mA) was performed on prostatic strips which were incubated with sildenafil 10(-6) M or vehicle before the successive addition of doxazosin (10(-7) M, 10(-6) M) or vehicle. Cavernosal strips were pre-incubated with doxazosin (10(-9) M, 10(-8) M) or vehicle, then CRC to sildenafil were constructed on NE (3.10(-6) M) precontracted cavernosal strips. RESULTS: Combination of sildenafil and doxazosin exerted a greater relaxing effect on CRC to Phe or NE compared with each compound alone in both tissues. Sildenafil significantly enhanced the relaxing effect of doxazosin on EFS-induced contractions in prostatic strips. Doxazosin significantly increased the ability of sildenafil to inhibit NE-induced contractions in cavernosal strips. CONCLUSIONS: Sildenafil and doxazosin reduced adrenergic tone of prostatic and cavernosal smooth muscle and their combination provided a significant benefit when targeting relaxation of both tissues. These experiments provide support for further clinical evaluation of the sildenafil and doxazosin combination in ED patients with LUTS/BPH.

Role of the neurokinin-1 receptors in ejaculation in anesthetized rats.

INTRODUCTION: Several lines of evidence indicate a role for substance P in the control of ejaculation, although its mode of action needs to be clarified. AIM: The effects and sites of action of a selective antagonist for the substance P-preferred receptor (neurokinin-1 receptor subtype; NK1) were investigated in a pharmacological model of ejaculation. METHODS: Ejaculation was induced in anesthetized rats by intracerebroventricular (i.c.v.) delivery of the dopamine D3 receptor preferring agonist 7-hydroxy-2-(di-N-propylamino)tetralin (7-OH-DPAT). The effects of the selective NK1 antagonist RP67580 on 7-OH-DPAT-induced ejaculation were measured following intraperitoneal (i.p.), i.c.v., or intrathecal (i.t.) (third lumbar spinal segment; L3) administration. MAIN OUTCOME MEASURES: Intraseminal vesicle pressure (SVP) and electromyogram of the bulbospongiosus muscle (BS) were recorded as physiological markers of emission and expulsion phases of ejaculation, respectively. RESULTS: Upon i.p., i.c.v., or i.t. administration, RP67580 significantly reduced the occurrence of ejaculation elicited by 7-OH-DPAT. A mild decrease in the occurrence of SVP and BS responses was observed in rats treated ip with RP67580, whereas only SVP responses were moderately affected following i.c.v. or i.t. administration. CONCLUSION: These results show the multilevel regulation of 7-OH-DPAT-induced ejaculation by NK1 receptors.

Impact of a long-term sildenafil treatment on pressor response in conscious rats with insulin resistance and hypertriglyceridemia.

BACKGROUND: Insulin resistance constitutes a risk factor for endothelial dysfunction and subsequent cardiovascular diseases including hypertension. Daily treatment with phosphodiesterase type 5 (PDE5) inhibitors has beneficial effects on endothelial function in men with increased cardiovascular risk. Endothelium-dependent vasomotor function is ultimately linked to blood pressure (BP) regulation. We postulated that sildenafil would ameliorate BP and biological markers of endothelial function in fructose-fed rats (FFRs). METHODS: Wistar rats were fed a standard chow or a 60% fructose-enriched diet containing 12% fat for 8 weeks (FFR). From week 6 through 8, sildenafil (twice a day subcutaneously, 20 mg/kg) was administered followed by a 1-week washout period. At the end of the washout period, BP was recorded using radiotelemetry following cumulative infusion of norepinephrine (from 50 to 400 ng/kg/min). RESULTS: FFR displayed both an impaired glucose tolerance and elevated triglyceridemia. The latter was corrected by sildenafil treatment. Resting BP was similar in all rats, whereas pressor responses were significantly enhanced in FFR (maximal increase in mean BP to norepinephrine: 25.6 +/- 3.8 vs. 40.8 +/- 4.0 mm Hg, P < 0.05) and normalized by sildenafil treatment (24.9 +/- 5.3 mm Hg, not significant vs. control). Urinary levels of 8-isoprostanes and thromboxane B(2) were increased in FFR and corrected by sildenafil treatment. CONCLUSION: Thus, chronic treatment with sildenafil normalized BP regulation in an experimental model of insulin resistance and hypertriglyceridemia while restoring normal excretion of urinary biological markers of oxidative stress and cyclooxygenase-derived vasoconstrictors. The modulation of ROS and cyclooxygenase-derived vasoconstrictors generation by a chronic treatment with sildenafil may represent an added benefit beyond PDE5 inhibition.