The Rho-kinase inhibitor Y27632 promotes ureteral relaxation in an in vivo rat model for partial ureteral obstruction.

PURPOSE: Aim of this study was to evaluate the effect of intravenous Y27632 (a ROK inhibitor) on intra-ureteral pressures and on blood pressure in an in vivo rat model for unilateral partial ureteral obstruction (PUO). METHODS: 15 Male Sprague Dawley rats were used. Under isofluran anesthesia, saline was continuously infused via polyethylene (PE)-10 catheters inserted in the ureters beneath the kidney pelvis. Left psoas muscle was sutured around the distal left ureter to create a partial obstruction. Carotid artery and femoral vein were cannulated with PE catheters for registration of mean arterial blood pressure (MAP) and for administration of drugs. Left and right ureter pressures and MAP were simultaneously recorded. Y27632 (0.03 and 0.1 mg/kg each n = 6-7) was given intravenously. T-test was used for comparisons. RESULTS: Spontaneous peristaltic pressure waves were recorded at baseline for both ureters. After the obstruction, Y27632 reduced maximum pressure (MaxP) by 10.5 ± 1.9% (0.03 mg/kg; p = 0.004) and 29.1 ± 4.8% (0.1 mg/kg; p < 0.001), minimum pressure (MinP) by 5.2 ± 2.3% (0.03 mg/kg; p = 0.02) and 12.2 ± 3.4% (0.1 mg/kg; p = 0.009), the area under the curve (AUC) by 7.8 ± 2.4% (0.03 mg/kg; p = 0.008) and 16.5 ± 3.7% (0.1 mg/kg;p = 0.007), the waves amplitude by 23.4 ± 11.3% (0.03 mg/kg; p = 0.098) and 38.7 ± 7.5% (0.1 mg/kg; p < 0.001), with no effect on contraction frequency. During simultaneous recordings from the normal ureter at the investigated doses, Y27632 reduced MaxP, MinP, AUC and waves amplitude by 1-7%. The MAP was reduced by 12.5 ± 5.3% (0.03 mg/kg; p = 0.07) and 15.8 ± 1.8% (0.1 mg/kg; p < 0.001). CONCLUSIONS: Y27632 decreased intra-ureteral pressures of a partially obstructed ureter with limited effect on blood pressure in an animal model of unilateral PUO.

Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human seminal vesicles.

Background and Aims: The transient receptor potential cationic channel ankyrin 1 (TRPA1), a channel protein permeable to most divalent cations, has been suggested to play a role in mechano-afferent/efferent signaling (including the release of neurotransmitters) in the human urinary tract (bladder, prostate, and urethra). To date, only a few studies have addressed the expression of this receptor in male and female reproductive tissues. The present study aimed to evaluate human seminal vesicles (SVs)  for the expression and localization of TRPA1. Methods: SV tissue was obtained from 5 males who had undergone pelvic surgery due to malignancies of the prostate or urinary bladder. The expression of messenger ribonucleic acid (mRNA) specifically encoding for the TRPA1 protein was elucidated by means of reverse transcriptase polymerase chain reaction (RT-PCR). Using immunohistochemical methods, the distribution of TRPA1 was examined in relation to the endothelial and neuronal nitric oxide synthases (eNOS, nNOS) and the neuropeptides calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). Results: RT-PCR revealed signals related to the expected molecular size of 656 bp. Immunohistochemistry demonstrated that TRPA1 is located in nerves running through the smooth muscle portion of the SV. Here, the protein is in part co-localized with nNOS and CGRP, whereas no co-localization with VIP was registered. Dot-like signals specific for TRPA1 were observed in the cytoplasm of epithelial cells lining the lumen of glandular spaces. The epithelial layer also presented staining for eNOS. The smooth musculature appeared free of immunosignals for TRPA1. Conclusion: The results convincingly show the expression of TRPA1 in nerve endings as well as in epithelial cells of the SV. Based on its location in epithelial cells, TRPA1 might be involved in the mechanism of the NO/cyclic guanosine monophosphate (GMP)-mediated signaling and also the control of secretory function (mediated by cyclic GMP) in the human SV.

Long-term consequences of bilateral cavernous crush injury in normal and diabetic rats: a functional study.

A recent statement from the European-Society-for-Sexual-Medicine has highlighted the limitations of using the rat model for nerve-sparing prostatectomy. The use of young rats with no comorbidities and the early evaluation of the erectile function (EF) are deemed a source of bias. Our aim was to evaluate the long-term consequences in EF of bilateral nerve cavernous crush- injury (BNCI) in type 1 diabetic (DM) rats 30-male/12-week-old rats were divided into four groups: Sham, BNCI, DM, and BNCI + DM. Sham group underwent an intraperitoneal injection (IP) of saline solution and after 1 month underwent a sham laparotomy. BNCI underwent an IP of saline solution and after 1 month to BNCI. DM underwent an IP of 60 mg/kg-1-streptozotocin (STZ) and after 1 month to a sham laparotomy. BNCI + DM underwent an IP of 60 mg/kg-1-STZ and after 1 month to BNCI. After 5 months from the induction of diabetes, all rats underwent measurement of intracorporeal pressure (ICP) and mean arterial pressure (MAP) during CN-electrostimulation. Multiple groups were compared using Kruskal-Wallis one-way analysis of variance followed by Mann-Whitney U test for post hoc comparisons. Blood glucose-level was higher (p < 0.05) in the groups with DM and BNCI + DM. After 5-months, DM and BNCI + DM also showed a lower weight compared to other groups (p < 0.05). No differences were noted in ICP/MAP between the sham and BNCI. BNCI + DM showed lower ICP/MAP compared to all the groups (p < 0.05). DM Showed lower ICP/MAP compared to Sham and BNCI (p < 0.05). BNCI in rats without comorbidities did not induce long-term erectile dysfunction (ED) suggesting a spontaneous EF recovery. BNCI in DM induced long-term ED. The results of previous short-term studies can only provide evidence on the time to recovery of spontaneous EF as to the actual EF recovery rate.

The transient receptor potential A1 ion channel (TRPA1) modifies in vivo autonomous ureter peristalsis in rats.

AIMS: The current study aimed to explore the expression of transient receptor potential A1 ion channels (TRPA1) in the rat ureter and to assess if TRPA1-active compounds modulate ureter function. METHODS: The expression of TRPA1 in rat ureter tissue was studied by immunofluorescence. The TRPA1 distribution was compared to calcitonin gene-related peptide (CGRP), α-actin (SMA1), anoctamin-1 (ANO1), and c-kit. For in vivo analyses, a catheter was implanted in the right ureter of 50 rats. Ureter peristalsis and pressures were continuously recorded by a data acquisition set-up during intraluminal infusion of saline (baseline), saline plus protamine sulfate (PS; to disrupt the urothelium), saline plus PS with hydrogen sulfide (NaHS) or cinnamaldehyde (CA). Comparisons were made between rats treated systemically with vehicle or a TRPA1-antagonist (HC030031). RESULTS: TRPA1-immunoreactive nerves co-expressed CGRP and were mainly located in the suburothelial region of the ureter. Immunoreactivity for TRPA1 was also encountered in c-kit-positive but ANO1-negative cells of the ureter suburothelium and wall. In vivo, HC030031-treated rats had elevated baseline peristaltic frequency (p < 0.05) and higher intraluminal pressures (p < 0.01). PS increased the frequency of ureter peristalsis versus baseline in vehicle-treated rats (p < 0.001) but not in HC030031-treated rats. CA (p < 0.001) and NaHS (p < 0.001) decreased ureter peristalsis. This was counteracted by HC030031 (p < 0.05 and p < 0.01). CONCLUSIONS: In rats, TRPA1 is expressed on cellular structures considered of importance for peristaltic and mechanoafferent functions of the ureter. Functional data indicate that TRPA1-mediated signals regulate ureter peristalsis. This effect was pronounced after mucosal disruption and suggests a role for TRPA1 in ureter pathologies involving urothelial damage.

Intratunical injection of autologous adipose stromal vascular fraction reduces collagen III expression in a rat model of chronic penile fibrosis.

Previous studies have shown that the injection of adipose stem cells and stromal vascular fraction(SVF) into the tunica albuginea (TA) during the inflammatory phase in a rat model of Peyronie's disease(PD) prevented the development of TA fibrosis. Our aim was to investigate whether local injection of SVF can reduce established fibrosis in a rat model of chronic phase of PD. Eighteen-male 12-wk-old Sprague-Dawley rats were divided in three equal groups: sham, PD without treatment (PD) and PD treated with SVF(PD-SVF). Sham rats underwent 2 injections of vehicle into the TA one month apart. PD rats underwent TGF-ß1 injection and injection of vehicle one month later. PD-SVF rats underwent TGF-ß1 injection followed by SVF (1-million cells) one month later. One month after the last treatment, the animals, n = 6 rats per group, underwent measurement of intracorporal and mean arterial pressure during electrostimulation of the cavernous nerve. Following euthanasia, penises were harvested for in-vitro study. Erectile function was not statistically significantly different between groups. PD animals developed subtunical areas of fibrosis and elastosis with upregulation of collagen III protein. These fibrotic changes were reversed after injection of SVF. We provide evidence that local injection of SVF reverses TA fibrosis in a rat model of chronic phase of PD.

Arginase enzymes in the human prostate: A molecular biological and immunohistochemical approach.

The nitric oxide (NO) pathway plays a role in maintaining the function of the prostate. An impairment in the activity of the NO system may have an impact in the manifestation of lower urinary tract symptomatology and benign prostatic hyperplasia. Arginase enzymes (Arg) counteract the generation of NO by depleting the intracellular pool of L-arginine, known to be the substrate of the NO synthases. This study investigated the expression of arginase type I and II in the human prostate. Nondiseased prostate tissue was obtained during pelvic surgeries (prostatectomy, cystoprostatectomy). Tissue sections were exposed to antibodies directed against Arg I and II, cGMP, the phosphodiesterase 5 and nNOS. The expression of mRNA transcripts encoding for Arg I and Arg II was investigated using molecular biology. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for Arg I and II, immunofluorescence specific for Arg I was seen in the stromal smooth musculature, and labelling for PDE5 and cyclic GMP was also observed. Nerve fibres containing nNOS were identified running across the smooth musculature. Immunostainings for Arg II did not yield signals. These findings are in support of the notion that, in the prostate, Arg is involved in the modulation of the activity of the NO system.

Intratunical injection of stromal vascular fraction prevents fibrosis in a rat model of Peyronie's disease.

OBJECTIVE: To investigate whether local injection of autologous adipose stromal vascular fraction (SVF) can prevent the development of fibrosis and elastosis in the tunica albuginea (TA) using a rat model of the acute phase of Peyronie's disease (PD). METHODS: A total of 24 male 12-week-old Sprague-Dawley rats were divided into three equal groups: sham; PD without treatment (transforming growth factor-ß [TGF -ß]); and PD treated with SVF 1 day after disease induction. Sham rats received two injections of vehicle into the TA 1 day apart. TGF -ß rats received TGF- ß1 injection and injection of vehicle 1 day later. SVF rats received TGF-ß1 injection, followed by SVF 1 day later. One month after treatment, all rats underwent measurement of intracavernosal pressure and mean arterial pressure during electrostimulation of the cavernous nerve. The rats were then killed and penises were harvested for histology and Western blot analysis. RESULTS: Erectile function was moderately reduced in the TGF-ß group and was significantly improved after SVF treatment (P < 0.05). PD rats developed areas of fibrosis with a significant upregulation of collagen III, collagen I and elastin protein expression. These fibrotic changes were prevented when treated with SVF. CONCLUSIONS: Local injection of SVF may represent treatment for the acute phase of PD.

Intratunical Injection of Human Adipose Tissue-Derived Stem Cells Restores Collagen III/I Ratio in a Rat Model of Chronic Peyronie's Disease.

INTRODUCTION: Previous studies have shown that the injection of adipose tissue-derived stem cells (ADSCs) into the tunica albuginea (TA) during the active phase of Peyronie's disease (PD) prevents the development of fibrosis. AIM: To investigate, using an animal model, whether local injection of human ADSCs (hADSCs) can alter the degree of fibrosis in the chronic phase of PD. METHODS: 27 male, 12-week-old rats were divided into 3 equal groups: sham, PD without treatment, and PD treated with hADSCs 1 month after disease induction. Sham rats underwent 2 injections of vehicle into the TA 1 month apart. PD rats underwent transforming growth factor ß1 (TGFß1) injection and injection of vehicle 1 month later. PD-hADSC rats underwent TGFß1 injection followed by 1 million hADSCs 1 month later. 1 week after treatment, n = 3 animals/group were euthanized, and the penises were harvested for quantitative polymerase chain reaction. 1 month after treatment, the other animals, n = 6 per group, underwent measurement of intracavernous pressure (ICP) and mean arterial pressure (MAP) during electrostimulation of the cavernous nerve. After euthanasia, penises were again harvested for histology and Western blot. MAIN OUTCOME MEASURE: The primary outcome measures included (a) gene expression at one week post-injection; (b) measurement of ICP/MAP upon cavernous nerve stimulation as a measure of erectile function; (c) elastin, collagen I and III protein expression; and (d) Histomorphometric analysis of the penis. Means where compared by analysis of variance (ANOVA) followed by a Student-Newman-Keuls test for post hoc comparisons or Mann-Whitney test when applicable. RESULTS: No significant difference was noted in ICP or ICP/MAP in response to cavernous nerve electrostimulation between the 3 groups at 2.5, 5, and 7.5 V (P > .05 for all voltages). PD animals developed tunical and subtunical areas of fibrosis with a significant upregulation of collagen III protein. The collagen III/I ratio was higher in the PD (4.6 ± 0.92) group compared with sham (0.66 ± 0.18) and PD-hADSC (0.86 ± 0.06) groups (P < .05) These fibrotic changes were prevented when treated with hADSCs. Compared with PD rats, PD-hADSC rats demonstrated a decreased expression of several fibrosis-related genes. CONCLUSION: Injection of hADSCs reduces collagen III expression in a rat model of chronic PD. Castiglione F, Hedlund P, Weyne E, et al. Intratunical Injection of Human Adipose Tissue-Derived Stem Cells Restores Collagen III/I Ratio in a Rat Model of Chronic Peyronie's Disease. Sex Med 2019;7:94-103.

Transient receptor potential a1 (TRPA1) agonists inhibit contractions of the isolated human ureter.

AIMS: Mechanoafferent and peristaltic mechanisms of the human ureter involve transient receptor potential V1 (TRPV1)- and purinoceptor-mediated functions. Hydrogen sulphide, an endogenous TRPA1 ligand, is linked to inhibitory neurotransmission of the pig ureter. No information is available on TRPA1 activity in the human ureter. We therefore examined the distribution and function of TRPA1 in the human ureter. METHODS: Expression of TRPA1 in human ureter tissue was studied by Western blot and immunofluorescence. The TRPA1 distribution was compared to TRPV1, calcitonin gene related peptide (CGRP), tyrosine hydroxylase (TH), and vimentin. Effects of the TRPA1 agonists allyl isothiocyanate (AI), cinnamaldehyde (CA), sodium hydrogen sulfide (NaHS), and capsaicin (TRPV1 agonist) on human ureter preparations were studied in organ baths. RESULTS: By Western blot, bands were detected at the expected molecular weight for TRPA1. TRPA1- and TRPV1-immunoreactivities were located on CGRP-positive nerves, but not on TH-positive nerves. TRPA1 was also located in vimentin-positive interstitial cells. In functional experiments, neither of the TRPA1-agonists (1-100 µM) had any direct effects on ureter tension (baseline/potassium-induced contractions). However, CA, AI, NaHS, and capsaicin (10 µM) decreased (P < 0.01-0.05) tetrodotoxin-sensitive electrically induced (2,4,8,16,32 Hz) contractions. Inhibitory activities were 50-61% (CA), 30-56% (AI), 30-40% (NaHS), and 37-67% (Capsaicin). CONCLUSIONS: In the human ureter, TRPA1 is located to sensory nerves and interstitial cells. TRPA1 agonists inhibited electrically induced contractions but had no direct effect on smooth muscle tension of the human ureter. A role for TRPA1 in modulating neurotransmission and possibly peristalsis of the human ureter is proposed.

Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism.

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (-50 ± 3%) and sphingosine 1-phosphate (S1P, -40 ± 4%), which ended up to reduction in cell motility (-46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

M1 Macrophages Are Predominantly Recruited to the Major Pelvic Ganglion of the Rat Following Cavernous Nerve Injury.

INTRODUCTION: Neurogenic erectile dysfunction is a common sequela of radical prostatectomy. The etiology involves injury to the autonomic cavernous nerves, which arise from the major pelvic ganglion (MPG), and subsequent neuroinflammation, which leads to recruitment of macrophages to the injury site. Currently, two macrophage phenotypes are known: neurotoxic M1 macrophages and neuroprotective M2 macrophages. AIM: To examine whether bilateral cavernous nerve injury (BCNI) in a rat model of erectile dysfunction would increase recruitment of neurotoxic M1 macrophages to the MPG. METHODS: Male Sprague-Dawley rats underwent BCNI and the MPG was harvested at various time points after injury. The corpora cavernosa was used to evaluate tissue myographic responses to electrical field stimulation ex vivo. Quantitative real-time polymerase chain reaction was used to examine the gene expression of global macrophage markers, M1 macrophage markers, M2 macrophage markers, and cytokines and chemokines in the MPG. Mathematical calculation of the M1/M2 index was used to quantify macrophage changes temporally. Western blot of MPG tissues was used to evaluate the protein amount of M1 and M2 macrophage markers quantitatively. Immunohistochemistry staining of MPGs for CD68, CD86, and CD206 was used to characterize M1 and M2 macrophage infiltration. MAIN OUTCOME MEASURES: Corpora cavernosa responsiveness ex vivo; gene (quantitative real-time polymerase chain reaction) and protein (western blot) expressions of M1 and M2 markers, cytokines, and chemokines; and immunohistochemical localization of M1 and M2 macrophages. RESULTS: BCNI impaired the corporal parasympathetic-mediated relaxation response to electrical field stimulation and enhanced the contraction response to electrical field stimulation. Gene expression of proinflammatory (Il1b, Il16, Tnfa, Tgfb, Ccl2, Ccr2) and anti-inflammatory (Il10) cytokines was upregulated in the MPG 48 hours after injury. M1 markers (CD86, inducible nitric oxide synthase, interleukin-1ß) and M2 markers (CD206, arginase-1, interleukin-10) were increased after BCNI in the MPG, with the M1/M2 index above 1.0 indicating that more M1 than M2 macrophages were recruited to the MPG. Protein expression of the M1 macrophage marker (inducible nitric oxide synthase) was increased in MPGs after BCNI. However, the protein amount of M2 macrophage markers (arginase-1) remained unchanged. Immunohistochemical characterization demonstrated predominant increases in M1 (CD68+CD86+) macrophages in the MPG after BCNI. CONCLUSION: These results suggest that an increase in M1 macrophage infiltration of the MPG after BCNI is associated with impaired neurogenically mediated erectile tissue physiology ex vivo and thus has significant implications for cavernous nerve axonal repair. Future studies are needed to demonstrate that inhibition of M1 macrophage recruitment prevents erectile dysfunction after CNI.

Impaired contraction and decreased detrusor innervation in a female rat model of pelvic neuropraxia.

INTRODUCTION AND HYPOTHESIS: Bilateral pelvic nerve injury (BPNI) is a model of post-radical hysterectomy neuropraxia, a common sequela. This study assessed the time course of changes to detrusor autonomic innervation, smooth muscle (SM) content and cholinergic-mediated contraction post-BPNI. METHODS: Female Sprague-Dawley rats underwent BPNI or sham surgery and were evaluated 3, 7, 14, and 30 days post-BPNI (n = 8/group). Electrical field-stimulated (EFS) and carbachol-induced contractions were measured. Gene expression was assessed by qPCR for muscarinic receptor types 2 (M2) and 3 (M3), collagen type 1α1 and 3α1, and SM actin. Western blots measured M2 and M3 protein expression. Bladder sections were stained with Masson's trichrome for SM content and immunofluorescence staining for nerve terminals expressing vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), and neuronal nitric oxide synthase (nNOS). RESULTS: Bilateral pelvic nerve injury caused larger bladders with less SM content and increased collagen type 1α1 and 3α1 gene expression. At early time points, cholinergic-mediated contraction increased, whereas EFS-mediated contraction decreased and returned to baseline by 30 days. Protein and gene expression of M3 was decreased 3 and 7 days post-BPNI, whereas M2 was unchanged. TH nerve terminals surrounding the detrusor decreased in all BPNI groups, whereas VAChT and nNOS terminals decreased 14 and 30 days post-BPNI. CONCLUSIONS: Bilateral pelvic nerve injury increased bladder size, impaired contractility, and decreased SM and autonomic innervation. Therapeutic strategies preventing nerve injury-mediated decline in neuronal input and SM content may prevent the development of a neurogenic bladder and improve quality of life after invasive pelvic surgery.

The endocannabinoid system - a target for the treatment of LUTS?

Lower urinary tract symptoms (LUTS) are common in all age groups and both sexes, resulting in tremendous personal suffering and a substantial burden to society. Antimuscarinic drugs are the mainstay of symptom management in patients with LUTS, although their clinical utility is limited by the high prevalence of adverse effects, which often limit patients' long-term adherence to these agents. Data from controversial studies in the 1990s revealed the positive effects of marijuana-based compounds on LUTS, and sparked an interest in the possibility of treating bladder disorders with cannabis. Increased understanding of cannabinoid receptor pharmacology and the discovery of endogenous ligands of these receptors has prompted debate and further research into the clinical utility of exogenous cannabinoid receptor agonists relative to the unwanted psychotropic effects of these agents. Currently, the endocannabinoid system is considered as a potential drug target for pharmacological management of LUTS, with a more favourable adverse event profile than antimuscarinic agents.

Adipose-derived Stem Cells Counteract Urethral Stricture Formation in Rats.

BACKGROUND: A medical treatment for urethral stricture (US) is not yet available. OBJECTIVE: To evaluate if local injection of human adipose tissue-derived stem cells (hADSC) prevents urethral fibrosis in a rat model of US. DESIGN, SETTING, AND PARTICIPANTS: Male rats were divided into three groups: sham, US, and hADSC (n=12 each). Sham rats received a vehicle injection in the urethral wall. US and hADSCs were incised and injected with the fibrosis-inducer transforming growth factor-ß1 in the urethral wall. INTERVENTION: One day later, hADSCs were injected in the urethral wall of hADSC rats whereas sham and US rats were injected with the vehicle. After 4 wk, the rats underwent cystometries and tissues were then harvested for functional and molecular analyses. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cystometry, microultrasound, histochemistry, organ bath studies, reverse transcription polymerase chain reaction, and western blot. RESULTS AND LIMITATIONS: US rats exhibited 49-51% shorter micturition intervals, 35-51% smaller micturition volumes and bladder capacity, 33-62% higher threshold pressures and flow pressures, and 35-37% lower bladder filling compliance compared with hADSC-treated rats and sham rats (p<0.05). By ultrasound, US rats had hyperechogenic and thick urethral walls with narrowed lumen compared with sham rats, whereas hADSC rats displayed less extensive urethral changes. Isolated detrusor from US rats exhibited 34-55% smaller contractions than detrusor from sham rats (p<0.05). Corresponding values were 11-35% for isolated detrusors from hADSC rats. Collagen and elastin protein expression were increased in the penile urethras of US rats compared with sham and hADSC groups (p<0.05). Endothelial and inducible nitric oxide synthase expressions were higher (p<0.05) in the hADSC group. Compared with US rats, hADSC rats demonstrated decreased expression of several fibrosis-related genes. Administration of hADSCs was performed at an early stage of US development, which we consider a limitation of the study. CONCLUSIONS: Local injection of hADSCs prevents stricture formation and urodynamic complications in a new rat model for US. PATIENT SUMMARY: Stem cell therapy is effective for preventing urethral stricture in an experimental setting.

Current Pharmacological Management of Premature Ejaculation: A Systematic Review and Meta-analysis.

CONTEXT: Premature ejaculation (PE) is the most prevalent male sexual dysfunction. In the last few years, several pharmacologic approaches for oral or topical treatment of PE have been studied. OBJECTIVE: To systematically review the literature on the outcome of pharmacologic interventions for PE on intravaginal ejaculation latency time (IELT) in comparison to placebo. EVIDENCE ACQUISITION: A systematic literature search of PubMed and Scopus using the term "premature ejaculation" was performed on 10 April 2015. Full-text articles on prospective randomized controlled trials (RCTs) investigating pharmacotherapy were included. The main outcome measure was IELT. EVIDENCE SYNTHESIS: Out of 266 unique records, a total of 22 were reviewed. The majority of RCTs were of unclear methodological quality because of limited reporting of methods. Pooled evidence suggests that selective serotonin reuptake inhibitors (SSRIs), topical anesthetic creams (TAs), tramadol, and phosphodiesterase type 5 inhibitors (PDE5is) are more effective than placebo at increasing IELT (all p<0.05). However, interpretation of the current meta-analyses may be impaired as a result of frequent heterogeneity in the pooled analyses (all I(2) > 70%). Only pooled analyses for dapoxetine 30mg and 60mg were characterized by homogeneous data (both I(2)<30%) while showing a modest but statistically significant improvement in IELT compared with placebo (mean difference 1.39min, 95% confidence interval 1.23-1.54min; p<0.00001). CONCLUSIONS: Meta-analysis revealed that treatment with dapoxetine significantly improves IELT in patients with PE but with modest efficacy. The efficacy of SSRIs, TAs, tramadol, and PDE5is remains unclear owing to high heterogeneity of the available RCT data. There is a persisting need for drug research and development in the field. PATIENT SUMMARY: Premature ejaculation is a condition for which the cause is not well understood. Several types of treatment with medium to low efficacy are available. More research is necessary to identify the ideal treatment.

DFL23448, A Novel Transient Receptor Potential Melastin 8-Selective Ion Channel Antagonist, Modifies Bladder Function and Reduces Bladder Overactivity in Awake Rats.

The transient receptor potential melastin 8 ion channel (TRPM8) is implicated in bladder sensing but limited information on TRPM8 antagonists in bladder overactivity is available. This study characterizes a new TRPM8-selective antagonist (DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol]) and evaluates it in cold-induced behavioral tests and tests on bladder function and experimental bladder overactivity in vivo in rats. DFL23448 displayed IC50 values of 10 and 21 nM in hTRPM8 human embryonic kidney 293 cells activated by Cooling Agent 10 or cold, but it had limited activity (IC50 > 10 µM) at transient receptor potential vanilloids TRPV1, TRPA1, or TRPV4 or at various G protein-coupled receptors. In rats, DFL23448 administered intravenously or orally had a half-life of 37 minutes or 4.9 hours, respectively. DLF23448 (10 mg/kg i.v.) reduced icilin-induced "wet dog-like" shakes in rats. Intravesical DFL23448 (10 mg/l), but not vehicle, increased micturition intervals, micturition volume, and bladder capacity. During bladder overactivity by intravesical prostaglandin E2 (PGE2), vehicle controls exhibited reductions in micturition intervals, micturition volumes, and bladder capacity by 37%-39%, whereas the same parameters only decreased by 12%-15% (P < 0.05-0.01 versus vehicle) in DFL23448-treated rats. In vehicle-treated rats, but not in DFL23448-treated rats, intravesical PGE2 increased bladder pressures. Intravenous DFL23448 at 10 mg/kg, but not 1 mg/kg DFL23448 or vehicle, increased micturition intervals, micturition volumes, and bladder capacity. During bladder overactivity by intravesical PGE2, micturition intervals, micturition volumes, and bladder capacity decreased in vehicle- and 1 mg/kg DFL23448-treated rats, but not in 10 mg/kg DFL23448-treated rats. Bladder pressures increased less in rats treated with DFL23448 10 mg/kg than in vehicle- or 1 mg/kg DFL23448-treated rats. DFL23448 (10 mg/kg i.v.), but not vehicle, prevented cold stress-induced bladder overactivity. Our results support a role for bladder TRPM8-mediated signals in experimental bladder overactivity.

Spinal neuronal cannabinoid receptors mediate urodynamic effects of systemic fatty acid amide hydrolase (FAAH) inhibition in rats.

AIMS: To test if urodynamic effects from systemic Fatty Acid Amide Hydrolase (FAAH) inhibition involve sacral spinal cannabinoid type 1 (CB1) or type 2 (CB2) receptors. METHODS: Male rats with or without partial urethral obstruction were used for cystometry or immunohistochemistry. Urodynamic effects of intravenous (IV) 0.3 mg/kg Oleoyl Ethyl Amide (OEtA; FAAH inhibitor), and intrathecal (IT) 5 µg rimonabant (CB1 antagonist) or 5 µg SR144528 (CB2 antagonist) were studied in awake rats. RESULTS: After administration of rimonabant or SR144528, non-obstructed rats with normal bladder function developed bladder overactivity (BO), which was counteracted by OEtA. OEtA also counteracted BO in obstructed rats. SR144528 did not affect bladder function in obstructed rats but counteracted the urodynamic effects of OEtA. Surprisingly, rimonabant (and AM251, another CB1 antagonist) reduced BO in obstructed rats, whereafter OEtA produced no additional urodynamic effects. CB1 expression increased in the sacral spinal cord of obstructed rats whereas no changes were observed for CB2 or FAAH. CONCLUSIONS: Urodynamic effects of systemic FAAH inhibition involve activities at spinal neuronal CB1 and CB2 receptors in normal and obstructed rats. Endogenous spinal CB receptor ligands seem to regulate normal micturition and BO. Altered spinal CB receptor functions may be involved in the pathogenesis of obstruction-induced BO. Neurourol. Urodynam. 35:464-470, 2016. © 2015 Wiley Periodicals, Inc.

URB937, a peripherally restricted inhibitor for fatty acid amide hydrolase, reduces prostaglandin E2 -induced bladder overactivity and hyperactivity of bladder mechano-afferent nerve fibres in rats.

OBJECTIVE: To determine if inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) can counteract the changes in urodynamic variables and bladder afferent activities induced by intravesical prostaglandin E2 (PGE2 ) instillation in rats. MATERIALS AND METHODS: In female Sprague-Dawley rats we studied the effects of URB937, a peripherally restricted FAAH inhibitor, on single-unit afferent activity (SAA) during PGE2 -induced bladder overactivity (BO). SAA measurements were made in urethane-anaesthetised rats and Aδ- and C-fibres were identified by electrical stimulation of the pelvic nerve and by bladder distention. Cystometry (CMG) in conscious animals and during SAA measurements was performed during intravesical instillation of PGE2 (50 or 100 µm) after intravenous administration of URB937 (0.1 and 1 mg/kg) or vehicle. In separate experiments, the comparative expressions of FAAH and cannabinoid receptors, CB1 and CB2 , in microsurgically removed L6 dorsal root ganglion (DRG) were studied by immunofluorescence. RESULTS: During CMG, 1 mg/kg URB937, but not vehicle or 0.1 mg/kg URB937, counteracted the PGE2 -induced changes in urodynamic variables. PGE2 increased the SAAs of C-fibres, but not Aδ-fibres. URB937 (1 mg/kg) depressed Aδ-fibre SAA and abolished the facilitated C-fibre SAA induced by PGE2 . The DRG nerve cells showed strong staining for FAAH, CB1 and CB2 , with a mean (sem) of 77 (2)% and 87 (3)% of FAAH-positive nerve cell bodies co-expressing CB1 or CB2 immunofluorescence, respectively. CONCLUSION: The present results show that URB937, a peripherally restricted FAAH inhibitor, reduces BO and C-fibre hyperactivity in the rat bladder provoked by PGE2 , suggesting an important role of the peripheral endocannabinoid system in BO and hypersensitivity.

Expression and distribution of phosphodiesterase isoenzymes in the human male urethra.

OBJECTIVE: To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. METHODS: Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. RESULTS: Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. CONCLUSION: The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.