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.

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 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.

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.

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.

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.

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.

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.

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.

Inhibition of peripheral FAAH depresses activities of bladder mechanosensitive nerve fibers of the rat.

PURPOSE: FAAH degrades endocannabinoids and fatty acid amides. FAAH inhibition reduces micturition frequency and counteracts bladder overactivity in rats. We studied the effects of the peripherally active selective FAAH inhibitor URB937, and the CB1 and CB2 receptor antagonists rimonabant and SR144528, respectively, on single unit afferent activity of primary bladder afferents in rats. MATERIALS AND METHODS: Female Sprague Dawley® rats were anesthetized. Single unit afferent activity of Aδ or C-fibers from the L6 dorsal roots was recorded during bladder filling before and after URB937 administration with or without rimonabant or SR144528. Drugs (1 mg/kg) were given intravenously. FAAH, CB1 and CB2 expression, and expression of the sensory marker CGRP in the L6 dorsal root ganglion were compared by immunofluorescence. RESULTS: A total of 102 single afferent fibers (48 Aδ and 54 C-fibers) were isolated from 57 rats. URB937 decreased single unit afferent activity of C-fibers to a mean ± SEM of 78% ± 9% and of Aδ-fibers to a mean of 67% ± 7% while increasing bladder compliance to a mean of 116% ± 3%. The effects of URB937 on single unit afferent activity and bladder compliance were counteracted by rimonabant or SR144528. Rimonabant increased single unit afferent activity of each fiber type but SR144528 affected only Aδ-fiber activity. CGRP positive L6 dorsal root ganglion neurons showed strong FAAH, CB1 and CB2 staining. CONCLUSIONS: To our knowledge we report for the first time that inhibiting peripheral FAAH depresses the Aδ and C-fiber activity of primary bladder afferents via CB1 and CB2 receptors. CB antagonists alone exerted facilitatory effects on single unit afferent activity during bladder filling in rats. The endocannabinoid system may be involved in physiological control of micturition as regulators of afferent signals.

Bladder function in a cannabinoid receptor type 1 knockout mouse.

OBJECTIVE: To evaluate bladder function in an established cannabinoid type 1 (CB1) receptor knockout (KO) mouse model via organ-bath (in vitro) and urodynamic (cystometric; in vivo) experiments. MATERIALS AND METHODS: In all, 20 8-week-old female wildtype (WT) mice (C57BL/6) and 20 age-matched CB1 KO mice were used. Six mice from each group were used for the organ-bath experiments, where the contractile responses of bladder tissue strips after carbachol exposure (carbachol concentration response curve [CCRC]; myogenic contraction) and during electrical field stimulation (EFS; neurogenic contraction) were assessed. In all, 14 mice per group were used for cystometric experiments without any anaesthesia, in which standard urodynamic variables were assessed 3 days after bladder catheterisation. RESULTS: The CCRCs of bladder strips from CB1 KO mice were similar to those of WT mice. However, during EFS the bladder strips from the CB1 KO mice had significantly lower contractile responses than WT preparations, indicating that in CB1 KO mice the neuronal component of bladder contraction was different. In cystometric experiments the CB1 KO mice had a higher micturition frequency (shorter mean [sem] inter-micturition interval of 3.24 [0.29] vs 7.32 [0.5] min), a lower bladder capacity (0.09 [0.01] vs 0.18 [0.01] mL) and micturition volume (0.07 [0.01] vs 0.14 [0.01] mL), a lower bladder compliance (0.007 [0.001] vs 0.02 [0.002] mL/cmH2 O), and higher spontaneous bladder activity (5.1 [0.5] vs 2.6 [0.6] cmH2 O) than WT mice (all P < 0.05, Student's t-test). In WT mice, systemic administration of rimonabant (SR141716), a CB1 receptor antagonist, resulted in urodynamic changes similar to those seen in the CB1 KO mice. CONCLUSIONS: In vitro, bladder strips from CB1 KO mice responded to muscarinic receptor stimulation similarly as the WT controls, but were less responsive to electrical stimulation of nerves. In vivo, CB1 KO mice had a higher micturition frequency and more spontaneous activity than WT mice. The present findings suggest that CB1 receptors are involved in peripheral and central nervous control of micturition.

Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats.

INTRODUCTION: Bilateral cavernous nerve injury (BCNI) causes profound penile changes such as apoptosis and fibrosis leading to erectile dysfunction (ED). Histone deacetylase (HDAC) has been implicated in chronic fibrotic diseases. AIMS: This study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI-induced ED and penile fibrosis. METHODS: Five groups of rats (8-10 weeks, n = 10/group) were utilized: (i) sham; (ii and iii) BCNI 14 and 30 days following injury; and (iv and v) BCNI treated with HDAC inhibitor valproic acid (VPA 250 mg/kg; 14 and 30 days). All groups underwent cavernous nerve stimulation (CNS) to determine intracavernosal pressure (ICP). Penile HDAC3, HDAC4, fibronectin, and transforming growth factor-ß1 (TGF-ß1) protein expression (Western blot) were assessed. Trichrome staining and the fractional area of fibrosis were determined in penes from each group. Cavernous smooth muscle content was assessed by immunofluorescence to alpha smooth muscle actin (α-SMA) antibodies. MAIN OUTCOME MEASURES: We measured ICP; HDAC3, HDAC4, fibronectin, and TGF-ß1 protein expression; penile fibrosis; penile α-SMA content. RESULTS: There was a voltage-dependent decline (P < 0.05) in ICP to CNS 14 and 30 days after BCNI. Penile HDAC3, HDAC4, and fibronectin were significantly increased (P < 0.05) 14 days after BCNI. There was a slight increase in TGF-ß1 protein expression after BCNI. Histological analysis showed increased (P < 0.05) corporal fibrosis after BCNI at both time points. VPA treatment decreased (P < 0.05) penile HDAC3, HDAC4, and fibronectin protein expression as well as corporal fibrosis. There was no change in penile α-SMA between all groups. Furthermore, VPA-treated BCNI rats had improved erectile responses to CNS (P < 0.05). CONCLUSION: HDAC-induced pathological signaling in response to BCNI contributes to penile vascular dysfunction. Pharmacological inhibition of HDAC prevents penile fibrosis, normalizes fibronectin expression, and preserves erectile function. The HDAC pathway may represent a suitable target in preventing the progression of ED occurring post-radical prostatectomy.

Increased expression of the neuroregenerative peptide galanin in the major pelvic ganglion following cavernous nerve injury.

INTRODUCTION: Erectile dysfunction (ED) remains a frequent complication of radical prostatectomy due to injury to the cavernous nerves (CNs). A recent microarray showed the neuropeptide galanin to be one of the most strikingly upregulated genes in the rat major pelvic ganglion (MPG) after bilateral CN crush injury (BCNI). AIM: The aim of this study is to evaluate the temporal regulation of galanin in the MPG after BCNI and its relationship to functional nerve regeneration. METHODS: Changes in galanin, galanin receptor (galR), and c-JUN mRNA expression were assessed in Sprague-Dawley rats after sham operation (n = 10) and at 48 hours (n = 10), 7 (n = 10), 14 (n = 5), 21 (n = 5), 30 (n = 5), and 60 (n = 5) days after BCNI using quantitative PCR. Erectile function was assessed by measuring intracavernous pressure (ICP) divided by mean arterial pressure (MAP) during CN electrostimulation. Immunohistochemistry was performed on the MPG in sham-operated animals and 5 days after BCNI. MAIN OUTCOME MEASURES: ICP/MAP upon CN stimulation; galanin, galR1, -2, -3, and c-JUN mRNA expression at various time points after BCNI; and nNOS, galanin, and galR distribution in the MPG of sham-operated rats and after BCNI. RESULTS: After BCNI, ICP/MAP values quickly deteriorate, while after 60 days, spontaneous restoration of erectile responses to CN stimulation is observed, reflecting CN regeneration. Galanin mRNA in the MPG is up to 186-fold upregulated compared with sham-operated rats at 48 hours and 7 days after BCNI and gradually declines with increasing time from injury, whereas galanin receptor expressions decrease and c-JUN gradually increases. Galanin expression shows a strong inverse correlation with erectile responses to CN stimulation with time from injury. Injured MPGs show a colocalization between galanin- and nNOS-positive neuronal cell population in the MPG. CONCLUSIONS: Galanin is upregulated in the MPG in the early phase after CN injury after which it gradually decreases and is present in nNOS-positive neurons of the ganglion. We hypothesize that galanin upregulation is an important factor in the endogenous neuroregenerative response to CN injury.

Cannabinoids and the endocannabinoid system in lower urinary tract function and dysfunction.

AIMS: To review knowledge on cannabinoids and the endocannabinoid system in lower urinary tract function and dysfunction. METHODS: Review of MEDLINE using defined search terms, and manual analysis. Articles published in English were included. RESULTS AND DISCUSSION: Components of the endocannabinoid system­cannabinoid (CB)receptor types 1 and 2, anandamide, and fatty acid amide hydrolase (FAAH), which degrades anandamide and related fatty-acid amides­have been located to lower urinary tract tissues of mice, rats, monkeys, and humans. Studies have located CB receptors in urothelium and sensory nerves and FAAH in the urothelium. CB receptor- and FAAH-related activities have also been reported in the lumbosacral spinal cord. Data on supraspinal CB functions in relation to micturition are lacking. Cannabinoids are reported to reduce sensory activity of isolated tissues, cause antihyperalgesia in animal studies of bladder inflammation, affect urodynamics parameters reflecting sensory functions in animals models, and appear to have effects on storage symptoms in humans. FAAH inhibitors have affected sensory bladder functions and reduced bladder overactivity in rat models. Cannabinoids may modify nerve-mediated functions of isolated lower urinary tract tissues. CONCLUSIONS: Evidence suggests components of the endocannabinoid system are involved in regulation of bladder function, possibly at several levels of the micturition pathway. It is unclear if either CB receptor has a dominant role in modification of sensory signals or if differences exist at peripheral and central nervous sites. Amplification of endocannabinoid activity by FAAH inhibitors may be an attractive drug target in specific pathways involved in LUTS.

Characterization of bladder function in a cannabinoid receptor type 2 knockout mouse in vivo and in vitro.

AIMS: The contribution of individual CB receptors (CB1 R and CB2 R) to normal micturition has not been clearly defined. Our goal was to study if differences in urodynamic parameters or in vitro bladder contractility can be demonstrated between CB2 R knockout (CB2 RKO) and C57BL/6J control (wild type, WT) mice. METHODS: Female WT and CB2 RKO mice underwent bladder catheterization and cystometry was performed after 2 and 3 days. Cystometric evaluations were performed in awake animals without drug administration, and WT were also given HU-308 (CB2 R agonist) followed by AM630 (CB2 R antagonist). Bladders were removed for in vitro assessment of contractile responses to carbachol and electrical field stimulation (EFS). RESULTS: CB2 RKO mice had significantly higher intercontraction intervals (ICIs), bladder capacity (BC) and compliance (Bcom) than WT controls (P < 0.05). In WT mice, BC and ICI were increased from baseline by HU-308 exposure, and then returned to baseline levels after AM630 administration (P < 0.05). There were no differences in contractility after carbachol or EFS between the groups. CONCLUSIONS: Lack of CB2 R was associated with longer ICI and higher BC and Bcom than its presence (WT controls). This was unexpected since in WT, an increase in BC and ICI from baseline was observed after CB2 R agonist administration, and this action was reversed by a CB2 R antagonist. Since there were no differences in the in vitro responses to carbachol and EFS in bladder strips, it may be speculated that the urodynamic differences are caused by a change in the central nervous micturition control in CB2 RKO animals. Neurourol. Urodynam. 33:566-570, 2014. © 2013 Wiley Periodicals, Inc.

The fatty acid amide hydrolase inhibitor oleoyl ethyl amide counteracts bladder overactivity in female rats.

AIMS: To study micturition and bladder overactivity in female rats after chronic treatment with the fatty acid amide hydrolase (FAAH) inhibitor oleoyl ethyl amide (OEtA). METHODS: Sprague-Dawley rats received daily subcutaneous injections of OEtA (0.3 mg/kg), or vehicle for 2 weeks. Cystometries, organ bath studies, Western blot, and immunofluorescence were then used. Expressions of FAAH, cannabinoid 1 and 2 receptors (CB1 and CB2), mitogen-activated protein kinase (MAPK), vesicular acetyl choline-transporter protein (VAChT), and calcitonin gene-related peptide (CGRP) were evaluated. RESULTS: At baseline, OEtA-treated rats had higher values (P < 0.05) of micturition intervals (MI) and volumes (MV), bladder capacity (BC), threshold pressure, and flow pressure than vehicle controls. Intravesical PGE2 reduced MI, MV, and BC, and increased basal pressure and the area under the curve in all rats. However, these urodynamic parameters were altered less by intravesical PGE2 in OEtA-treated rats (P < 0.05 vs. vehicle controls). Compared to vehicle controls, detrusor from OEtA-treated rats had larger contractions to carbachol at 10-0.1 µM, but no difference in Emax was recorded. FAAH, CB1, CB2, VAChT, or CGRP was similarly expressed in bladders from all rats. In separate experiments, intravesical OEtA increased mucosal expression of phosphorylated MAPK. CONCLUSIONS: Chronic FAAH inhibition altered sensory urodynamic parameters and reduced bladder overactivity. Even if it cannot be excluded that OEtA may act on central nervous sensory pathways to contribute to these effects, the presence of FAAH and CB receptors in the bladder and activation of intracellular signals for CB receptors by intravesical OEtA suggest a local role for FAAH in micturition control.

Inhibition of Rho-kinase improves erectile function, increases nitric oxide signaling and decreases penile apoptosis in a rat model of cavernous nerve injury.

PURPOSE: Bilateral cavernous nerve injury results in up-regulation of ROCK signaling in the penis. This is linked to erectile dysfunction in an animal model of post-prostatectomy erectile dysfunction. We evaluated whether daily treatment with the ROCK inhibitor Y-27632 (Tocris Bioscience, Ellisville, Missouri) would prevent erectile dysfunction in a rat model of bilateral cavernous nerve injury. MATERIALS AND METHODS: Sprague-Dawley® rats underwent surgery to create sham (14) or bilateral (27) cavernous nerve injury. In the injury group 13 rats received treatment with Y-27632 (5 mg/kg twice daily) and 14 received vehicle. At 14 days after injury, rats underwent cavernous nerve stimulation to determine erectile function. Penes were assessed for neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase. ROCK2 was assessed by Western blot. Cyclic guanosine monophosphate was determined by enzyme-linked immunosorbent assay. Cavernous homogenates were tested for ROCK and protein kinase G enzymatic activity. Penile apoptosis was evaluated using the Apostain technique (Alexis, San Diego, California). Data were analyzed on ROCK using ANOVA and the t test. RESULTS: While erectile function was decreased in rats with bilateral cavernous nerve injury, daily administration of Y-27632 improved erectile responses. Injury decreased neuronal and nitric oxide synthase membrane-endothelial nitric oxide synthase but ROCK2 was significantly increased. Y-27632 treatment restored neuronal nitric oxide synthase, nitric oxide synthase membrane-endothelial nitric oxide synthase and cyclic guanosine monophosphate levels, and protein kinase G activity. Treatment significantly decreased ROCK2 protein and ROCK activity. There were significantly fewer apoptotic cells after treatment than in injured controls. CONCLUSIONS: These results provide evidence for up-regulation of the RhoA/ROCK signaling pathway with detrimental effects on erectile function after bilateral cavernous nerve injury. ROCK inhibition improved erectile dysfunction associated with bilateral cavernous nerve injury by preserving penile nitric oxide bioavailability and decreasing penile apoptosis.

Spinal cord FAAH in normal micturition control and bladder overactivity in awake rats.

PURPOSE: We assessed whether spinal inhibition of the cannabinoid degrading enzyme FAAH would have urodynamic effects in normal rats and rats with bladder overactivity induced by partial urethral obstruction or prostaglandin E2. We also determined the expression of FAAH, and the cannabinoid receptors CB1 and CB2 in the sacral spinal cord. MATERIALS AND METHODS: We used 44 rats for functional (cystometry) and Western blot experiments. The FAAH inhibitor oleoyl ethyl amide (3 to 300 nmol) was administered intrathecally (subarachnoidally) or intravenously. The expression of FAAH and CB1/CB2 receptors was determined by Western blot. RESULTS: Oleoyl ethyl amide given intrathecally affected micturition in normal rats and rats with bladder overactivity but effects were more pronounced in the latter. In normal rats oleoyl ethyl amide only decreased micturition frequency, while it decreased frequency and bladder pressures in rats with bladder overactivity. Intravenous oleoyl ethyl amide (3 to 300 nmol) had no urodynamic effect. FAAH and CB1/CB2 receptors were expressed in the rat sacral spinal cord. The expression of CB1/CB2 receptors but not FAAH was higher in obstructed than in normal rats. CONCLUSIONS: FAAH inhibition in the sacral spinal cord by oleoyl ethyl amide resulted in urodynamic effects in normal rats and rats with bladder overactivity. The spinal endocannabinoid system may be involved in normal micturition control and it appears altered when there is bladder overactivity.

Nerve-sparing approach during radical prostatectomy is strongly associated with the rate of postoperative urinary continence recovery.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Urinary incontinence and erectile dysfunction are the most bothersome sequelae affecting health-related quality of life in patients treated with radical prostatectomy for prostate cancer. While it has been widely reported that a nerve-sparing approach significantly improves postoperative erectile function, the impact of neurovascular bundle preservation on urinary continence recovery is still a matter of controversy. Our study clearly demonstrates that patients treated with nerve-sparing radical prostatectomy have higher chances of recovering full continence after surgery. The results indicate that, when technically and oncologically feasible, an attempt at a nerve-sparing approach should be planned in order to increase the probability of achieving full continence after radical prostatectomy. OBJECTIVE: To demonstrate that nerve-sparing radical prostatectomy (NSRP) is associated with higher rates of urinary continence (UC) recovery compared with non-nerve-sparing procedures in patients with surgically treated organ-confined prostate cancer. PATIENTS AND METHODS: The study included 1249 patients treated with radical prostatectomy between 2003 and 2010. Patients were divided into three preoperative risk groups: low (PSA < 10 ng/mL, cT1, biopsy Gleason sum ≤ 6), high (cT3 or biopsy Gleason 8-10 or PSA > 20 ng/mL) and intermediate (all the remaining). Postoperative UC recovery was defined as the absence of any protection device. The association between nerve-sparing status and UC recovery was assessed in univariable and multivariable Cox regression analyses after accounting for age at surgery, Charlson Comorbidity Index and preoperative risk group. RESULTS: At a mean follow-up of 42.2 months (range 1-78), 993 patients (79.5%) recovered UC. Overall, UC recovery rate at 1 and 2 years was 76% and 79%, respectively. On univariable Cox regression analysis, age at surgery, preoperative risk group, medical comorbidities and nerve-sparing status were significantly associated with UC recovery (all P ≤ 0.001). On multivariable analysis, age, risk group and nerve-sparing status were also independently associated with UC recovery (all P < 0.003). Patients treated with bilateral NSRP had a 1.8-fold higher chance of full UC recovery. CONCLUSIONS: Patients treated with bilateral NSRP have significantly higher chances of recovering full continence. Therefore, when oncologically and technically feasible, a nerve-sparing procedure should be attempted.