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Purpose We systematically reviewed preclinical and clinical studies on bladder chemodenervation with onabotulinumtoxin A to highlight current limitations and future drug delivery approaches. Materials and Methods We identified peer reviewed basic and clinical research studies of onabotulinumtoxin A in the treatment of neurogenic bladder and refractory idiopathic overactive bladder published between March 2000 and March 2016. Paired investigators independently screened 125 English language articles to identify controlled studies on onabotulinumtoxin A administration in the MEDLINE® database and abstracts presented at annual American Urological Association meetings. The review yielded an evidence base of more than 50 articles relevant to the approach of injection-free onabotulinumtoxin A chemodenervation. Results The efficacy and safety of intradetrusor injection of onabotulinumtoxin A for the treatment of overactive bladder are sensitive to injection volume and depth, and this issue has motivated researchers to study injection-free modes of drug delivery into the bladder. Urothelial denudation with protamine sulfate or dimethyl sulfoxide, liposome encapsulated onabotulinumtoxin A and other physical approaches are being studied to increase toxin permeability and avoid intradetrusor injections. Liposome encapsulated onabotulinumtoxin A enhances toxin activity while reducing its toxin degradation. The safety and efficacy of liposome encapsulated onabotulinumtoxin A were tested in a multicenter, placebo controlled study. Although this treatment successfully reduced urinary frequency and urgency, it did not significantly reduce urgency urinary incontinence episodes. Conclusions Intradetrusor injection of onabotulinumtoxin A is a safe and effective treatment as reported in several large multicenter, randomized controlled trials. Injection of the toxin into the bladder wall impairs afferent and efferent nerves, but injection-free drug delivery approaches only impair the bladder afferent nerves. Further studies are needed to develop better drug delivery platforms that overcome the drawbacks of intradetrusor injection, increase patient acceptance and reduce treatment costs. [ABSTRACT FROM AUTHOR]

Purpose We examined the functional role of endogenous IGF-1 (insulin-like growth factor-1) in the recovery phase of stress urinary incontinence induced by simulated childbirth trauma using an IGF-1 receptor inhibitor. Materials and Methods Simulated birth trauma was induced by vaginal distension in female Sprague Dawley® rats. The IGF-1 receptor antagonist JB-1 (10 and 100 μg/kg per day) or vehicle was continuously delivered from 1 day before vaginal distension for 7 days using subcutaneous osmotic pumps. Seven, 14 and 21 days after vaginal distension the effect of JB-1 treatment was examined by functional analyses, including leak point and urethral baseline pressure, and urethral responses during passive increments in intravesical pressure, as well as molecular analyses in urethral tissues, including phosphorylation of Akt, apoptotic changes and peripheral nerve density using Western blot and immunohistochemistry. Results On functional analyses vehicle treated rats with vaginal distension had significantly decreased leak point and urethral baseline pressure, and urethral responses at 7 days, which recovered to the normal level 14 and 21 days after vaginal distension. In the JB-1 treated vaginal distension group leak point and urethral baseline pressure, and urethral responses were still significantly reduced 21 days after vaginal distension. On molecular analyses JB-1 treatment increased apoptotic cells, induced a significant decrease in phosphorylated Akt and prolonged the decrease of peripheral nerve density in urethral tissues. Conclusions Suppression of endogenous IGF-1 activity delayed recovery from stress urinary incontinence induced by simulated childbirth trauma in rats. Thus, IGF-1 is likely to be an important endogenous mediator for functional recovery from childbirth related stress urinary incontinence. This suggests that IGF-1 could be an effective target for treating stress urinary incontinence in women. [ABSTRACT FROM AUTHOR]

Purpose Pelvic organ cross sensitization is considered to contribute to overlapping symptoms in chronic pelvic pain syndrome. Nerve growth factor over expression in the bladder is reportedly involved in the symptom development of bladder pain syndrome/interstitial cystitis. We examined whether a reduction of over expressed nerve growth factor in the bladder by intravesical treatment with liposome and oligonucleotide conjugates would ameliorate bladder hypersensitivity in a rat colitis model. Materials and Methods Adult female rats were divided into 1) a control group, 2) a colitis-oligonucleotide group with intracolonic TNBS (2,4,6-trinitrobenzene sulfonic acid) enema and intravesical liposome-oligonucleotide treatments, 2) a colitis-saline group with intracolonic TNBS and intravesical saline treatments, 4) a sham oligonucleotide group with intravesical liposome-oligonucleotide treatment without colitis and 5) a sham-saline group with intravesical saline treatment without colitis. Liposomes conjugated with nerve growth factor antisense oligonucleotide or saline solution were instilled in the bladder and 24 hours later colitis was induced by TNBS enema. Effects of nerve growth factor antisense treatment were evaluated by pain behavior, cystometry, molecular analyses and immunohistochemistry 10 days after TNBS treatment. Results In colitis-oligonucleotide rats nerve growth factor antisense treatment ameliorated pain behavior and decreased a reduction in the intercontraction interval in response to acetic acid stimulation as well as nerve growth factor expression in the bladder mucosa. All were enhanced in colitis-saline rats compared to sham rats. Conclusions Nerve growth factor over expression in the bladder mucosa and bladder hypersensitivity induced after colitis were decreased by intravesical application of liposome-oligonucleotide targeting nerve growth factor. This suggests that local antinerve growth factor therapy could be effective treatment of bladder symptoms in chronic pelvic pain syndrome. [ABSTRACT FROM AUTHOR]

Purpose In the current study we examined dynamic changes in neural activity of the anterior cingulate cortex and the midbrain periaqueductal gray during the micturition reflex in a Parkinson’s disease model as well as the effects of direct stimulation of the anterior cingulate cortex on the micturition reflex. Materials and Methods Electrodes were inserted in the anterior cingulate cortex or the periaqueductal gray. The effects of intravenous administration of the adenosine A2A receptor antagonist ZM24138 on pelvic nerve evoked field potentials were examined. The effect of electrical stimulation of the anterior cingulate cortex was also examined. Results Rats with Parkinson’s disease showed bladder overactivity as evidenced by a significant decrease in the intercontraction interval compared with sham operated rats. Intravenous administration of ZM24138 increased the intercontraction interval in both groups with the inhibitory effects greater in rats with Parkinson’s disease. It dose dependently increased the amplitude of evoked potentials in the anterior cingulate cortex of rats with Parkinson’s disease but not in sham operated rats. Intravenous administration of ZM24138 decreased evoked potential amplitude in the periaqueductal gray of both groups with the inhibitory effects greater in Parkinson’s disease vs sham operated rats. Electrical stimulation of the anterior cingulate cortex significantly increased the intercontraction interval. Conclusions These results suggest that anterior cingulate cortex neurons have an inhibitory role in bladder control. Neural activity in the anterior cingulate cortex was significantly increased along with suppression of bladder overactivity after ZM241385 administration in the Parkinson’s disease model and the stimulation of the anterior cingulate cortex inhibited the micturition reflex. Understanding the roles of the anterior cingulate cortex in the modulation of micturition could provide further insights into the pathophysiology of overactive bladder. [ABSTRACT FROM AUTHOR]

Purpose We investigated the effect of duloxetine, a norepinephrine and serotonin reuptake inhibitor, on the sneeze induced continence reflex and on bladder function in rats with cerebral infarction. Materials and Methods Using urethane anesthesia the effect of duloxetine (1 mg/kg intravenously) on the amplitude of urethral responses during sneezing as well as urethral baseline pressure at the mid urethra was evaluated in normal female adult rats and cerebral infarction rats. Tilt leak point pressure was also measured. In normal and cerebral infarction rats continuous cystometry was evaluated before and after duloxetine injection. Results In cerebral infarction rats urethral baseline pressure was 43% lower than in normal rats but the amplitude of urethral responses during sneezing did not differ in the 2 groups. Duloxetine increased the amplitude of urethral responses during sneezing and urethral baseline pressure by 31% and 21%, respectively, in normal rats but did not affect either in cerebral infarction rats. Also, in cerebral infarction rats leak point pressure was 29% lower compared with normal rats. Duloxetine increased leak point pressure in normal rats but not in cerebral infarction rats. Cerebral infarction reduced intercontraction intervals without affecting the amplitude of bladder contractions compared with normal rats. Duloxetine prolonged intercontraction intervals in cerebral infarction rats but not in normal rats. Conclusions These results suggest that cerebral infarction induces not only bladder overactivity but also stress urinary incontinence, which may account for mixed incontinence in patients with cerebral infarction. After cerebral infarction duloxetine reduced bladder overactivity but failed to enhance active urethral closure mechanisms during sneezing, suggesting that disorganization of the brain network after cerebral infarction might influence the effect of duloxetine on lower urinary tract function. [ABSTRACT FROM AUTHOR]

Purpose: We examined the effect of IGF-1 in a rat model of stress urinary incontinence induced by simulated childbirth trauma. Materials and Methods: Simulated birth trauma was induced by vaginal distension in female Sprague Dawley® rats. Four, 7, 14 and 28 days after distension we performed functional assessment by measuring leak point pressure, urethral baseline pressure and the urethral response during a passive increment in intravesical pressure. The expression of IGF-1 and IGF1R mRNA and protein in damaged tissues was examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. Thereafter hrIGF-1 (50 and 150 μg/kg per day) was continuously delivered from 1 day before distension using subcutaneous osmotic pumps. Four and 7 days after distension the effect of hrIGF-1 treatment was examined by functional analysis of leak point pressure, urethral baseline pressure and the urethral response as well as Western blot and histological analysis. Results: After 4 and 7 days rats with vaginal distension had significantly decreased leak point pressure, urethral baseline pressure and urethral responses. IGF-1 and IGF1R mRNA and protein levels were significantly increased in urethral and pudendal nerves 4 and 7 days after distension. IGF-1 treated groups showed significant improvement in leak point pressure, urethral baseline pressure and urethral responses 4 and 7 days after distension. Moreover, IGF-1 treatment increased Akt phosphorylation and induced cellular proliferation and antiapoptotic effects in the urethra. Conclusions: IGF-1 treatment accelerated recovery from stress urinary incontinence induced by simulated childbirth trauma in association with activation of the Akt signal transduction pathway in rats. This suggests that IGF-1 has therapeutic potential for stress urinary incontinence in women. [ABSTRACT FROM AUTHOR]

Purpose: To clarify the functional and molecular mechanisms inducing hyperexcitability of C-fiber bladder afferent pathways after spinal cord injury we examined changes in the electrophysiological properties of bladder afferent neurons, focusing especially on voltage-gated K channels. Materials and Methods: Freshly dissociated L6-S1 dorsal root ganglion neurons were prepared from female spinal intact and spinal transected (T9-T10 transection) Sprague Dawley® rats. Whole cell patch clamp recordings were performed on individual bladder afferent neurons. Kv1.2 and Kv1.4 α-subunit expression levels were also evaluated by immunohistochemical and real-time polymerase chain reaction methods. Results: Capsaicin sensitive bladder afferent neurons from spinal transected rats showed increased cell excitability, as evidenced by lower spike activation thresholds and a tonic firing pattern. The peak density of transient A-type K+ currents in capsaicin sensitive bladder afferent neurons from spinal transected rats was significantly less than that from spinal intact rats. Also, the KA current inactivation curve was displaced to more hyperpolarized levels after spinal transection. The protein and mRNA expression of Kv1.4 α-subunits, which can form transient A-type K+ channels, was decreased in bladder afferent neurons after spinal transection. Conclusions: Results indicate that the excitability of capsaicin sensitive C-fiber bladder afferent neurons is increased in association with reductions in transient A-type K+ current density and Kv1.4 α-subunit expression in injured rats. Thus, the Kv1.4 α-subunit could be a molecular target for treating overactive bladder due to neurogenic detrusor overactivity. [ABSTRACT FROM AUTHOR]

Purpose: Nerve growth factor over expression in the bladder has a role in overactive bladder symptoms via the mediation of functional changes in bladder afferent path- ways. We studied whether blocking nerve growth factor over expression in bladder urothelium by a sequence specific gene silencing mechanism would suppress bladder overactivity and chemokine expression induced by acetic acid. Materials and Methods: Female Sprague-Dawley@ rats anesthetized with isoflurane TM were instilled with 0.5 ml saline, scrambled or TYE 563 labeled antisense ohgonu otide targeting nerve growth factor (12/μM) alone or complexed with cationic liposomes for 30 minutes. The efficacy of nerve growth factor antisense treatments for acetic acid induced bladder overactivity was assessed by cystometry. Bladder nerve growth factor expression levels and cellular distribution were quantified by immunofluorescence stain- ing and enzyme-linked immunosorbent assay. Effects on bladder chemokine expression were measured by Luminex@ xMA_P@ analysis. Results: Liposomes were needed for bladder uptake of ohgonucleotide, as seen by the absence of bright red TYE 563 fuorescence in rats instilled with oligonucleotide alone. At 24 hours afar liposome-oligonucleotide treatment baseline bladder activity during sa- line infusion was indistinct in the sham and ant/sense treated groups with a mean +_ SEM intercontraction interval of 348 ± 55 and 390 ± 120 seconds, respectively. Acetic acid induced bladder overactivity was shown by a decrease in the intercontraction interval to a mean of 33.2% ± 4.0% of baseline in sham treated rats. However, the reduction was blunted to a mean of 75.8% ± 3.4% of baseline in rats treated with liposomal antisense oligonucleotide (p <0.05). Acetic acid induced increased nerve growth factor in the urothehum of sham treated rats, which was decreased by ant/sense treatment, as shown by enzyme-linked immunosorbent assay and reduced nerve growth factor immunoreactivity in the urothelium. Increased nerve growth factor in bladder tissue was associated with sICAM-1, sE-selectin, CXCL-IO and 1, leptin, MCP-1 and vascular endothelial growth factor over expression, which was significantly decreased by nerve growth factor antisense treatment (p <0.01). Conclusions: Acetic acid induced bladder overactivity is associated with nerve growth factor over expression in the urothelium and with chemokine up-regula- tion. Treatment with liposomal antisense suppresses bladder overactivity, and nerve growth factor and chemokine expression. Local suppression of nerve growth factor in the bladder could be an attractive approach for overactive bladder. It would avoid the systemic side effects that may be associated with nonspecific blockade of nerve growth factor expression. [ABSTRACT FROM AUTHOR]

Purpose: We examined the effects of tumor necrosis factor-α blockade on bladder overactivity and nociception using replication defective HSV vectors expressing tumor necrosis factor-α soluble receptor. Materials and Methods: HSV vectors expressing tumor necrosis factor-α soluble receptor or β-galactosidase/green fluorescent protein as the control were injected into the bladder wall of female Sprague-Dawley® rats. Green fluorescent protein was observed with fluorescent microscopy in the bladder and L6 dorsal root ganglia. mRNA and protein expression of tumor necrosis factor-α, and interleukin-1β and 6 as well as myeloperoxidase activity in the bladder were determined by quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay 4 hours after intravesical resiniferatoxin administration. c-Fos positive neurons were counted in the L6 spinal dorsal horn. Cystometry and behavioral analyses were also performed. Results: Green fluorescent protein expression was confirmed in the bladder and L6 dorsal root ganglia. Resiniferatoxin administration significantly increased tumor necrosis factor-α mRNA and protein levels in the bladder in controls. Tumor necrosis factor-α mRNA was also increased in the tumor necrosis factor-α soluble receptor group, although tumor necrosis factor-α protein up-regulation was suppressed. The up-regulation of interleukin-1β and 6 mRNA and protein levels, and the myeloperoxidase activity seen in controls were suppressed in the tumor necrosis factor-α soluble receptor group. c-Fos positive cells in the L6 spinal dorsal horn were less prominent in the tumor necrosis factor-α soluble receptor group than in controls. On cystometry the significant decrease in intercontraction intervals after resiniferatoxin infusion detected in controls was not seen in the tumor necrosis factor-α soluble receptor group. On behavioral analyses freezing behavior was significantly decreased in the tumor necrosis factor-α soluble receptor group without affecting licking behavior. Conclusions: HSV vector mediated tumor necrosis factor-α blockade gene therapy in the bladder and bladder afferent pathways decreases the bladder pain and overactivity induced by nociceptive bladder stimuli. [Copyright &y& Elsevier]

Purpose: To determine the mechanisms of botulinum neurotoxin A (Metabiologics, Madison, Wisconsin) induced inhibition of bladder activity we examined the effect of botulinum neurotoxin A on detrusor contractile responses to the activation of L-type voltage-gated Ca2+ channels, and efferent and afferent nerve terminals in the rat bladder. Materials and Methods: Rat bladder strips were incubated for 3 hours with different concentrations of botulinum neurotoxin A (0.3 to 100 nM). We examined the effect of botulinum neurotoxin A on detrusor contractility in response to activation of L-type voltage-gated Ca2+ channels, and efferent and afferent nerve terminals induced by 70 mM KCl, electrical field stimulation and 1 μM capsaicin, respectively. Results: Botulinum neurotoxin A inhibited electrical field stimulation induced contractions at a concentration of 10 nM or higher. The maximal inhibition at 100 nM was 70% compared to that of control strips. KCl induced contractions, which were sensitive to nifedipine, were significantly inhibited by incubation with botulinum neurotoxin A at a concentration of 3 nM or higher. Maximal inhibition at 100 nM was 30% compared to that of control strips. Capsaicin induced contractions were not inhibited by 3-hour incubation but they were significantly inhibited by overnight incubation with 100 nM botulinum neurotoxin A (30% compared to control strips). Carbachol induced contractions were not altered by incubation with botulinum neurotoxin A. Conclusions: The order of inhibitory potency of botulinum neurotoxin A was efferent nerve terminals >L-type voltage-gated Ca2+ channels >afferent nerve terminals. Since the inhibitory effects on L-type voltage-gated Ca2+ channels and efferent nerve terminals were observed at similar botulinum neurotoxin A concentrations, the inhibitory effect of botulinum neurotoxin A on L-type voltage-gated Ca2+ channels may have an important role in regulating and stabilizing bladder activity. [Copyright &y& Elsevier]

Purpose: Overactive bladder is highly prevalent among patients with Parkinson disease. Adenosine is an important neurotransmitter in the central nervous system but it is not fully clarified how adenosine receptors regulate the micturition reflex. Thus, we examined the effect of an adenosine A2A receptor antagonist on the micturition reflex in a rat model of Parkinson disease. Materials and Methods: In a rat model of Parkinson disease induced by 6-hydroxydopamine (Tocris Bioscience, Ellisville, Missouri) injection we examined the effects of the adenosine A2A receptor antagonist ZM241385, the dopamine D1 receptor agonist SKF38393 and the dopamine D2 receptor agonist quinpirole on bladder activity. Results: Intravenous administration of ZM241385 increased the intercontraction interval in a dose dependent manner in rats with Parkinson disease and sham operated rats but the inhibitory effect was greater in the Parkinson disease group. Intrathecal and intracerebroventricular administration of ZM241385 increased the intercontraction interval in each group. However, in rats with Parkinson disease the inhibitory effects induced by intracerebroventricular administration of ZM241385 were greater than in sham operated rats. Intravenous administration of SKF38393 increased the intercontraction interval in rats with Parkinson disease and subsequent administration of ZM further increased the intercontraction interval. However, SKF38393 did not increase the intercontraction interval after ZM241385 application. Also, ZM241385 increased the intercontraction interval without being affected by pre-administration or post-administration of quinpirole, which decreased the intercontraction interval. Conclusions: Results indicate that the adenosine A2A receptor mediated excitatory mechanism is enhanced at a supraspinal site to induce bladder overactivity and A2A receptor inhibition effectively suppresses bladder overactivity in rats with Parkinson disease. Thus, adenosine A2A receptor antagonists could be useful for bladder dysfunction in Parkinson disease cases. [Copyright &y& Elsevier]

Purpose: Stress urinary incontinence is often seen in postmenopausal women but limited information is available on hormone dependent changes of urethral function. Thus, we examined how ovariectomy and estrogen replacement affect urethral continence mechanisms. Materials and Methods: In female nulliparous Sprague-Dawley® rats under urethane anesthesia after ovariectomy with or without estrogen replacement we measured urethral response amplitude during sneezing, urethral baseline pressure and sneeze induced leak point pressure. Whole urethras were tested for ex vivo urethral properties. Results: Urethral response amplitude during sneezing was significantly decreased in 3 and 6-week ovariectomized rats. Urethral baseline pressure was significantly decreased only in 6-week ovariectomized rats. After estrogen replacement urethral baseline pressure but not urethral response amplitude during sneezing was significantly increased. Neither 3-week ovariectomized nor sham operated rats leaked during sneezing but fluid leakage was observed in 63% of 6-week ovariectomized rats. Estrogen replacement decreased the stress urinary incontinence incidence to 25%. Ex vivo testing revealed a significant increase in middle urethral compliance and a decrease in β stiffness at the proximal and middle urethras in 6-week ovariectomized rats. Conclusions: Results indicate that ovariectomy significantly impairs urethral function from the early stage (3 weeks) but does not induce stress urinary incontinence until the late stage (6 weeks). Also, estrogen replacement restores only the urethral baseline pressure parameter, leading to partial prevention of stress urinary incontinence. Since urethral baseline pressure and urethral response amplitude during sneezing parameters are related to urethral smooth and striated muscle activity, respectively, based on our previous studies, hormone replacement therapy may be partially effective for stress urinary incontinence by enhancing smooth muscle mediated urethral activity under stress conditions such as sneezing. [Copyright &y& Elsevier]

Purpose: We investigated whether replication defective herpes simplex virus vectors encoding genes of glutamic acid decarboxylase, the γ-aminobutyric acid synthesis enzyme, could suppress detrusor-sphincter dyssynergia in rats with spinal cord injury. Materials and Methods: One week after spinalization herpes simplex virus vectors expressing glutamic acid decarboxylase and green fluorescent protein were injected into the bladder wall. Spinal cord injured rats without herpes simplex virus injection (sham treated) and those injected with LacZ encoding herpes simplex virus vectors served as controls. Three weeks after viral injection we simultaneously recorded urethral and intravesical pressure in awake rats. Results: In the glutamic acid decarboxylase group the urethral pressure increase during bladder contraction was significantly decreased by 77% to 79% compared with that in the sham treated and LacZ groups. Bladder activity and urethral baseline pressure did not differ among the 3 groups. Intrathecal application of the γ-aminobutyric acid-A receptor antagonist bicuculline almost completely reversed the decrease in the urethral pressure increase during bladder contractions while intrathecal saclofen (Tocris Cookson, Ellisville, Missouri), a γ-aminobutyric acid-B receptor antagonist, partially reversed it. In the glutamic acid decarboxylase group the mRNA of glutamic acid decarboxylase 67 was significantly increased in L6-S1 dorsal root ganglia, which is where bladder afferents originate, compared with that in the LacZ group. Conclusions: Herpes simplex virus based glutamic acid decarboxylase gene transfer to bladder afferent pathway may represent a novel approach to detrusor-sphincter dyssynergia in cases of spinal cord injury. [ABSTRACT FROM AUTHOR]

Purpose: Serotonin and norepinephrine reuptake inhibitors such as duloxetine have clinical efficacy for stress urinary incontinence. However, the therapeutic dose of duloxetine is often associated with unwanted side effects. We examined whether combined α2-adrenoceptor antagonists could decrease the duloxetine dose due to synergistic effects. Materials and Methods: We used normal female rats and rats with stress urinary incontinence induced by vaginal distention. Urethral responses were measured using a microtransducer tipped catheter. We evaluated the effect of low dose duloxetine (0.1 mg/kg), α2-adrenoceptor antagonists (idazoxan or yohimbine) and sequential administration of drugs on urethral pressure response amplitude during sneezing and urethral baseline pressure. Sneeze induced leak point pressure was also measured. Results: In normal and vaginally distended rats low dose duloxetine did not alter urethral pressure response amplitude during sneezing but it significantly increased urethral baseline pressure. When low dose duloxetine was co-applied with α2-adrenoceptor antagonists, urethral pressure response amplitude during sneezing was significantly increased. In all 7 vaginally distended rats leakage was observed during sneezing. After low dose duloxetine administration fluid leakage still occurred during sneezing. However, after low dose duloxetine and idazoxan co-administration fluid leakage disappeared in 2 of 7 rats and sneeze induced leak point pressure was significantly increased in the remaining incontinent rats with vaginal distention. Conclusions: Findings support the concept that combination therapy with α2-adrenoceptor antagonists may be an effective, novel strategy to reinforce the clinical efficacy of serotonin and norepinephrine reuptake inhibitors for stress urinary incontinence. [Copyright &y& Elsevier]

Purpose: Botulinum toxin A (Allergan, Irvine, California) is a high molecular weight neurotoxin used to treat hypersensitive bladder by direct injection to pass the urothelial barrier. We investigated the feasibility of intravesical botulinum toxin A delivery using liposomes (Lipella Pharmaceuticals, Pittsburgh, Pennsylvania), which are phospholipid bilayered vesicles, and evaluated the urodynamic and immunohistochemical effect on acetic acid induced bladder hyperactivity in rats. Materials and Methods: Liposomes (1 ml), botulinum toxin A (20 U/1 ml saline) or botulinum toxin A encapsulated in liposomes (lipotoxin, that is 20 U botulinum toxin A plus 1 ml liposomes) was administered in the bladder and retained for 1 hour on day 1 after baseline cystometrogram. Continuous cystometrogram was performed on day 1 by filling the bladder with saline and on day 8 by filling the bladder with saline, followed by 0.3% acetic acid. The bladder was then harvested. Cystometrogram parameters, histology, SNAP25 and calcitonin gene-related peptide expression were measured by Western blotting or immunostaining. Results: The intercontraction interval was decreased 57.2% and 56.0% after intravesical acetic acid instillation in liposome and botulinum toxin A pretreated rats, respectively. However, rats that received lipotoxin showed a significantly decreased intercontraction interval response (21.1% decrease) to acetic acid instillation but without compromised voiding function. Also, lipotoxin pretreated rats had a better decrease in the inflammatory reaction and SNAP-25 expression, and increase in calcitonin gene-related peptide immunoreactivity than those in liposome or botulinum toxin A pretreated rats. Conclusions: Intravesical lipotoxin administration cleaved SNAP-25, inhibited calcitonin gene-related peptide release from afferent nerve terminals and blocked the acetic acid induced hyperactive bladder. These results support liposomes as an efficient vehicle for delivering botulinum toxin A without injection. [Copyright &y& Elsevier]

Purpose: Although cannabinoid receptor expression has been demonstrated in human brain and other peripheral neuronal tissues, definitive expression of these receptors in the human bladder has not been reported. Consequently we investigated the expression of functional cannabinoid 1 and 2 receptors in human bladder detrusor and urothelium. Materials and Methods: Human bladders were micro-dissected for detrusor (6) and urothelium (8), and analyzed for cannabinoid 1 and 2 mRNA expression using real-time quantitative polymerase chain reaction, and for protein expression using immunohistochemistry and Western blot. Functional response of these receptors was tested by studying the effect of selective cannabinoid 1 and 2 agonists on nerve evoked smooth muscle contraction. Results: Quantitative polymerase chain reaction analysis revealed differential expression of cannabinoid 1 and 2 receptors in detrusor and urothelium. The expression of cannabinoid 1 and 2 receptor mRNA in urothelium was approximately 2-fold higher than in detrusor, although this was not significant (p >0.05). Cannabinoid 1 receptor mRNA expression was significantly higher than cannabinoid 2 receptor expression in the 2 tissue subtypes (p ≤0.05). Expression at mRNA level was confirmed at the protein level by immunoreactivity and Western blot analysis. Activation of cannabinoid 1 and 2 receptors in human bladder attenuated the electrically evoked contraction of detrusor strips. Conclusions: Together these findings suggest a physiological role of cannabinoid 1 and 2 receptors in the human bladder. Moreover, these results confirm the presence of functional cannabinoid 1 and 2 receptors in the human bladder, which can serve as a target for drugs acting on symptoms of interstitial cystitis/painful bladder syndrome. [Copyright &y& Elsevier]

Purpose: We investigated the role of α2-adrenoceptors and glutamate mechanisms in the urethral continence reflex in response to abdominal pressure increases. Materials and Methods: Under urethane anesthesia external urethral sphincter electromyogram activity was evaluated in spinal cord transected (T8–T9) female rats during lower abdominal wall compression before and after intravenous application of test drugs. The effects of the N-methyl-D-aspartate glutamate receptor antagonist MK-801 (Sigma®) or the α2-adrenoceptor agonist medetomidine (Tocris Cookson, Ellisville, Missouri) (each 0.03, 0.3 and 3 mg/kg intravenously) on external urethral sphincter activity were examined. A 0.3 mg/kg intravenous dose of the α2-adrenoceptor antagonist idazoxan (Sigma) was then administered before or after the application of 1 mg/kg MK-801 intravenously. In addition, 0.3 mg/kg idazoxan were administered intravenously following the application of 1 mg/kg of the serotonin/norepinephrine reuptake inhibitor duloxetine (Kemprotec, Middlesbrough, United Kingdom) intravenously. Results: MK-801 and medetomidine dose dependently decreased external urethral sphincter activity. Idazoxan significantly increased external urethral sphincter activity by 64% but the increase in activity after idazoxan was abolished by MK-801. On the other hand, idazoxan did not reverse the inhibitory effects of MK-801. In addition, idazoxan significantly potentiated the duloxetine effects on external urethral sphincter activity by 120%. Conclusions: These results indicate that 1) glutamate is a major excitatory neurotransmitter in the urethral continence reflex response to abdominal pressure increases, 2) α2-adrenoceptor activation suppresses external urethral sphincter activity, probably via presynaptic inhibition of glutamate release and 3) the effects of serotonin/norepinephrine reuptake inhibitors are enhanced by α2-adrenoceptor inhibition. Therefore, α2-adrenoceptor antagonists could be beneficial for treating stress urinary incontinence. [Copyright &y& Elsevier]

Purpose: Cyclooxygenase-2 is a key enzyme in the conversion of arachidonic acid to prostaglandins, which are important mediators of inflammation and pain. We investigated the effect of intraprostatic botulinum toxin A administration on pain reaction and cyclooxygenase-2 expression in a capsaicin induced prostatitis model in rats. Materials and Methods: Adult male Sprague-Dawley rats were injected with vehicle or capsaicin (10 mM, 0.1 cc) into the prostate. The nociceptive effects of capsaicin were evaluated for 30 minutes using a behavior approach. The prostate and L6 spinal cord were then removed for histology and cyclooxygenase-2 expression using Western blotting or immunostaining. A second set of animals was injected with botulinum toxin A (5 to 20 U) into the prostate 1 week before intraprostatic injection of capsaicin. Results: Capsaicin induced increased pain behavior and inflammatory reaction. Botulinum toxin A 1 week before treatment dose dependently decreased inflammatory cell accumulation, cyclooxygenase-2 expression and prostatic pain. Botulinum toxin A (20 U) significantly decreased inflammatory cell accumulation, and cyclooxygenase-2 expression in the prostate, ventral horn and dorsal horn of the L6 spinal cord (93.5%, 89.4%, 90.5% and 77.5%, respectively). It decreased pain behavior for eye and locomotion scores (59.5% and 40.0%, respectively). Conclusions: Intraprostatic capsaicin injection activates cyclooxygenase-2 expression in the prostate, and spinal sensory and motor neurons, and it induces prostatic pain. Botulinum toxin A pretreatment could inhibit capsaicin induced cyclooxygenase-2 expression from the peripheral organ to the L6 spinal cord and inhibit prostatic pain and inflammation. This finding suggests a potential clinical benefit of botulinum toxin A for the treatment of nonbacterial prostatitis. [Copyright &y& Elsevier]

Purpose: We investigated the effects of intrathecal application of γ-aminobutyric acid A and B receptor agonists on detrusor overactivity in spinal cord injured rats. Materials and Methods: Adult female Sprague-Dawley rats were used. At 4 weeks after Th9-10 spinal cord transection awake cystometry and recordings of external urethral sphincter electromyogram were performed to examine the effect of intrathecal application of the γ-aminobutyric acid A and B agonists muscimol and baclofen or the γ-aminobutyric acid A and B antagonists bicuculline and saclofen (Tocris Cookson, Ellisville, Missouri), respectively, at the level of the L6-S1 spinal cord. The expression of glutamate decarboxylase 67 mRNA in the L6-S1 spinal cord and dorsal root ganglia was also assessed. Results: Muscimol and baclofen produced a dose dependent inhibition of the number (51% to 73% decrease) and amplitude (35% to 93% decrease) of nonvoiding bladder contractions and a decrease in micturition pressure. The effects of muscimol and baclofen were antagonized by bicuculline and saclofen, respectively. Bursting activity of external urethral sphincter electromyogram was inhibited, corresponding to the inhibition of bladder activity by muscimol and baclofen. Glutamate decarboxylase 67 mRNA levels in the spinal cord and dorsal root ganglia were decreased after spinal cord transection (55% and 84%, respectively). Conclusions: These results indicate that γ-aminobutyric acid A and B receptor activation in the spinal cord inhibits detrusor overactivity. The decrease in glutamate decarboxylase 67 mRNA suggests hypofunction of GABAergic inhibitory mechanisms in the spinal cord. Therefore, stimulation of spinal GABAergic mechanisms could be effective for the treatment of detrusor overactivity after spinal cord injury. [Copyright &y& Elsevier]

Purpose: A new animal model in which to concurrently evaluate bladder function and nociceptive behavior was developed using freely moving, noncatheterized, conscious rats to assess the nociceptive behavior responses induced by intravesical instillation of resiniferatoxin (Sigma®) and its relationship with bladder dysfunction. Materials and Methods: In female Sprague-Dawley rats resiniferatoxin (0, 0.3 and 3 μM) was instilled via a catheter that was temporarily inserted into the bladder through the urethra. After removing the catheter the incidence of nociceptive behavior (lower abdominal licking and freezing) was scored. Voided urine was collected continuously to measure bladder capacity. In some rats the pudendal nerves were transected bilaterally to eliminate the activation of urethral afferents by resiniferatoxin. Results: Intravesical instillation of resiniferatoxin induced decreased bladder capacity and increased nociceptive behaviors, such as licking and freezing, which were blocked by the transient receptor potential vanilloid receptor 1 antagonist BCTC (Biomol™). In rats with pudendal nerve transection the early phase of resiniferatoxin induced licking was decreased without affecting the resiniferatoxin induced decrease in bladder capacity and late phase licking behavior. Resiniferatoxin induced late phase licking in the water unloaded group was observed to a lesser extent than in the water loaded diuresis group. Conclusions: The intravesical instillation of resiniferatoxin, which decreases bladder capacity, acts by at least 3 distinct mechanisms to induce licking behavior, including 1) an immediate response mediated by the activation of urethral afferents in the pudendal nerve, 2) a late response evoked by the direct stimulation of C-fiber afferents in the bladder and 3) gradual facilitation of the response elicited by the bladder wall distention induced by rapid bladder filling. [Copyright &y& Elsevier]

Purpose: Interstitial cystitis remains a poorly understood urological condition characterized by chronic pelvic pain and increased urinary frequency in the absence of any known etiology. Urothelial dysfunction and other abnormalities are presumed to be involved in the disease. Uroplakins that are expressed by urothelial cells are thought to have an important role as major barrier proteins on the apical surface of the urothelium. Materials and Methods: Gene expression of uroplakin Ia, Ib, II, III and III-delta4 was quantitatively measured in bladder biopsy samples from 29 patients with interstitial cystitis and 16 control subjects using real-time reverse transcriptase-polymerase chain reaction. Results: The mRNA levels of the uroplakin Ia, Ib and II genes were relatively low and uroplakin III was relatively high in interstitial cystitis bladders compared to normal controls, although not significantly. Uroplakin III-delta4, a splicing variant of uroplakin III, was significantly up-regulated in interstitial cystitis samples (p <0.001). When patients with interstitial cystitis were divided into those with and without ulcerative changes, the uroplakin III and III-delta4 genes were significantly up-regulated only in patients with nonulcerative interstitial cystitis. Even more interesting was the finding that up-regulation of uroplakin III-delta4 was much more prominent than that of uroplakin III, that is 26.5 vs 5.6-fold compared to the median values of normal subjects. Conclusions: Although the clinical implications of the over expression of uroplakin III and III-delta4 in nonulcerative interstitial cystitis bladders remains to be clarified, from the diagnostic viewpoint uroplakin III-delta4 is a potential marker for identifying nonulcerative interstitial cystitis. [Copyright &y& Elsevier]

Purpose: We evaluated changes in the urethral closure mechanism under a sneeze induced stress condition in a rat model of birth trauma. Materials and Methods: Four days after vaginal distention induced by balloon catheter inflation in the vagina sneezing was induced while recording intravesical pressure with the rat under urethane anesthesia to evaluate sneeze induced leak point pressure, defined as the lowest pressure inducing fluid leakage from the urethral meatus during sneezing. Sneeze induced responses in the bladder and proximal or mid urethra were also measured using microtip transducer catheters. Results: In 5 sham operated rats no leakage was observed from the urethral meatus during sneezing, which produced an increase in intravesical pressure of up to 34 cm H2O. However, in 5 of 6 rats with vaginal distention leakage during sneezing was observed with a sneeze leak point pressure of 26.2 cm H2O. In the mid urethra microtip transducer catheters revealed that pressure increases during sneezing were greater than those in the bladder but they were significantly decreased in the 5 incontinent vaginal distention rats. However, sneeze induced responses at the proximal urethra, which were similar to those in the bladder, were not different in sham operated and incontinent vaginal distention rats. Conclusions: Sneeze induced stress urinary incontinence in a rat model of birth trauma was caused by decreased active closure mechanisms at the mid urethra without affecting the passive transmission of abdominal pressure in the proximal urethra. [Copyright &y& Elsevier]

Purpose: We compared the complete spectrum of receptor subtypes expressed by human detrusor and its primary culture with the expression profile in a human urothelium immortalized cell line, and in fresh urothelium tissue and its primary cell culture. Materials and Methods: The levels of mRNA expressed for receptor subtypes M1 through M5 were determined with reverse transcriptase-polymerase chain reaction and quantitative polymerase chain reaction in total RNA extracted individually from different human bladder specimens, including fresh tissue of human urothelium and detrusor, and their respective primary cultures, as well as from the UROtsa cell line. Results: All 5 muscarinic receptors were detected in fresh human bladder tissue by reverse transcriptase-polymerase chain reaction RNA. The same was true in separated urothelium and detrusor tissue except for the lack of the M5 receptor transcript. Receptor subtype mRNA expression in the UROtsa cell line paralleled expression in fresh human bladder. Quantitative polymerase chain reaction data further corroborated these results and showed comparable mRNA expression for M2 and M3 in primary detrusor cultures. Primary cultures also had a decreased copy number of receptor genes than native tissue. The decrease was even more pronounced in primary urothelium culture and the UROtsa cell line in the presence of high calcium. M2 and M3 receptors were also detected in urothelium and detrusor by immunoreactivity. Conclusions: We identified all 5 existing muscarinic receptor subtypes in detrusor and urothelium, and transcripts levels of M2 and M3 were comparable in detrusor. These results support an alternative site of action in urothelium for anti-muscarinic drugs. Urothelial receptors should be considered in the design of future drugs for overactive bladder. [Copyright &y& Elsevier]

Purpose: We investigated the effects of nicotinic acetylcholine receptor activation in the bladder and central nervous system on the micturition reflex in urethane anesthetized rats. Materials and Methods: The effects of nicotinic acetylcholine receptor activation on bladder activity were examined during continuous infusion cystometrogram. Nicotine with or without the nicotinic acetylcholine receptor antagonist mecamylamine (Sigma Chemical Co., St. Louis, Missouri) was administered intravesically, intrathecally or intracerebroventricularly in normal or capsaicin pretreated rats. We also examined nicotine induced responses in dissociated bladder afferent neurons from L6 to S1 dorsal root ganglia that were sensitive to capsaicin using whole cell patch clamp recordings. Results: Intravesical nicotine (1 to 10 mM) significantly decreased intercontraction intervals in dose dependent fashion. This excitatory effect was abolished by co-application of mecamylamine (3 mM) as well as by capsaicin pretreatment. On patch clamp recordings 300 μM nicotine evoked rapid inward currents that were antagonized by mecamylamine in capsaicin sensitive bladder afferent neurons. Intrathecal and intracerebroventricular administration of nicotine (10 μg) decreased and increase intercontraction intervals, respectively. Each effect was antagonized by mecamylamine (50 μg) administered intrathecally and intracerebroventricularly. The spinal excitatory effect was significantly inhibited by the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (20 μg) given intrathecally or by capsaicin pretreatment, although the effects of capsaicin pretreatment were significantly smaller than those of (+)-MK-801 hydrogen maleate. Conclusions: These results indicate that nicotinic acetylcholine receptor activation in capsaicin sensitive C-fiber afferents in the bladder can induce detrusor overactivity. In the central nervous system nicotinic acetylcholine receptor activation in the spinal cord and brain has an excitatory and an inhibitory effect on the micturition reflex, respectively. In addition, the nicotine induced spinal excitatory effect may be mediated by the activation of glutamatergic mechanisms. [Copyright &y& Elsevier]

Purpose: Intraprostatic injection of BTX-A has demonstrated clinical improvement in men with bladder outlet obstruction. We investigated the mechanisms of action of BTX-A on the prostate. Materials and Methods: Adult male Sprague-Dawley rats were injected with varying doses of BTX-A into the prostate and the prostates were harvested after 1 or 2 weeks. The effects of BTX-A on prostate histology, and the proliferative and apoptotic indexes were determined using hematoxylin and eosin staining, proliferative cell nuclear antigen staining and TUNEL staining, respectively. Changes in α1A adrenergic receptor and androgen receptor were evaluated by Western blotting. Results: One week after BTX-A injection generalized prostate atrophy was observed. There was a significant increase in apoptotic cells (12, 16 and 22-fold), and decrease in proliferative cells (38%, 77% and 80%) and α1A adrenergic receptor (13%, 80% and 81%) for 5, 10 and 20 U, respectively. There was no significant change in androgen receptors. The effects were decreased 2 weeks after BTX-A treatment. Conclusions: BTX-A injection into the prostate alters cellular dynamics by inducing apoptosis, inhibiting proliferation and down-regulating α1A adrenergic receptors. BTX-A may potentially be the drug that has dual actions on the static and dynamic components of benign prostatic hyperplasia. [Copyright &y& Elsevier]

ABSTRACT: Purpose:: We investigated the contribution of α1-adrenoceptor mechanisms to urethral dysfunction associated with diabetes mellitus (DM) in rats. Materials and Methods:: Eight weeks after streptozotocin injection (65 mg/kg intraperitoneally) the effects of DM on urethral relaxation mechanisms were evaluated with subjects under urethane anesthesia by simultaneous recordings of intravesical pressure in isovolumetric conditions and urethral perfusion pressure (UPP). Results:: In diabetic rats the intravesical pressure thresholds for inducing urethral relaxation and the lowest urethral pressure (UPP nadir) during urethral relaxation were significantly higher by 142% and 86%, respectively, than in normal rats, while baseline UPPs were not significantly different. The mean rate of high frequency oscillations of urethral striated muscle in diabetic rats was also significantly lower by 23% than in normal rats. After α-bungarotoxin treatment (333 μg/kg intravenously) to eliminate striated muscle sphincter contractions the SD of baseline UPPs was significantly larger by 93% than in normal rats. Intravenous administration of terazosin (0.4 mg/kg), an α1-adrenoceptor antagonist, significantly decreased the UPP nadir, intravesical pressure thresholds inducing urethral relaxation and the SD by 41%, 87% and 138%, respectively, in diabetic rats but not in normal rats. In the 2 groups of animals after α-bungarotoxin treatment urethral relaxation during a reflex bladder contraction was inhibited by Nω-nitro-L-arginine (40 mg/kg intravenously), a nitric oxide synthase inhibitor. Conclusions:: During reflex bladder contractions streptozotocin induced diabetic rats showed smooth and striated muscle dysfunctions of the urethra. The inhibition of α1-adrenoceptors, which decreased the UPP nadir and UPP fluctuation, may be useful for treating urethral dysfunction in DM. [Copyright &y& Elsevier]