Targeting neurotrophin and nitric oxide signaling to promote recovery and ameliorate neurogenic bladder dysfunction following spinal cord injury - Mechanistic concepts and clinical implications.

This review summarizes the presentations made to a workshop entitled "Targeting Neurotrophin and Nitric Oxide Signaling to Promote Recovery and Ameliorate Neurogenic Bladder Dysfunction following Spinal Cord Injury - Mechanistic Concepts and Clinical Implications" at the International Continence Society (ICS) 2022 Vienna Meeting. Spinal cord injury (SCI; T8-T9 contusion/transection) causes impaired mobility, neurogenic detrusor overactivity (NDO), detrusor sphincter dyssynergia (DSD) and subsequent decreased quality of life. This workshop discussed the potential of future therapeutic agents that manage the lesion and its consequences, in particular possibilities to reduce the lesion itself and manage pathophysiological changes to the lower urinary tract (LUT). Attenuation of the spinal cord lesion itself was discussed with respect to the potential of a trio of agents: LM11A-3, a p75 neurotrophin receptor modulator to counter activation of local apoptotic pathways; LM22B-10 to promote neuronal growth by targeting tropomyosin-related kinase (Trk) receptors; and cinaciguat, a soluble guanylate cyclase (sGC) activator as an agent promoting angiogenesis at the injury site. The workshop also discussed targets on the bladder to block selectivity sites associated with detrusor overactivity and poor urinary filling profiles, such as purinergic pathways controlling excess contractile activity and afferent signaling, as well as excess fibrosis. Finally, the importance of increased mechanosensitive signaling as a contributor to DSD was considered, as well as potential drug targets. Overall, an emphasis was placed on targets that help restore function and reduce pathological LUT consequences, rather than downregulate normal function.

Soluble Guanylate Cyclase Activators to Treat Benign Prostatic Hyperplasia and associated LUTS.

This review summarises the presentations during a workshop session entitled "The Use of Soluble Guanylate Cyclase Activators to Treat Benign Prostatic Hyperplasia, Obstruction and Fibrosis - Mechanistic Concepts and Clinical Implications" at the International Continence Society (ICS) 2021 Melbourne Virtual meeting. Benign prostatic hyperplasia (BPH) is a highly prevalent condition that can result in bladder outflow obstruction (BOO) and development of lower urinary tract symptoms (LUTS), and by 80 years of age is present in about 75% of men. Current pharmacological therapies include α-adrenoceptor antagonists, 5α-reductase inhibitors, and the phosphodiesterase type 5 (PDE5) inhibitor, tadalafil. The efficacy of tadalafil suggests a role for nitric oxide (NO•) through activation of soluble guanylate cyclase (sGC) and production of cyclic guanosine 3'5'-monophosphate (cGMP), a cyclic nucleotide that relaxes smooth muscle, reduces neurotransmitter release and also acts as an antifibrotic agent. Patient refractoriness to tadalafil may be, for example, due to sGC inactivation due to oxidative stress. The workshop discussed the superiority of cinaciguat, an sGC activator that functions even when the enzyme is oxidised, over PDE5 inhibitors, and potentially its use in combination with agents that reduce formation of reactive oxygen species.

Serotonergic paraneurones in the female mouse urethral epithelium and their potential role in peripheral sensory information processing.

AIM: The mechanisms underlying detection and transmission of sensory signals arising from visceral organs, such as the urethra, are poorly understood. Recently, specialized ACh-expressing cells embedded in the urethral epithelium have been proposed as chemosensory sentinels for detection of bacterial infection. Here, we examined the morphology and potential role in sensory signalling of a different class of specialized cells that express serotonin (5-HT), termed paraneurones. METHODS: Urethrae, dorsal root ganglia neurones and spinal cords were isolated from adult female mice and used for immunohistochemistry and calcium imaging. Visceromotor reflexes (VMRs) were recorded in vivo. RESULTS: We identified two morphologically distinct groups of 5-HT+ cells with distinct regional locations: bipolar-like cells predominant in the mid-urethra and multipolar-like cells predominant in the proximal and distal urethra. Sensory nerve fibres positive for calcitonin gene-related peptide, substance P, and TRPV1 were found in close proximity to 5-HT+ paraneurones. In vitro 5-HT (1 µm) stimulation of urethral primary afferent neurones, mimicking 5-HT release from paraneurones, elicited changes in the intracellular calcium concentration ([Ca2+ ]i ) mediated by 5-HT2 and 5-HT3 receptors. Approximately 50% of 5-HT responding cells also responded to capsaicin with changes in the [Ca2+ ]i . In vivo intra-urethral 5-HT application increased VMRs induced by urethral distention and activated pERK in lumbosacral spinal cord neurones. CONCLUSION: These morphological and functional findings provide insights into a putative paraneurone-neural network within the urethra that utilizes 5-HT signalling, presumably from paraneurones, to modulate primary sensory pathways carrying nociceptive and non-nociceptive (mechano-sensitive) information to the central nervous system.

A novel LSD1 inhibitor NCD38 ameliorates MDS-related leukemia with complex karyotype by attenuating leukemia programs via activating super-enhancers.

Lysine-specific demethylase 1 (LSD1) regulates gene expression by affecting histone modifications and is a promising target for acute myeloid leukemia (AML) with specific genetic abnormalities. Novel LSD1 inhibitors, NCD25 and NCD38, inhibited growth of MLL-AF9 leukemia as well as erythroleukemia, megakaryoblastic leukemia and myelodysplastic syndromes (MDSs) overt leukemia cells in the concentration range that normal hematopoiesis was spared. NCD25 and NCD38 invoked the myeloid development programs, hindered the MDS and AML oncogenic programs, and commonly upregulated 62 genes in several leukemia cells. NCD38 elevated H3K27ac level on enhancers of these LSD1 signature genes and newly activated ~500 super-enhancers. Upregulated genes with super-enhancer activation in erythroleukemia cells were enriched in leukocyte differentiation. Eleven genes including GFI1 and ERG, but not CEBPA, were identified as the LSD1 signature with super-enhancer activation. Super-enhancers of these genes were activated prior to induction of the transcripts and myeloid differentiation. Depletion of GFI1 attenuated myeloid differentiation by NCD38. Finally, a single administration of NCD38 causes the in vivo eradication of primary MDS-related leukemia cells with a complex karyotype. Together, NCD38 derepresses super-enhancers of hematopoietic regulators that are silenced abnormally by LSD1, attenuates leukemogenic programs and consequently exerts anti-leukemic effect against MDS-related leukemia with adverse outcome.

Fgfr2 is integral for bladder mesenchyme patterning and function.

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2BM-/-). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic (E) day 16.5 (E16.5) Fgfr2BM-/- bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal (P) day 1 (P1) to P30, Fgfr2BM-/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2BM-/- bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2BM-/- bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2BM-/- bladders compared with controls. Moreover, E16.5Fgfr2BM-/- bladders exhibit higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling.

ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Fgfr2 is integral for bladder mesenchyme patterning and function.

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor (Fgfr)2 is necessary for kidney and ureter mesenchymal development. The objective of the present study was to determine the role of Fgfr2 in the bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in the bladder mesenchyme (Fgfr2(BM-/-)). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblot analysis, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with control bladders, embryonic day 16.5 (E16.5) Fgfr2(BM-/-) bladders had thin muscle layers with less α-smooth muscle actin and thickened lamina propria with increased collagen type Ia and IIIa that intruded into the muscle. The reciprocal changes in mutant layer thicknesses appeared partly due to a cell fate switch. From postnatal days 1 to 30, Fgfr2(BM-/-) bladders demonstrated progressive muscle loss and increased collagen expression. Postnatal Fgfr2(BM-/-) bladder sheets exhibited decreased agonist-mediated contractility and increased passive stretch tension versus control bladder sheets. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2(BM-/-) versus control bladders. Mechanistically, whereas Shh expression appeared normal, mRNA and protein readouts of hedgehog activity were increased in E16.5 Fgfr2(BM-/-) versus control bladders. Moreover, E16.5 Fgfr2(BM-/-) bladders exhibited higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, compared with control bladders. In conclusion, loss of Fgfr2 in the bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility.

Emergence of resistant variants detected by ultra-deep sequencing after asunaprevir and daclatasvir combination therapy in patients infected with hepatitis C virus genotype 1.

Daclatasvir (DCV) and asunaprevir (ASV) are NS5A and NS3 protease-targeted antivirals respectively, currently under development for the treatment of chronic hepatitis C virus (HCV) infection. We analysed the relationship between pre-existing drug-resistant variants and clinical outcome of the combination treatment with DCV and ASV. Ten patients with HCV genotype 1b were orally treated with a combination of ASV and DCV for 24 weeks. The frequencies of amino acid (aa) variants at NS3 aa positions 155, 156 and 168 and at NS5A aa31 and 93 before and after treatment were analysed by ultra-deep sequencing. We established a minimum variant frequency threshold of 0.3% based on plasmid sequencing. Sustained virological response (SVR) was achieved in 8 out of 10 patients (80%), and relapse of HCV RNA after cessation of the treatment and viral breakthrough occurred in the other two patients. Pre-existing DCV-resistant variants (L31V/M and/or Y93H; 0.9-99.4%) were detected in three out of eight patients who achieved SVR. Pre-existing DCV-resistant variants were detected in a relapsed patient (L31M, Y93H) and in a patient with viral breakthrough (Y93H); however, no ASV-resistant variants were detected. In these patients, HCV RNA rebounded with ASV- and DCV- double resistant variants (NS3 D168A/V plus NS5A L31M and Y93H). While pre-existing DCV-resistant variants might contribute to viral breakthrough in DCV and ASV combination therapy, the effectiveness of prediction of the outcome of therapy based on ultra-deep sequence analysis of pre-existing resistant variants appears limited.

Modulation of spontaneous activity in the overactive bladder: the role of P2Y agonists.

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca(2+) and membrane potential (E(m)) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y(6)-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca(2+)/E(m) waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca(2+)/E(m) waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.

Endothelial and inflammatory markers in relation to progression of ischaemic cerebral small-vessel disease and cognitive impairment: a 6-year longitudinal study in patients with type 2 diabetes mellitus.

BACKGROUND: Progression of silent brain infarctions (SBIs) and white-matter lesions (WMLs) seen on brain MRI is associated with an increased risk of cognitive impairment, but their relation to endothelial and inflammatory markers is unknown in type 2 diabetes mellitus. METHODS: In 190 type 2 diabetic outpatients (mean age 62.7 years), the authors related baseline levels of soluble intercellular adhesion molecule-1 (sICAM-1) and high-sensitivity C-reactive protein (hs-CRP) to subsequent brain MRI findings and cognitive function. The authors assessed incident SBIs and changes in periventricular and subcortical WMLs (PVWMLs and SCWMLs) on MRI performed at baseline and 3 and 6 years. Neuropsychological tests were administered to 83 patients older than 65 years at 6 years. This present study represents an extension of the authors' previously published study. RESULTS: SBIs were observed in 46 patients (24.2%), PVWMLs in 93 (48.9%) and SCWMLs in 87 (45.8%) on baseline MRI. After adjustment for age, gender, hypertension, duration of diabetes, baseline MRI findings and medication use, the relative odds associated with a 1SD increase in sICAM-1 levels at baseline were 1.67 (95% CI 1.02 to 3.05) for SBI progression and 2.17 (95% CI 1.29 to 3.62) for PVWML progression at 6 years. In contrast, baseline hs-CRP levels were significantly associated with SBI progression only at 3 years. Significant trends were observed between quartiles of sICAM-1 at baseline and scores in Digit Symbol substitution (p for trend=0.01). CONCLUSIONS: The findings suggest that higher sICAM-1 levels are associated with SBI and PVWML progression, and may predict impairment in psychomotor function in type 2 diabetes.

Animal models and their use in understanding lower urinary tract dysfunction.

This review will highlight appropriate animal models for the study of a number of disorders involving changes to lower urinary tract function. A major hurdle to the development of animal models for human lower urinary tract disorders is that the clinical pathophysiology of the latter mostly remain idiopathic. Acute injury/inflammation of otherwise healthy animals has often been used to study effects on a target tissue/organ. However, these "acute" models may not adequately address the characteristics of "chronic" visceral disorders. In addition, the relevance of observed changes following acute injury/inflammation, in terms of possible therapeutic targets, may not reflect that which occurs in the human condition. We have therefore emphasized the situations when animal models are required to investigate lower urinary tract disorders and what they should set out to achieve. In particular we have discussed the merits and disadvantages of a number of paradigms that set out to investigate specific lower urinary tract disorders or situations associated with these conditions. These include animal models of overactive bladder, stress urinary incontinence, ageing and congenital defects of the urinary tract and bladder pain syndrome.

Is the urothelium intelligent?

The urothelium separates the urinary tract lumen from underlying tissues of the tract wall. Previously considered as merely an effective barrier between these two compartments it is now recognized as a more active tissue that senses and transduces information about physical and chemical conditions within the urinary tract, such as luminal pressure, urine composition, etc. To understand this sensory function it is useful to consider the urothelium and suburothelium as a functional unit; containing uroepithelial cells, afferent and efferent nerve fibers and suburothelial interstitial cells. This structure responds to alterations in its external environment through the release of diffusible agents, such as ATP and acetylcholine, and eventually modulates the activity of afferent nerves and underlying smooth muscles. This review considers different stresses the urothelium/suburothelium responds to; the particular chemicals released; the cellular receptors that are consequently affected; and how nerve and muscle function is modulated. Brief consideration is also to regional differences in the urothelium/suburothelium along the urinary tract. The importance of different pathways in relaying sensory information in the normal urinary tract, or whether they are significant only in pathological conditions is also discussed. An operational definition of intelligence is used, whereby a system (urothelium/suburothelium) responds to external changes, to maximize the possibility of the urinary tract achieving its normal function. If so, the urothelium can be regarded as intelligent. The advantage of this approach is that input-output functions can be mathematically formulated, and the importance of different components contributing to abnormal urinary tract function can be calculated.

Mucosal muscarinic receptors enhance bladder activity in cats with feline interstitial cystitis.

PURPOSE: Interstitial cystitis is a chronic pelvic pain syndrome of which the origin and mechanisms involved remain unclear. In this study Ca(2+) transients in the bladder wall of domestic cats diagnosed with naturally occurring feline interstitial cystitis were examined. MATERIALS AND METHODS: Cross-sections of full-thickness bladder strips from normal cats and cats with feline interstitial cystitis were examined by optically mapping Ca(2+) transients and recording tension. Responses of Ca(2+) activity and detrusor contractions to pharmacological interventions were compared. In addition, pharmacological responses were compared in mucosa denuded preparations. RESULTS: Optical mapping showed that feline interstitial cystitis bladders had significantly more spontaneous Ca(2+) transients in the mucosal layer than control bladders. Optical mapping also demonstrated that feline interstitial cystitis bladders were hypersensitive to a low dose (50 nM) of the muscarinic receptor agonist arecaidine when the mucosal layer was intact. This hypersensitivity was markedly decreased in mucosa denuded bladder strips. CONCLUSIONS: In feline interstitial cystitis cat bladders there is increased Ca(2+) activity and sensitivity of muscarinic receptors in the mucosal layer, which can enhance smooth muscle spontaneous contractions.

Urotheliogenic modulation of intrinsic activity in spinal cord-transected rat bladders: role of mucosal muscarinic receptors.

We examined the modulation of intrinsic (i.e., spontaneous) detrusor contractions by the urothelium and the lamina propria through optical mapping approaches. Normal adult and spinal cord-transected (SCT) rat bladders were stained with Ca2+- and voltage-sensitive dyes, and optical activity generated from intrinsic contractions was mapped from the mucosal surface of whole bladder sheets. Both normal adult and SCT rat bladders displayed intrinsic contractions, where normal bladders showed low-amplitude, high-frequency contractions with disorganized patterns of activity. In contrast, in the SCT animals there were high-amplitude, low-frequency contractions that displayed an organized spread of membrane potential and intracellular Ca2+. The difference in contractile activity was mirrored in the Ca2+ and membrane potential maps of bladder sheets. Normal bladders showed multiple initiation sites across the mucosal surface, whereas SCT bladders showed only one or two fixed initiation sites localized to the dome. The magnitude of intrinsic contractions could be enhanced by stretch or low-dose arecaidine (50 nM), a muscarinic-specific agonist. Partial removal of the mucosa decreased the amplitude of the intrinsic contractions and decreased the response to stretch or arecaidine. Optical mapping of mucosa-denuded sheets, where enhanced spontaneous activity was abolished, or application of 1 microM nifedipine to remove smooth muscle signals, but not the mucosal signals, shows that intrinsic activity in pathological bladders is driven by the mucosal layer. In summary, we suggest an urotheliogenic origin for intrinsic activity, where structures within the mucosal layer organize and thereby enhance intrinsic detrusor contractions.

The function of suburothelial myofibroblasts in the bladder.

The properties of suburothelial myofibroblasts are described, and their possible role in shaping sensory responses from the bladder wall are discussed. Suburothelial myofibroblasts consist of long spindle-shaped cells that form a distinctive layer below the urothelium and are connected to each other through connexin 43 gap junctions. Isolated cells from guinea pig or human bladders display spontaneous fluctuations of membrane potential and intracellular [Ca(2+)], and respond in a similar way to exogenous application of adenosine triphosphate (ATP) or lowering of extracellular pH. ATP generates an intracellular Ca(2+) transient via activation of a P2Y receptor, which in turn initiates a Ca(2+)-sensitive Cl(-) current inward at the normal membrane potential of -50 to -60 mV. Of the P2Y receptor subtypes identified by immunolabeling, the most prominent was the P2Y(6) receptor. Cell pairs, without the formation of gap junctions, elicit augmented responses to exogenous agonists. Mechanical stimulation of the suburothelial layer in intact cross-sections of the bladder elicited Ca(2+) waves that propagated across the suburothelial layer before invading the detrusor layer. This indicates that the suburothelial layer forms a discrete functional layer of cells capable of propagating signals over many cell lengths. A function for suburothelial myofibroblasts is proposed whereby they act as an amplification stage in the sensory response to bladder-wall stretch, as occurs during bladder filling.

Role of gap junctions in spontaneous activity of the rat bladder.

Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca(2+)-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibroblasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18beta-glycyrrhetinic acid, 1 microM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.

Origin of spontaneous activity in neonatal and adult rat bladders and its enhancement by stretch and muscarinic agonists.

This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and Ca(2+)-sensitive dyes. Intracellular Ca(2+) and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37 degrees C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous Ca(2+) and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities (Ca(2+): 3.7 +/- 0.4 mm/s; membrane potential: 46.2 +/- 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10-20 cmH(2)O). By contrast, in adult bladders spontaneous Ca(2+) and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2-5 cmH(2)O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 muM) but were blocked by nifedipine (10 muM). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20-35 cmH(2)O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that Ca(2+) and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity.

Intranasal lidocaine 8% spray for second-division trigeminal neuralgia.

BACKGROUND: Trigeminal nerve block has been widely used for trigeminal neuralgia. This may induce paraesthesia. The second division of the trigeminal nerve passes through the sphenopalatine ganglion, which is located posterior to the middle turbinate and is covered by a mucous membrane. We examined the effectiveness of intranasal lidocaine 8% spray on paroxysmal pain in second-division trigeminal neuralgia. METHODS: Twenty-five patients with second-division trigeminal neuralgia were randomized to receive two sprays (0.2 ml) of either lidocaine 8% or saline placebo in the affected nostril using a metered-dose spray. After a 7 day period, patients were crossed over to receive the alternative treatment. The paroxysmal pain triggered by touching or moving face was assessed with a 10 cm visual analogue scale (VAS) before and 15 min after treatment. Patients used a descriptive scale to grade pain outcome, and were asked to note whether the pain returned and how long after therapy it recurred. RESULTS: Intranasal lidocaine 8% spray significantly decreased VAS [baseline: 8.0 (2.0) cm, 15 min postspray: 1.5 (1.9) cm, mean (SD)], whereas the placebo spray did not [7.9 (2.0) cm, 7.6 (2.0) cm]. Moreover, pain was described as moderate or better by 23 patients of the lidocaine spray and 1 of the placebo group. The effect of treatment persisted for 4.3 h (range 0.5-24 h). CONCLUSIONS: Intranasal lidocaine 8% administered by a metered-dose spray produced prompt but temporary analgesia without serious adverse reactions in patients with second-division trigeminal neuralgia.

Symposium report on urothelial dysfunction: pathophysiology and novel therapies.

PURPOSE: The basic premise of this symposium (Workshop 7) at the 2004 International Continence Society meeting in Paris was to elucidate different mechanisms of urothelial cell pathology, explore their impact on bladder function and discuss novel therapeutic interventions. RESULTS: The topics included 1) urothelial structure and function, 2) the role of adenosine triphosphate in urothelial signaling and cystitis, 3) lamina propria myofibroblasts and purinergic receptors, 4) antiproliferative factor involvement in interstitial cystitis, 5) the urothelium as a reservoir for bacterial infections, 6) radiation cystitis, nitric oxide and gene therapy, and 7) intravesical treatments. DISCUSSION: It was agreed that the urothelium can no longer be regarded merely as a passive barrier separating urine from the underlying tissues. The epithelial cells of the urothelium form part of an integrated network that also includes afferent and possibly efferent nerves, and suburothelial myofibroblasts. It has a central role in several functions, including bladder wall sensation, local blood flow modulation, pathogen removal and active barrier provision. These functions are achieved through several autocrine and paracrine pathways that involve transmitter release from the urothelium and its ability to integrate incoming signals through its battery of membrane receptors. Several pathological processes were discussed using this knowledge, including the role of small glycoproteins released during interstitial cystitis, the molecular basis of radiation induced urothelial damage, the origin of recurrent urinary tract infections and the mode of action of potential intravesical treatments for overactive bladder. CONCLUSIONS: Overall it was concluded that the urothelium has a key role in regulating lower urinary tract physiology and pathology.

Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis.

PURPOSE: Alterations in nitric oxide (NO) levels have been demonstrated in some humans with interstitial cystitis (IC) as well as in chemically induced animal models of cystitis. Thus, in the current study we investigated whether inducible NO synthase (iNOS) mediated NO production is altered in the bladder of cats with a naturally occurring model of IC termed feline IC (FIC). MATERIALS AND METHODS: We examined iNOS expression using Western immunoblotting and baseline NO production using an NO microsensor from smooth muscle and mucosal bladder strips in 9 healthy cats and 6 diagnosed with FIC. RESULTS: There was a significant increase in baseline NO production in cats with FIC compared with that in healthy cats in smooth muscle and mucosal strips. This production was not ablated in the absence of extracellular Ca (100 microM egtazic acid) or following incubation with the calmodulin antagonist trifluoroperazine (20 microM), indicating iNOS mediated Ca independent NO production. Release was significantly decreased following incubation with the NOS antagonist L-NAME (N-nitro-L-arginine methyl ester) (100 microM). Furthermore, immunoblotting revealed a trend toward increased iNOS expression in smooth muscle and mucosal strips from FIC cats but not from healthy cats. CONCLUSIONS: In light of previous findings that the barrier property of the urothelial surface is disrupted in FIC and iNOS mediated increase in NO alters barrier function in other types of epithelium our findings suggest that iNOS dependent NO production may have a role in epithelial barrier dysfunction in FIC.