Enhancing Therapeutic Efficacy and Safety of Immune Checkpoint Inhibition for Bladder Cancer: A Comparative Analysis of Injectable vs. Intravesical Administration.

Bladder cancer (BC) presents a significant global health burden, characterized by high recurrence rates post-initial treatment. Gender differences in BC prevalence and response to therapy emphasize the importance of personalized treatment strategies. While Bacillus Calmette-Guérin (BCG) remains a cornerstone of BC therapy, resistance poses a challenge, necessitating alternative strategies. Immune checkpoint inhibitors (ICIs) have shown promise, yet systemic toxicity raises concern. Intravesical administration of ICIs offers a potential solution, with recent studies demonstrating the feasibility and efficacy of intravesical pembrolizumab. Although systemic toxicity remains a concern, its localized administration may mitigate adverse events. Additionally, liposomal delivery of ICIs exhibits promises in enhancing drug penetration and reducing toxicity. Novel imaging modalities compatible with Vesical Imaging-Reporting and Data System (VI-RADS) and capable of predicting high-grade bladder cancer can aid the pre-operative shared decision making of patient and surgeon. Future research should focus on refining treatment approaches, optimizing dosing regimens, and leveraging advanced imaging techniques to improve patient outcomes. In conclusion, intravesical immunotherapy presents a promising avenue for BC treatment, offering enhanced therapeutic effectiveness while minimizing systemic toxicity. Continued research efforts are essential to validate these findings and optimize intravesical immunotherapy's role in BC management, ultimately improving patient outcomes.

Benign prostatic hyperplasia/obstruction ameliorated using a soluble guanylate cyclase activator.

Benign prostatic hyperplasia (BPH) is a feature of ageing males. Up to half demonstrate bladder outlet obstruction (BOO) with associated lower urinary tract symptoms (LUTS) including bladder overactivity. Current therapies to reduce obstruction, such as α1-adrenoceptor antagonists and 5α-reductase inhibitors, are not effective in all patients. The phosphodiesterase-5 inhibitor (PDE5I) tadalafil is also approved to treat BPH and LUTS, suggesting a role for nitric oxide (NO• ), soluble guanylate cyclase (sGC), and cGMP signalling pathways. However, PDE5I refractoriness can develop for reasons including nitrergic nerve damage and decreased NO• production, or inflammation-related oxidation of the sGC haem group, normally maintained in a reduced state by the cofactor cytochrome-b5-reductase 3 (CYB5R3). sGC activators, such as cinaciguat (BAY 58-2667), have been developed to enhance sGC activity in the absence of NO• or when sGC is oxidised. Accordingly, their effects on the prostate and LUT function of aged mice were evaluated. Aged mice (≥24 months) demonstrated a functional BPH/BOO phenotype, compared with adult animals (2-12 months), with low, delayed voiding responses and elevated intravesical pressures as measured by telemetric cystometry. This was consistent with outflow tract histological and molecular data that showed urethral constriction, increased prostate weight, greater collagen deposition, and cellular hyperplasia. All changes in aged animals were attenuated by daily oral treatment with cinaciguat for 2 weeks, without effect on serum testosterone levels. Cinaciguat had only transient (1 h) cardiovascular effects with oral gavage, suggesting a positive safety profile. The benefit of cinaciguat was suggested by its reversal of an overactive cystometric profile in CYB5R3 smooth muscle knockout mice that mirrors a profile of oxidative dysfunction where PDE5I may not be effective. Thus, the aged male mouse is a suitable model for BPH-induced BOO and cinaciguat has a demonstrated ability to reduce prostate-induced obstruction and consequent effects on bladder function. © 2021 The Pathological Society of Great Britain and Ireland.

Temporally complex inflammatory networks in an animal model reveal signatures for interstitial cystitis and bladder pain syndrome phenotype.

INTRODUCTION AND OBJECTIVE: Interstitial cystitis and bladder pain syndrome (IC/BPS) presents with symptoms of debilitating bladder pain and is typically a diagnosis of exclusion. The cystoscopic detection of Hunner's lesions increases the likelihood of detecting tissue inflammation on bladder biopsy and increases the odds of therapeutic success with anti-inflammatory drugs. However, the identification of this subgroup remains challenging with the current lack of surrogate biomarkers of IC/BPS. On the path towards identifying biomarkers of IC/BPS, we modeled the dynamic evolution of inflammation in an experimental IC/BPS rodent model using computational biological network analysis of inflammatory mediators (cytokines and chemokines) released into urine. The use of biological network analysis allows us to identify urinary proteins that could be drivers of inflammation and could therefore serve as therapeutic targets for the treatment of IC/BPS. METHODS: Rats subjected to cyclophosphamide (CYP) injection (150 mg/kg) were used as an experimental model for acute IC/BPS (n = 8). Urine from each void was collected from the rats over a 12-h period and was assayed for 13 inflammatory mediators using Luminex™. Time-interval principal component analysis (TI-PCA) and dynamic network analysis (DyNA), two biological network algorithms, were used to identify biomarkers of inflammation characteristic of IC/BPS over time. RESULTS: Compared to vehicle-treated rats, nearly all inflammatory mediators were elevated significantly (p < 0.05) in the urine of CYP treated rats. TI-PCA highlighted that GRO-KC, IL-5, IL-18, and MCP-1 account for the greatest variance in the inflammatory response. At early time points, DyNA indicated a positive correlation between IL-4 and IL-1ß and between TNF-α and IL-1ß. Analysis of TI-PCA and DyNA at later time points showed the emergence of IL-5, IL-6, and IFNγ as additional key mediators of inflammation. Furthermore, DyNA network complexity rose and fell before peaking at 9.5 h following CYP treatment. This pattern of inflammation may mimic the fluctuating severity of inflammation associated with IC/BPS flares. CONCLUSIONS: Computational analysis of inflammation networks in experimental IC/BPS analysis expands on the previously accepted inflammatory signatures of IC by adding IL-5, IL-18, and MCP-1 to the prior studies implicating IL-6 and GRO as IC/BPS biomarkers. This analysis supports a complex evolution of inflammatory networks suggestive of the rise and fall of inflammation characteristic of IC/BPS flares.

Time-dependent progression of neurogenic lower urinary tract dysfunction after spinal cord injury in the mouse model.

This study evaluated the time-course changes in bladder and external urinary sphincter (EUS) activity and the expression of mechanosensitive channels in lumbosacral dorsal root ganglia (DRG) after spinal cord injury (SCI). Female C57BL/6N mice in the SCI group underwent transection of the Th8/9 spinal cord. Spinal intact mice and SCI mice at 2, 4, and 6 wk post-SCI were evaluated by single-filling cystometry and EUS-electromyography (EMG). In another set of mice, the bladder and L6-S1 DRG were harvested for protein and mRNA analyses. In SCI mice, nonvoiding contractions were confirmed at 2 wk post-SCI and did not increase over time to 6 wk. In 2-wk SCI mice, EUS-EMG measurements revealed detrusor sphincter dyssynergia, but periodic EMG reductions during bladder contraction were hardly observed. At 4 wk, SCI mice showed increases of EMG activity reduction time with increased voiding efficiency. At 6 wk, SCI mice exhibited a further increase in EMG reduction time. RT-PCR of L6-S1 DRG showed increased mRNA levels of transient receptor potential vanilloid 1 and acid-sensing ion channels (ASIC1-ASIC3) in SCI mice with a decrease of ASIC2 and ASIC3 at 6 wk compared with 4 wk, whereas Piezo2 showed a slow increase at 6 wk. Protein assay showed SCI-induced overexpression of bladder brain-derived neurotrophic factor with a time-dependent decrease post-SCI. These results indicate that detrusor overactivity is established in the early phase, whereas detrusor sphincter dyssynergia is completed later at 4 wk with an improvement at 6 wk post-SCI, and that mechanosensitive channels may be involved in the time-dependent changes.NEW & NOTEWORTHY This is the first paper to evaluate the time-course changes of bladder dysfunction associated with mechanosensitive channels in a mouse model.

Therapeutic effects of nerve growth factor-targeting therapy on bladder overactivity in rats with prostatic inflammation.

BACKGROUND: The present study examined the effect of liposomes conjugated with antisense oligonucleotide of nerve growth factor (NGF-OND) on local overexpression of NGF and bladder overactivity using rats with prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups: (1) Control group; intact rats, (2) PI-NS group; rats with PI and intravesical instillation of normal saline (NS), (3) PI-OND group; rats with PI and intravesical instillation of NGF-OND. On Day 0, PI was induced by intraprostatic 5%-formalin injection. On Day 14, NGF-OND or NS was instilled directly into the bladder after laparotomy. On Day 28, therapeutic effects of NGF-OND were evaluated by awake cystometry and histological analysis as well as reverse-transcription polymerase chain reaction measurements of messenger RNA (mRNA) levels of NGF in the bladder and prostate, inflammatory markers in the prostate, C-fiber afferent markers, and an A-type K+ channel α-subunit (Kv 1.4) in L6-S1 dorsal root ganglia (DRG). RESULTS: Intravesical NFG-OND treatment reduced PI-induced overexpression of NGF in both bladder and prostate, and reduced PI-induced bladder overactivity evident as longer intercontraction intervals in association with reductions of TRPV1 and TRPA1 mRNA expression levels in DRG. mRNA expression of Kv1.4 in DRG was reduced after PI, but improved in the PI-OND group. CONCLUSIONS: These results indicate that NGF locally expressed in the bladder is an important mediator inducing bladder overactivity with upregulation of C-fiber afferent markers and downregulation of an A-type K+ channel subunit in DRG following PI, and that liposome-based, local NGF-targeting therapy could be effective for not only bladder overactivity and afferent sensitization, but also PI. Thus, local blockade of NGF in the bladder could be a therapeutic modality for male LUTS due to BPH with PI.

Virtual measurements of paracellular permeability and chronic inflammation via color coded pixel-wise T1 mapping.

To assess whether quantitative T1 relaxometry can measure permeability, chronic inflammation and mural thickening of mouse bladder wall. Adult female C57BL6 mice unexposed to radiation (controls) or 40 wk postirradiation of 10 Gy were scanned at 9.4 T before and after instillation (0.1 mL) of aqueous, novel contrast mixture (NCM) containing 4 mM gadobutrol and 5 mM ferumoxytol. Rapid acquisition with refocused echo (RARE) sequence was used with variable repetition times (TR). Pixel-wise maps of T1 relaxation times for the segmented bladder wall layers were generated from voxel-wise, nonlinear least square data fitting of TR-dependent signal intensity acquired with TR array of 0.4-10 s followed by the histology of harvested bladder. Significant differences between precontrast and postcontrast T1 (ΔT1) were noted in urothelium and lamina propria of both groups but only in detrusor of irradiated group (P < 0.001; 2-way ANOVA). Nearly twofold higher gadobutrol permeability (550 ± 73 vs. 294 ± 160 µM; P < 0.01) derived as per 1/ΔT1 = r1. [C] in urothelium of irradiated group. Inflammation and bladder wall thickening (0.75 ± 0. vs. 0.44 ± 0.08 mm; P < 0.001) predicted by MRI was subsequently confirmed by histology and altered expression of CD45 and zonula occludens-1 (ZO-1) relative to controls. NCM enhanced MRI relies on the retention of large molecular weight ferumoxytol in lumen for negative contrast, while permeation of the non-ionic, small molecular weight gadobutrol through ZO-1 generates positive contrast in bladder wall for virtual measurement of paracellular permeability and assessment of chronic inflammation in thin and distensible bladder wall, which is also defined by its variable shape and location within pelvis.

The early, long-term inhibition of brain-derived neurotrophic factor improves voiding, and storage dysfunctions in mice with spinal cord injury.

AIMS: We examined the time course of urodynamic changes and the effect of the short or long-term inhibition of brain-derived neurotrophic factor (BDNF) from the early phase after spinal cord injury (SCI) in mice. METHODS: The spinal cord of female C57BL/6N mice was completely transected. We examined filling cystometry and bladder BDNF levels at 10, 20, and 30 days after SCI, with an additional day-5 measurement of BDNF. In a separate group of mice, anti-BDNF antibody (Ab) (10 µg/kg/h) was subcutaneously administered using osmotic pumps from day 3 after SCI, and single-filling cystometry was performed at 10 and 30 days (7 and 27 days of treatment, respectively) after SCI. RESULTS: Compared to spinal intact mice, bladder mucosal BDNF was increased at each time point after SCI with the maximal level at day 5 after SCI. Voiding efficiency was lower at each time point after SCI than that of spinal intact mice. The number of non-voiding contractions (NVC) during bladder filling was gradually increased with time. In both 10- and 30-day SCI groups treated with anti-BDNF Ab, voiding efficiency was improved, and the duration of notch-like intravesical pressure reductions during voiding bladder contractions was prolonged. The number of NVC was significantly decreased only in 30-day SCI mice with 27-day anti-BDNF treatment. CONCLUSIONS: Overexpression of BDNF is associated with the deterioration of voiding efficiency after SCI. The early-started, long-term inhibition of BDNF improved voiding dysfunction and was also effective to reduce the later-phase development of detrusor overactivity after SCI.

Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation.

KEY POINTS: There is clinical evidence showing that prostatic inflammation contributes to overactive bladder symptoms in male patients; however, little is known about the underlying mechanisms In this study, we investigated the mechanism that prostatic inflammation causes detrusor overactivity by using a rat model of chemically induced prostatic inflammation. We observed a significant number of dorsal root ganglion neurons with dichotomized afferents innervating both prostate and bladder. We also found that prostatic inflammation induces bladder overactivity and urothelial NGF overexpression in the bladder, both dependent on activation of the pelvic nerve, as well as changes in ion channel expression and hyperexcitability of bladder afferent neurons. These results indicate that the prostate-to-bladder cross-sensitization through primary afferent pathways in the pelvic nerve, which contain dichotomized afferents, could be an important mechanism contributing to bladder overactivity and afferent hyperexcitability induced by prostatic inflammation. ABSTRACT: Prostatic inflammation is reportedly an important factor inducing lower urinary tract symptoms (LUTS) including urinary frequency, urgency and incontinence in patients with benign prostatic hyperplasia (BPH). However, the underlying mechanisms inducing bladder dysfunction after prostatic inflammation are not well clarified. We therefore investigated the effects of prostatic inflammation on bladder activity and afferent function using a rat model of non-bacterial prostatic inflammation. We demonstrated that bladder overactivity, evident as decreased voided volume and shorter intercontraction intervals in cystometry, was observed in rats with prostatic inflammation versus controls. Tissue inflammation, evident as increased myeloperoxidase activity, and IL-1α, IL-1ß, and IL-6 levels inside the prostate, but not in the bladder, following intraprostatic formalin injection induced an increase in NGF expression in the bladder urothelium, which depended on activation of the pelvic nerve. A significant proportion (18-19%) of dorsal root ganglion neurons were double labelled by dye tracers injected into either bladder or prostate. In rats with prostatic inflammation, TRPV1, TRPA1 and P2X2 increased, and Kv1.4, a potassium channel α-subunit that can form A-type potassium (KA ) channels, decreased at mRNA levels in bladder afferent and double-labelled neurons vs. non-labelled neurons, and slow KA current density decreased in association with hyperexcitability of these neurons. Collectively, non-bacterial inflammation localized in the prostate induces bladder overactivity and enhances bladder afferent function. Thus, prostate-to-bladder afferent cross-sensitization through primary afferents in the pelvic nerve, which contain dichotomized afferents, could underlie storage LUTS in symptomatic BPH with prostatic inflammation.

Therapeutic effects of inhibition of brain-derived neurotrophic factor on voiding dysfunction in mice with spinal cord injury.

We investigated the involvement of brain-derived neurotrophic factor (BDNF) in bladder and urethral dysfunction using spinal cord-injured mice. We evaluated bladder and urethral function of female mice with 4-wk spinal cord injury (SCI) by filling cystometry and electromyography (EMG) of the external urethral sphincter (EUS) under a conscious condition. Anti-BDNF antibodies (10 µg·kg-1·h-1) were administered in some mice for 1 wk before the evaluation. Bladder and spinal (L6-S1) BDNF protein levels were examined by ELISA. Transcript levels of transient receptor potential channels or acid-sensing ion channels (Asic) in L6-S1 dorsal root ganglia were evaluated by RT-PCR. Voided volume and voiding efficiency were significantly increased without any changes in nonvoiding contractions, and the duration of reduced EMG activity during the voiding phase was significantly prolonged in anti-BDNF antibody-treated SCI mice. Compared with spinal cord-intact mice, SCI mice showed increased concentrations of bladder and spinal BDNF. Anti-BDNF antibody treatment decreased bladder and spinal BDNF protein concentrations of SCI mice. Asic2 and Asic3 transcripts were significantly increased after SCI but decreased after anti-BDNF antibody administration. These results indicate that upregulated expression of bladder and spinal BDNF is involved in the emergence of inefficient voiding in SCI mice. Thus, BDNF-targeting treatment could be an effective modality for the treatment of voiding problems, including inefficient voiding and detrusor sphincter dyssynergia after SCI.

Long-lasting bladder overactivity and bladder afferent hyperexcitability in rats with chemically-induced prostatic inflammation.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the major causes of lower urinary tract symptoms (LUTS), including storage LUTS such as urinary frequency and urgency. Recently, a growing number of clinical studies indicate that prostatic inflammation could be an important pathophysiological mechanism inducing storage LUTS in patients with BPH. Here we aimed to investigate whether nonbacterial prostatic inflammation in a rat model induced by intraprostatic formalin injection can lead to long-lasting bladder overactivity and changes in bladder afferent neuron excitability. METHODS: Male Sprague-Dawley rats were divided into four groups (n = 12 each): normal control group, 1-week prostatic inflammation group, 4-week inflammation group, and 8-week inflammation group. Prostatic inflammation was induced by formalin (10%; 50 µL per lobe) injection into bilateral ventral lobes of the prostate. Voiding behavior was evaluated in metabolic cages for each group. Ventral lobes of the prostate and the bladder were then removed for hematoxylin and eosin (HE) staining to evaluate inflammation levels. Continuous cystometrograms (CMG) were recorded to measure intercontraction intervals (ICI) and voided volume per micturition. Whole-cell patch clamp recordings were performed on dissociated bladder afferent neurons labeled by fluorogold injected into the bladder wall, to examine the electrophysiological properties. RESULTS: Results of metabolic cage measurements showed that formalin-treated rats exhibited significantly (P < 0.05) increases in micturition episodes/12 hours and decrease in voided volume per micturition at every time point post injection. Continuous CMG illustrated the significant ( P < 0.05) higher number of nonvoiding contractions per void and shorter ICI in formalin-treated rats compared with control rats. HE staining showed significant prostatic inflammation, which declined gradually, in prostate tissues of formalin-induced rats. In patch clamp recordings, capsaicin-sensitive bladder afferent neurons from rats with prostatic inflammation had significantly ( P < 0.05) lower thresholds for spike activation and a "multiple" firing pattern compared with control rats at every time point post injection. CONCLUSIONS: Formalin-induced prostatic inflammation can lead to long-lasting bladder overactivity in association with bladder afferent neuron hyperexcitability. This long-lasting model could be a useful tool for the study of inflammation-related aspects of male LUTS pathophysiology.

Low Energy Shock Wave Therapy Inhibits Inflammatory Molecules and Suppresses Prostatic Pain and Hypersensitivity in a Capsaicin Induced Prostatitis Model in Rats.

The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the eyes, hypolocomotion, and tactile allodynia, which effects were ameliorated by LESW treatment. LESW therapy (2Hz, energy flux density of 0.12 mJ/mm2) at 200 and 300 shocks significantly decreased capsaicin-induced inflammatory reactions, reflected by a reduction of tissue edema and inflammatory cells, COX-2 and TNF-α stained positive cells, however, the therapeutic effects were not observed at 100 shocks treated group. Capsaicin-induced IL-1ß, COX-2, IL-6, caspase-1, and NGF upregulation on day 3 and 7, while NALP1 and TNF-α upregulation was observed on day 7. LESW significantly suppressed the expression of IL-1ß, COX-2, caspase-1, NGF on day 3 and IL-1ß, TNF-α, COX-2, NALP1, caspase-1, NGF expression on day 7 in a dose-dependent fashion. LESW has no significant effect on IL-6 expression. Intraprostatic capsaicin injection activates inflammatory molecules and induces prostatic pain and hypersensitivity, which effects were suppressed by LESW. These findings might be the potential mechanisms of LESW therapy for nonbacterial prostatitis in humans.

BDNF overexpression in the bladder induces neuronal changes to mediate bladder overactivity.

Elevated levels of brain-derived neurotrophic factor (BDNF) in urine of overactive bladder (OAB) patients support the association of BDNF with OAB symptoms, but the causality is not known. Here, we investigated the functionality of BDNF overexpression in rat bladder following bladder wall transfection of either BDNF or luciferase (luciferase) transgenes (10 µg). One week after transfection, BDNF overexpression in bladder tissue and elevation of urine BDNF levels were observed together with increased transcript of BDNF, its cognate receptors (TrkB and p75NTR), and downstream PLCγ isoforms in bladder. BDNF overexpression can induce the bladder overactivity (BO) phenotype which is demonstrated by the increased voiding pressure and reduced intercontractile interval during transurethral open cystometry under urethane anesthesia. A role for BDNF-mediated enhancement of prejunctional cholinergic transmission in BO is supported by the significant increase in the atropine- and neostigmine-sensitive component of nerve-evoked contractions and upregulation of choline acetyltransferase, vesicular acetylcholine transporter, and transporter Oct2 and -α1 receptors. In addition, higher expression of transient receptor channels (TRPV1 and TRPA1) and pannexin-1 channels in conjunction with elevation of ATP and neurotrophins in bladder and also in L6/S1 dorsal root ganglia together support a role for sensitized afferent nerve terminals in BO. Overall, genomic changes in efferent and afferent neurons of bladder induced by the overexpression of BDNF per se establish a mechanistic link between elevated BDNF levels in urine and dysfunctional voiding observed in animal models and in OAB patients.

Molecular correlates in urine for the obesity and prostatic inflammation of BPH/LUTS patients.

PURPOSE: Benign prostatic hyperplasia (BPH) is strongly associated with obesity and prostatic tissue inflammation, but the molecular underpinning of this relationship is not known. Here, we examined the association between urine levels of chemokines/adipokines with histological markers of prostate inflammation, obesity, and lower urinary tract symptoms LUTS in BPH patients. METHODS: Frozen urine specimens from 207 BPH/LUTS patients enrolled in Nashville Men's Health Study were sent for blinded analysis of 11 analytes, namely sIL-1RA, CXC chemokines (CXCL-1, CXCL-8, CXCL-10), CC chemokines (CCL2, CCL3, CCL5), PDGF-BB, interleukins IL-6, IL-17, and sCD40L using Luminex™ xMAP® technology. After adjusting for age and medication use, the urine levels of analytes were correlated with the scales of obesity, prostate inflammation grade, extent, and markers of lymphocytic infiltration (CD3 and CD20) using linear regression. RESULTS: sIL-1RA levels were significantly raised with higher BMI, waist circumference and waist-hip ratio in BPH patients after correction for multiple testing (P = 0.02). Men with greater overall extent of inflammatory infiltrates and maximal CD3 infiltration were marginally associated with CXCL-10 (P = 0.054) and CCL5 (P = 0.054), respectively. CCL3 in 15 patients with moderate to severe grade inflammation was marginally associated with maximal CD20 infiltration (P = 0.09), whereas CCL3 was undetectable in men with mild prostate tissue inflammation. There was marginal association of sCD40L with AUA-SI scores (P = 0.07). CONCLUSIONS: Strong association of sIL-1RA in urine with greater body size supports it as a major molecular correlate of obesity in the urine of BPH patients. Increased urine levels of CXCL-10, CCL5, and CCL3 were marginally associated with the scores for prostate tissue inflammation and lymphocytic infiltration. Overall, elevated urinary chemokines support that BPH is a metabolic disorder and suggest a molecular link between BPH/LUTS and prostatic inflammation.

Nerve growth factor-dependent hyperexcitability of capsaicin-sensitive bladder afferent neurones in mice with spinal cord injury.

NEW FINDINGS: What is the central question of this study? Nerve growth factor (NGF) is reportedly a mediator inducing urinary bladder dysfunction. Is NGF directly involved in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent pathways after spinal cord injury (SCI)? What is the main finding and its importance? Neutralization of NGF by anti-NGF antibody treatment reversed the SCI-induced increase in the number of action potentials and the reduction in spike thresholds and A-type K+ current density in mouse capsaicin-sensitive bladder afferent neurones. Thus, NGF plays an important and direct role in hyperexcitability of capsaicin-sensitive C-fibre bladder afferent neurones attributable to the reduction in A-type K+ channel activity in SCI. ABSTRACT: Nerve growth factor (NGF) has been implicated as an important mediator in the induction of C-fibre bladder afferent hyperexcitability, which contributes to the emergence of neurogenic lower urinary tract dysfunction after spinal cord injury (SCI). In this study, we determined whether NGF immunoneutralization using an anti-NGF antibody (NGF-Ab) normalizes the SCI-induced changes in electrophysiological properties of capsaicin-sensitive C-fibre bladder afferent neurones in female C57BL/6 mice. The spinal cord was transected at the Th8/Th9 level. Two weeks later, continuous administration of NGF-Ab (10 µg kg-1  h-1 , s.c. for 2 weeks) was started. Bladder afferent neurones were labelled with Fast-Blue (FB), a fluorescent retrograde tracer, injected into the bladder wall 3 weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurones were prepared. Whole-cell patch-clamp recordings were then performed in FB-labelled neurones. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neurone to capsaicin was evaluated. In capsaicin-sensitive FB-labelled neurones, SCI significantly reduced the spike threshold and increased the number of action potentials during membrane depolarization for 800 ms. These SCI-induced changes were reversed by NGF-Ab. Densities of slow-decaying A-type K+ (KA ) and sustained delayed rectifier-type K+ currents were significantly reduced by SCI. The NGF-Ab treatment reversed the SCI-induced reduction in the KA current density. These results indicate that NGF plays an important role in hyperexcitability of mouse capsaicin-sensitive C-fibre bladder afferent neurones attributable to a reduction in KA channel activity. Thus, NGF-targeting therapies could be effective for treatment of afferent hyperexcitability and neurogenic lower urinary tract dysfunction after SCI.

MicroRNAs as potential biomarkers to predict the risk of urinary retention following intradetrusor onabotulinumtoxin-A injection.

AIMS: MicroRNAs (miRs) control post-transcriptional gene expression, and this is relevant in understanding better chronic diseases and treatment outcomes. The role of miRs in the pathology and treatment outcomes of overactive bladder (OAB) is unknown. In this study, we assessed the differential expression of miRs in OAB patients responding with either normal or elevated post-void residual volumes (PVRs) ≥200 mL following intradetrusor injection of onabotulinumtoxin-A (onaBoNT-A). METHODS: Female OAB patients refractory to OAB drugs were consented for this study. Cystoscopic-guided punch bladder biopsy was obtained at the time of injection of onaBoNT-A 100 units. The expression of 13 miR species, selected for their known effect on neurotrophin expression and smooth muscle function, was measured. PVRs and urine nerve growth factor (NGF) levels were measured at baseline and at the follow-up visit. RESULTS: Fourteen patients with mean age of 66 years were consented. Of these patients, nine maintained PVRs <200 mL after onaBoNT-A injection to comprise the low PVR group. The other five patients with PVRs ≥200 mL comprised the high PVR group. The expression of miR221 and miR125b was upregulated by 11- and 2-fold, respectively, in patients who responded with low PVRs after onaBoNT-A (P < 0.05). Urine NGF levels at baseline were not different between the two groups. CONCLUSIONS: This study suggests that deficiency in the pretreatment expression of miR221 and miR125b may predispose OAB patients to high PVRs following intradetrusor onaBoNT-A. Additional studies are needed to better understand the role of miRs in OAB.

Urodynamic effects of intravenous and intrathecal administration of E-series prostaglandin 1 receptor antagonist on detrusor overactivity in rats with spinal cord injury.

AIMS: We examined the effect of an E-series prostaglandin 1 (EP1) receptor antagonist on bladder activity using a rat model of spinal cord injury (SCI). METHODS: Female Sprague-Dawley rats were used. Six weeks after spinal cord transection, conscious-filling cystometry was performed. We evaluated the urodynamic parameters before and after intravenous (0.1 and 1.0 mg/kg) or intrathecal (0.1 and 1.0 µg) administration of SC51089, an EP1 antagonist. Spinal prostaglandin E2 (PGE2) concentration and EP1 receptor transcripts in the spinal cord and dorsal root ganglia (DRG) were measured by enzyme-linked immunosorbent assay and RT-PCR, respectively. RESULTS: The time to the first non-voiding contraction (NVC) was significantly prolonged after both 0.1 and 1.0 mg/kg of intravenous administration of SC51089 (75% prolongation at 1.0 mg/kg) whereas other parameters were not significantly changed compared to vehicle treatment. In addition, the time to the first NVC was also significantly prolonged after 1.0 µg of intrathecal administration of SC51089 (18% prolongation at 1.0 µg) whereas other parameters were not significantly changed. The spinal PGE2 concentration in SCI rats was significantly higher than that in spinal intact rats. The mRNA expressions of EP1 receptors in the both spinal cord and DRG from SCI rats were significantly higher than those from spinal intact rats. CONCLUSIONS: The PGE2-induced activation of EP1 receptors in the spinal cord contributes to the initiation of detrusor overactivity in SCI. Thus, the EP1 receptor could be a therapeutic target for the treatment of neurogenic detrusor overactivity due to SCI.

The effect of neutralization of nerve growth factor (NGF) on bladder and urethral dysfunction in mice with spinal cord injury.

AIMS: To investigate the role of nerve growth factor (NGF) in lower urinary tract dysfunction in mice with spinal cord injury (SCI). METHODS: Using 4-week SCI mice, single-filling cystometry and external urethral sphincter (EUS)-electromyography were performed under an awake condition. In some SCI mice, anti-NGF antibodies (10 µg/kg/h) were administered for 1 or 2 weeks before the urodynamic study. NGF levels in the bladder and L6/S1 spinal cord were assayed by ELISA. The transcript levels of P2X receptors and TRP channels in L6/S1 dorsal root ganglia (DRG) were measured by RT-PCR. RESULTS: In SCI mice, the area under the curve of non-voiding contractions (NVCs) during the storage phase was significantly decreased in both 1- and 2-week anti-NGF antibody-treated SCI groups. However, EUS-electromyogram parameters during voiding were not altered by the treatment. Bladder mucosal and spinal NGF levels were decreased after 2 weeks of anti-NGF antibody treatment. TRPA1 and TRPV1 transcripts in L6/S1 DRG were significantly decreased after 1- or 2-week anti-NGF treatment. CONCLUSIONS: In SCI mice, NGF is involved in the emergence of NVCs in association with increased expression of TRP receptors that are predominantly found in C-fiber afferent pathways. Thus, NGF targeting treatments could be effective for treating storage problems such as detrusor overactivity after SCI.

Urodynamic and molecular characteristics of detrusor underactivity in a rat cryoinjury model and effects of low energy shock wave therapy.

AIMS: Low energy shock wave (LESW) has been shown to facilitate tissue regeneration and reduce inflammation. We investigated the effects of LESW in an underactive (DU) model induced by cryoinjury of rat detrusor. METHODS: Forty-six female Sprague-Dawley rats were divided into sham, cryoinjury with or without LESW (0.12 mJ/mm2 ; 200 pulses). Under halothane anesthesia, a low midline incision was made and a cryoinjury of detrusor was induced by placing an aluminum rod (chilled with dry ice) for 30 s on the serosal side of the bladder filled with 1 mL sterile saline bilaterally. Awake cystometrogram (CMG), molecular and histopathology studies were performed on Day 8 or 15 after cryoinjury. RESULTS: Significant urodynamic, histological, and molecular changes induced by cryoinjury of rat detrusor were detected on Day 8 and decrease in the contraction amplitude (54.3%), a significant increase in wet bladder weight (64.1%), edematous changes, muscle thinning and downregulation of α-SMA, IL-6, and upregulation of COX-2. LESW reversed the cryoinjury induced histological and COX-2 expression to cause a 49.0% increase in the contraction amplitude (P < 0.05). LESW induced cell proliferation was revealed by increased CD31 and Ki67 immunostaining. The effect of cryoinjury on urodynamic and histological changes was maintained till Day 15. CONCLUSION: The cryoinjury of rat detrusor models myogenic DU, which is partially reversed by LESW. LESW may afford a simple, non-invasive modality to facilitate tissue regeneration and improve voiding function in myogenic detrusor underactivity.

Relaxin-2 therapy reverses radiation-induced fibrosis and restores bladder function in mice.

AIM: To determine the efficacy of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD). Radiation cystitis is a consequence of radiotherapy for pelvic malignancies. Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation. Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions. In severe cases, cystectomies are performed as current therapies do not reverse fibrosis. METHODS: We developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) resulting in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence. Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with hRLX2 (400 µg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps. Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements, Western blots, histology, and immunohistochemistry. RESULTS: hRLX2 reversed fibrosis, decreased collagen content, improved bladder wall architecture, and increased bladder compliance, detrusor smooth muscle Cav1.2 expression and detrusor contractility in mice with chronic radiation cystitis. hRLX2 treatment outcomes were likely caused by the activation of RXFP1/2 receptors which are expressed on the detrusor. CONCLUSION: hRLX2 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.

Targeting p75 neurotrophin receptors ameliorates spinal cord injury-induced detrusor sphincter dyssynergia in mice.

AIMS: To determine the role of p75 neurotrophin receptor (p75NTR ) and the therapeutic effect of the selective small molecule p75NTR modulator, LM11A-31, in spinal cord injury (SCI) induced lower urinary tract dysfunction (LTUD) using a mouse model. METHODS: Adult female T8 -T9 transected mice were gavaged daily with LM11A-31 (100 mg/kg) for up to 6 weeks, starting 1 day before, or 7 days following injury. Mice were evaluated in vivo using urine spot analysis, cystometrograms (CMGs), and external urethral sphincter (EUS) electromyograms (EMGs); and in vitro using histology, immunohistochemistry, and Western blot. RESULTS: Our studies confirm highest expression of p75NTRs in the detrusor layer of the mouse bladder and lamina II region of the dorsal horn of the lumbar-sacral (L6 -S1 ) spinal cord which significantly decreased following SCI. LM11A-31 prevented or ameliorated the detrusor sphincter dyssynergia (DSD) and detrusor overactivity (DO) in SCI mice, significantly improving bladder compliance. Furthermore, LM11A-31 treatment blocked the SCI-related urothelial damage and bladder wall remodeling. CONCLUSION: Drugs targeting p75NTRs can moderate DSD and DO in SCI mice, may identify pathophysiological mechanisms, and have therapeutic potential in SCI patients.