L6-S1 Dorsal Root Ganglia Research Articles

Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased,and bladder MAGL was decreased after MAGL inhibitor treatment. MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury

AimsNerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. Main methodsWe used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 μg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. Key findingsIn capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SignificanceP38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

MP60-08 COMPARISON OF LOWER URINARY TRACT DYSFUNCTION AFTER SPINAL CORD INJURY (SCI) BETWEEN FEMALE AND MALE MICE

You have accessJournal of UrologyCME1 Apr 2023MP60-08 COMPARISON OF LOWER URINARY TRACT DYSFUNCTION AFTER SPINAL CORD INJURY (SCI) BETWEEN FEMALE AND MALE MICE Mamoru Hashimoto, Kang Jun Cho, Stephanie L Daugherty, PA Pittsburgh, Nobutaka Shimizu, Pradeep Tyagi, Akihide Hirayama, Hirotsugu Uemura, Jonathan M Beckel, Sergei Karnup, and Naoki Yoshimura Mamoru HashimotoMamoru Hashimoto More articles by this author , Kang Jun ChoKang Jun Cho More articles by this author , Stephanie L DaughertyStephanie L Daugherty More articles by this author , PA PittsburghPA Pittsburgh More articles by this author , Nobutaka ShimizuNobutaka Shimizu More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Akihide HirayamaAkihide Hirayama More articles by this author , Hirotsugu UemuraHirotsugu Uemura More articles by this author , Jonathan M BeckelJonathan M Beckel More articles by this author , Sergei KarnupSergei Karnup More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In humans, voiding occurs during synergic relaxation of the external urethral sphincter (EUS), and SCI induces detrusor-sphincter dyssynergia (DSD) shown by tonic EUS contraction during voiding. However, EUS bursting activity is known to be an important mechanism for efficient voiding in spinal intact (SI) and SCI rats while such data are less available in mice. Thus, we evaluated gender-specific EUS function during voiding in male and female mice with or without SCI. METHODS: C57BL/6N mice at 9-10-weeks old were used. SCI mice underwent complete transection of the Th8-9 spinal cord. Six weeks after SCI, cystometrograms (CMGs) and EUS- electromyography (EMG) were performed under an awake condition. Thereafter, L6-S1 dorsal root ganglia (DRG) were harvested to evaluate the transcripts of TRPV1 and Piezo2 using qPCR. RESULTS: In female SI mice, voiding occurred during an EUS relaxation period. Female SCI mice voided urine during notch like reductions in bladder pressure, which corresponded to EMG signal reduction periods during tonic EMG activity (DSD). On the other hand, male SI and SCI mice voided during EUS bursting activity characterized by alternating active and silent phases of EMG activity, with the longer active phase and low voiding efficiency in SCI vs. SI mice (Table and Figure). The mRNA levels of TRPV1 and Piezo2 in L6-S1 DRG were significantly higher in SCI male mice (n=4) and female mice (n=4) vs. SI mice (n=4). CONCLUSIONS: Lower urinary tract function during the voiding phase was different between male and female mice. Female SI mice, in which voiding occurs during EUS relaxation, and female SCI mice, which exhibit DSD with periodic EUS relaxations, seem to be more suitable for the study of SCI-induced EUS dysfunction in humans, compared to SI and SCI male mice that exhibit EUS bursting activity during voiding. Source of Funding: NIH R01DK129194 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e846 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mamoru Hashimoto More articles by this author Kang Jun Cho More articles by this author Stephanie L Daugherty More articles by this author PA Pittsburgh More articles by this author Nobutaka Shimizu More articles by this author Pradeep Tyagi More articles by this author Akihide Hirayama More articles by this author Hirotsugu Uemura More articles by this author Jonathan M Beckel More articles by this author Sergei Karnup More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

Involvement of acid sensing ion channel (ASIC)-3 in an acute urinary bladder-colon cross sensitization model in rodent.

Irritable bowel syndrome and bladder pain syndrome are both characterized by pain in response to organ distension. Epidemiologic studies showed that these two syndromes are often overlapped. Such overlap may be due to sharing of common extrinsic innervations between the colorectum and the urinary bladder, where cross-sensitization of the urinary bladder and the colon would occur in response to mechanical distension of either organ. The aim of this project was to develop and characterize a rodent model of urinary bladder-colon sensitization and to assess the role of the acid sensing ion channel (ASIC)-3. Double retrograde labelling was performed to identify extrinsic primary afferent neurons innervating both the colon (Fluororuby) and urinary bladder (Fluorogold) in the L6-S1 dorsal root ganglia (DRG) in Sprague Dawley rats. The phenotype of the colon/urinary bladder co-innervating primary afferent neurons was assessed using immunohistochemistry directed against ASIC-3. Cross-organ sensitization was induced in Sprague Dawley rats by using an echography-guided intravesical administration of acetic acid (0.75%) under brief isoflurane anesthesia. Colonic sensitivity was assessed in conscious rats by measuring abdominal contraction during isobaric colorectal distension (CRD). Measurement of urinary bladder and colonic paracellular permeabilities and tissue myeloperoxidase assay were performed. The involvement of ASIC-3 was assessed by use of S1 intrathecal administration of the ASIC-3 blocker, APETx2 (2.2 µM). Immunohistochemistry showed that 73.1% of extrinsic primary afferent neurons co-innervating the colon and the urinary bladder express ASIC-3. By contrast, extrinsic primary afferent neurons innervating the colon only or the urinary bladder only were positive for ASIC-3 in 39.3% and 42.6%, respectively. Echography-guided intravesical administration of acetic acid resulted in colonic hypersensitivity to colorectal distension. This effect started 1 h post-injection and lasted up to 24 h, and was not longer seen after 3 days after injection. No colonic hyperpermeability and no difference in urinary bladder and colon MPO activity was observed between control and acetic acid-treated rats. Colonic sensitization by intravesical acetic acid administration was prevented by S1 intrathecal administration of APETx2. We developed an acute pelvic cross-organ sensitization model in conscious rat. In this model, cross-organ sensitization is likely to involve S1-L6 extrinsic primary afferents co-innervating the colon and urinary bladder through an ASIC-3 pathway.

Effect of Bladder Injection of OnabotulinumtoxinA on the Central Expression of Genes Associated with the Control of the Lower Urinary Tract: A Study in Normal Rats.

To investigate a possible central mechanism of action of Botulinum toxin A (BoNT/A) following injection in the bladder, complementary to the acknowledged peripheral bladder effect, we studied changes in the expression of neuropeptides and receptors involved in lower urinary tract function in the spinal cord (SC) and dorsal root ganglia (DRG) of normal rats following BoNT/A bladder injection. Thirty-six Sprague-Dawley rats, divided into three groups of n = 12, received bladder injections of 2U or 5U OnabotulinumtoxinA (BOTOX®), or saline. Six animals from each group were sacrificed on days 7 and 14. Expression of Tachykinin 1 (Tac1), capsaicin receptor (TRPV1), neuropeptide Y (NPY), proenkephalin (PENK) and muscarinic receptors M1, M2, M3, was evaluated in the bladder, L6-S1 DRG, and SC segments using real-time PCR and Western blotting. Real-time PCR revealed increased expression of NPY in all tissues except for SC, and increased TRPV1 and PENK expression in DRG and SC, whereas expression of Tac1, M1 and M2 was decreased. Less significant changes were noted in protein levels. These findings suggest that bladder injections of OnabotulinumtoxinA may be followed by changes in the expression of sensory, sympathetic and cholinergic bladder function regulators at the DRG/SC level.

Therapeutic effects of a soluble guanylate cyclase activator, BAY 60-2770, on lower urinary tract dysfunction in mice with spinal cord injury.

We aimed to evaluate the effects of a soluble guanylate cyclase (sGC) activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury (SCI). Mice were divided into the following three groups: spinal cord intact (group A), SCI + vehicle (group B), and SCI + BAY 60-2770 (group C). SCI mice underwent Th8-Th9 spinal cord transection and treatment with BAY 60-2770 (10 mg/kg/day) once daily for 2-4 wk after SCI. We evaluated urodynamic parameters using awake cystometry and external urethral sphincter electromyograms (EMG); mRNA levels of mechanosensory channels, nitric oxide (NO)-, ischemia-, and inflammation-related markers in L6-S1 dorsal root ganglia, the urethra, and bladder tissues; and protein levels of cGMP in the urethra at 4 wk after SCI. With awake cystometry, nonvoiding contractions, postvoid residual, and bladder capacity were significantly larger in group B than in group C. Voiding efficiency (VE) was significantly higher in group C than in group B. In external urethral sphincter EMGs, the duration of notch-like reductions in intravesical pressure and reduced EMG activity time were significantly longer in group C than in group B. mRNA expression levels of transient receptor potential ankyrin 1, transient receptor potential vanilloid 1, acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and Piezo2 in the dorsal root ganglia, and hypoxia-inducible factor-1α, VEGF, and transforming growth factor-β1 in the bladder were significantly higher in group B than in groups A and C. mRNA levels of neuronal NO synthase, endothelial NO synthase, and sGCα1 and protein levels of cGMP in the urethra were significantly lower in group B than in groups A and C. sGC modulation might be useful for the treatment of SCI-related neurogenic lower urinary tract dysfunction.NEW & NOTEWORTHY This is the first report to evaluate the effects of a soluble guanylate cyclase activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury.

Continuous administration of mirabegron has advantages in inhibition of central sensitization compared with short-term treatment cessation in a mouse model of overactive bladder.

There is no clear pathophysiologic evidence determining how long overactive bladder (OAB) medication should be continued. We, therefore, investigated the effect of mirabegron using cessation (CES) or continuation (CON) treatment in an OAB animal model. Female C57BL/6 mice were divided into four groups (N = 8 each): Sham, OAB, CES, and CON groups. The OAB-like condition was induced by three times weekly intravesical instillations of KCl mixture with hyaluronidase. After the last intravesical instillation for inducing OAB, mirabegron (2 mg/kg/day) was administered in CES and CON groups for 10 and 20 days, respectively. Final experiments were carried out on 20 days from the last intravesical instillation in all groups. After cystometry, mRNA levels of bladder muscarinic, β-adrenergic, and P2X purinergic receptors were measured to investigate bladder efferent and afferent activity. In addition, mRNA levels of CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG) were measured to assess afferent sensitization. Immunofluorescent staining of CX3CR1, GFAP, and CCR2 in the L6 spinal cord was also conducted to investigate glial activation and central sensitization. OAB mice showed bladder overactivity evidenced by decreased intercontraction interval (3.56 ± 0.51vs. 5.76 ± 0.95 min in sham mice), increased non-voiding contractions (0.39 ± 0.11vs. 0.13 ± 0.07/min in sham mice), and inefficient voiding (72.1 ± 8.6% vs. 87.1 ± 9.5% in sham mice). Increased M2, M3, β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG indicate bladder efferent and afferent hyperexcitability. In addition, CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in OAB mice. However, theCON group exhibited reduced β2, β3, P2X2 , P2X3 , P2X4 , and P2X7 levels in the bladder, reduced CCL2 and CCR2 in DRG, which are markers of afferent hyperexcitability, and reduced immunoreactivities of CX3CR1, GFAP, and CCR2 in the L6 spinal cord, which are markers of the central sensitization. Moreover, theCON group showed better improvements in nonvoiding contractions (0.16 ± 0.09vs. 0.44 ± 0.17/min) and voiding efficiency (93.9 ± 7.4% vs. 76.5 ± 13.1%) and reductions in bladder β3 receptors and CCL2 of L6-S1 DRG, and immunoreactivities of CX3CR1 and GFAP in the L6 spinal cord compared to theCES group. Continuous mirabegron treatment seems to prevent central sensitization and, thus, might be desirable for long-term disease control of OAB.

MP49-15 OVERACTIVE BLADDER MEDICATION; HOW LONG SHOULD IT BE CONTINUED? - MIRABEGRON INTERFERES WITH CENTRAL SENSITIZATION IN A MOUSE MODEL OF OVERACTIVE BLADDER

You have accessJournal of UrologyCME1 May 2022MP49-15 OVERACTIVE BLADDER MEDICATION; HOW LONG SHOULD IT BE CONTINUED? - MIRABEGRON INTERFERES WITH CENTRAL SENSITIZATION IN A MOUSE MODEL OF OVERACTIVE BLADDER Joonbeom Kwon, Eun-Ju Lee, Hye-Ri Park, Hyun-Jung Cho, Ji-Ae Jang, Donghwi Park, and Naoki Yoshimura Joonbeom KwonJoonbeom Kwon More articles by this author , Eun-Ju LeeEun-Ju Lee More articles by this author , Hye-Ri ParkHye-Ri Park More articles by this author , Hyun-Jung ChoHyun-Jung Cho More articles by this author , Ji-Ae JangJi-Ae Jang More articles by this author , Donghwi ParkDonghwi Park More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The etiology of overactive bladder (OAB) is multifactorial, and bladder afferent hyperexcitability has been proposed to be one of the contributing factors. In addition, chronic afferent hyperexcitability may induce neural remodeling in the central nervous system (CNS). However, there has been no clear pathophysiologic evidence determining how long OAB treatments should be continued. We, therefore, investigated the CNS effect of mirabegron using an OAB animal model and compared the therapeutic effect between treatment cessation (CES) and continuation (CON) groups. METHODS: Female C57BL/6 mice were divided into 4 groups (N=8 in each): Sham, OAB, CES, and CON groups. OAB was induced by 3-times weekly intravesical instillations of KCl and hyaluronidase. Mirabegron (2mg/kg) was administered for 10 and 20 days in CES and CON groups, respectively. Cystometry was performed, and mRNA levels of bladder muscarinic, β-adrenergic, and P2X purinergic receptors were measured. CCL2 and CCR2 in L6-S1 dorsal root ganglia (DRG), as afferent markers, and CX3CR1, GFAP, and CCR2 in the L6 spinal cord, as markers of glial activation and central sensitization, were also evaluated by RT-PCR and immunohistology. RESULTS: OAB mice showed bladder overactivity and inefficient voiding (Fig 1-a) with increased M2, M3, P2X2, P2X3, P2X4, and P2X7 levels in the bladder and increased CCL2 and CCR2 in DRG, indicating afferent hyperexcitability (Fig 1-b). CX3CR1, GFAP, and CCR2 in the L6 spinal cord were upregulated in the OAB group (Fig 1-c). However, CON mice exhibited improvements of all these parameters of afferent hyperexcitability and central sensitization. Also, CON mice showed better voiding efficiency and reduced CNS changes compared with CES mice. CONCLUSIONS: Central sensitization may be an important pathophysiological mechanism of OAB. Continuous treatment of OAB with mirabegron seems to prevent the process of central sensitization via improvement of CNS neural remodeling. Therefore, continuous OAB medication may be desirable for long-term disease control. Source of Funding: Astellas Pharma. Co.Ltd. (ISR005728) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e859 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joonbeom Kwon More articles by this author Eun-Ju Lee More articles by this author Hye-Ri Park More articles by this author Hyun-Jung Cho More articles by this author Ji-Ae Jang More articles by this author Donghwi Park More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF DownloadLoading ...

PD08-07 ANTIFIBROSIS TREATMENT IMPROVES BLADDER DYSFUNCTION AND PAIN PERCEPTION IN A MOUSE MODEL OF BLADDER PAIN WITH CENTRAL SENSITIZATION MIMICKING INTERSTITIAL CYSTITIS

You have accessJournal of UrologyCME1 May 2022PD08-07 ANTIFIBROSIS TREATMENT IMPROVES BLADDER DYSFUNCTION AND PAIN PERCEPTION IN A MOUSE MODEL OF BLADDER PAIN WITH CENTRAL SENSITIZATION MIMICKING INTERSTITIAL CYSTITIS Joonbeom Kwon, Hye-Ri Park, Eun-Ju Lee, Ji-Ae Jang, Hyun-Jung Cho, and Naoki Yoshimura Joonbeom KwonJoonbeom Kwon More articles by this author , Hye-Ri ParkHye-Ri Park More articles by this author , Eun-Ju LeeEun-Ju Lee More articles by this author , Ji-Ae JangJi-Ae Jang More articles by this author , Hyun-Jung ChoHyun-Jung Cho More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002527.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies reported the possibility that the interruption of fibrosis may improve interstitial cystitis (IC). In addition, our previous study with nintedanib, which has an antifibrotic effect and inhibits the receptors of VEGF, FGF, and PDGF, demonstrated the improvement of bladder overactivity and C-fiber hyperexcitability in spinal cord injured mice. Therefore, we investigated whether antifibrotic therapy using nintedanib can improve the IC-like pathologic conditions. METHODS: Female C57BL/6 mice were divided into 3 groups (each N=8): (A) Sham, (B) IC mice treated with vehicle, and (C) IC mice treated with nintedanib. For inducing an IC-like model, intravesical instillation of hyaluronidase mixed with lipopolysaccharide was conducted 3 times once a week. At the last instillation, vehicle or nintedanib (50mg/kg) was administered daily for 3 weeks. Then, pain assessment using Von-Frey filaments and cystometry was conducted. Trichrome staining, RT-PCR in the bladder, and immunohistochemistry of L6-S1 dorsal root ganglia (DRG) and L6 spinal cord were performed to investigate the improvement of bladder fibrosis, inflammation, nociception, and central sensitization. RESULTS: In pain assessment, 50% thresholds of target force were significantly reduced in group B vs. A, but recovered in group C vs. B. In cystometry, non-voiding contractions and voiding efficiency were worsened in group B, but restored in group C (Fig 1-a). Bladder fibrosis increased in group B were improved in group C in trichrome staining (Fig.1-b). mRNA levels in the bladder related to inflammation (IFN-r, CCR2), nociception (TACR2), P2X purinergic (P2X3, P2X4, P2X7), muscarinic (M2), and β-adrenergic receptors (β2, β3) were increased in group B, but decreased in group C (Fig 1-c). In immunohistochemistry, TRPV1 in L6-S1 DRG and CX3CR1, GFAP, and CCR2 in L6 spinal cord were upregulated in group B, but reduced in group C (Fig 1-d). CONCLUSIONS: Antifibrosis treatment using nintedanib improved bladder pain and bladder overactivity in the IC model. Therapeutic effects seem to be mediated by inhibitions of bladder afferent hyperexcitability as well as central sensitization. Source of Funding: National Research Foundation of Korea (2020R1C1C1012208) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e107 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joonbeom Kwon More articles by this author Hye-Ri Park More articles by this author Eun-Ju Lee More articles by this author Ji-Ae Jang More articles by this author Hyun-Jung Cho More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF DownloadLoading ...

Nerve growth factor-mediated Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury

AimTo investigate the effect of nerve growth factor (NGF) neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury (SCI). Main methodsFemale C57/BL6 mice were randomly divided into spinal intact (SI) group, SCI group and SCI + NGF-Ab group. SCI was induced by spinal cord transection at the Th8/9 level. In SCI + NGF-Ab group, anti-NGF antibodies (10 μg·kg-1 per hour) were continuously administered for 2 weeks using osmotic pumps. Bladder afferent neurons were labelled with Fluoro‑gold (FG) injected into the bladder wall. L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole-cell patch clamp recordings were performed on FG-labelled neurons. Expression of Nav1.7 and Nav1.8 was examined by immunofluorescent staining. Key findingsWhole-cell patch clamp recordings showed that TTX only partially inhibited action potentials (AP) and Na+ currents of bladder afferent neurons in SI mice, but it almost completely inhibited them in SCI mice. Total and TTX-sensitive Na+ currents were increased and TTX-resistant currents were decreased in bladder afferent neurons from SCI mice vs. SI mice. These changes in SCI mice were significantly reversed by NGF-antibody treatment. Immunostaining results showed the increased and decreased levels of Nav1.7 and Nav1.8, respectively, in FG-labelled bladder afferent neurons in SCI mice vs. SI mice, which was significantly reversed in SCI + NGF-Ab mice. SignificanceNGF mediates the Na+ channel plasticity with a shift from TTX-resistant Nav1.8 to TTX-sensitive Nav1.7 in bladder afferent neurons, which could be a possible underlying mechanism of bladder afferent hyperexcitability and detrusor overactivity after SCI.

Upregulation of transient receptor potential cation channel subfamily M member-3 in bladder afferents is involved in chronic pain in cyclophosphamide-induced cystitis.

The transient receptor potential cation channel subfamily M member-3 (TRPM3) channel is a recently recognized noxious heat sensor that is involved in inflammatory thermal hyperalgesia. To examine its involvement in the development of hyperalgesia in interstitial cystitis/painful bladder syndrome (IC/PBS), rats with cyclophosphamide (CYP)-induced chronic cystitis were used as a model of IC/PBS. Mechanical and thermal hyperalgesia in lower abdominal region overlying the bladder in CYP rats were measured using von Frey filaments and radiant heat, respectively. Transient receptor potential cation channel subfamily M member-3 expression at the mRNA, protein, and functional levels in dorsal root ganglion neurons innervating the bladder was detected using RNA in situ hybridization (RNAscope), Western blotting, immunohistochemistry, and Ca 2+ imaging, respectively. Transient receptor potential cation channel subfamily M member-3 channels were expressed on most of the bladder primary afferent nerve terminals containing calcitonin gene-related peptide and their cell bodies in L6-S1 dorsal root ganglion. Activation of TRPM3 in the bladder wall by its specific agonist pregnenolone sulphate or CIM0216 induced spontaneous bladder pain, calcitonin gene-related peptide release, and neurogenic inflammation that was evidenced by edema, plasma extravasation, inflammatory cell accumulation, and mast cell infiltration. In CYP rats, pretreatment with the TRPM3 antagonist primidone (2 mg/kg, i.p.) significantly alleviated the mechanical and thermal hyperalgesia, bladder submucosal edema, mast cell infiltration, and bladder hyperactivity. Cyclophosphamide-induced cystitis was associated with TRPM3 upregulation at the mRNA, protein, and functional levels in bladder afferent neurons. Our results suggest that upregulation of TRPM3 channels is involved in the development of chronic pain in CYP-induced cystitis, and targeting TRPM3 may be a pharmacological strategy for treating bladder pain in IC/PBS.

Inhibition of CXCR4 in Spinal Cord and DRG with AMD3100 Attenuates Colon-Bladder Cross-Organ Sensitization.

BackgroundCross-sensitization of pelvic organs is one theory for why symptoms of gut sickness and interstitial cystitis/bladder pain syndrome overlap. Experimental colitis has been shown to trigger bladder hyperactivity and hyperalgesia in rats. The chemokine receptor CXCR4 plays a key role in bladder function and central sensitization. We aim to study the role of CXCR4 and its inhibitor AMD3100 in colon-bladder cross-organ sensitization.MethodsThe colitis model was established by rectal infusion of trinitrobenzene sulfonic acid. Western blot and immunofluorescence were used to assess the expression and distribution of CXCR4. Intrathecal injection of AMD3100 (a CXCR4 inhibitor) and PD98059 (an ERK inhibitor) were used to inhibit CXCR4 and downstream extracellular signal-regulated kinase (ERK) in the spinal cord and dorsal root ganglion (DRG). Intravesical perfusion of resiniferatoxin was performed to measure the pain behavior counts of rats, and continuous cystometry was performed to evaluate bladder voiding function.ResultsCompared to the control group, CXCR4 was expressed more in bladder mucosa and colon mucosa, L6-S1 dorsal root ganglion (DRG), and the corresponding segment of the spinal dorsal horn (SDH) in rats with colitis. Moreover, intrathecal injection of the AMD3100 suppressed bladder overactivity, bladder hyperalgesia, and mastocytosis symptoms caused by colitis. Furthermore, AMD3100 effectively inhibited ERK activation in the spinal cord induced by experimental colitis. Finally, treatment with PD98059 alleviated bladder overactivity and hyperalgesia caused by colitis.ConclusionIncreased CXCR4 in the DRG and SDH contributes to colon inflammation-induced bladder overactivity and hyperalgesia partly via the phosphorylation of spinal ERK. Treatment targeting the CXCR4/ERK pathway might provide a potential new approach for the comorbidity between the digestive system and the urinary system.

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

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). 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). 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. 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.

Efficacy of vibegron, a novel β3-adrenoreceptor agonist, for lower urinary tract dysfunction in mice with spinal cord injury.

To investigate the effect of vibegron, a new clinically approved β3-adrenoceptor agonist in lower urinary tract dysfunction in mice with spinal cord injury. Investigators performed cystometry under awake conditions in 4-week spinal cord injury female mice. Two weeks after spinal cord injury, saline or vibegron (30mg/kg) was orally administered for 2weeks prior to the urodynamic study. Investigators removed L6-S1 dorsal root ganglia from the saline- or vibegron-treated spinal cord injury mice as well as from saline-treated normal (spinal intact) mice to evaluate the levels of transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, activating transcription factor 3, and inducible nitric oxide synthase transcripts using real-time polymerase chain reaction. In vibegron-treated spinal cord injury mice, nonvoiding contractions during bladder filling, which were increased in spinal cord injury compared to spinal intact mice, were significantly decreased. Micturition pressure or voiding efficiency was not significantly increased in comparison to measurements in saline-treated spinal cord injury mice. The expression of transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, activating transcription factor 3, and inducible nitric oxide synthase messenger RNA was increased in spinal cord injury mice compared to spinal intact mice, but significantly decreased after vibegron treatment. Vibegron improves spinal cord injury-induced detrusor overactivity in addition to significantly reducing C-fiber afferent receptors such as transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily A member 1, and inflammatory cytokines/markers, such as activating transcription factor 3 and inducible nitric oxide synthase, in spinal cord injury mice. Thus, vibegron might be effective in the treatment of storage lower urinary tract dysfunction induced by C-fiber afferent activation after spinal cord injury.

Antifibrosis treatment by inhibition of VEGF, FGF, and PDGF receptors improves bladder wall remodeling and detrusor overactivity in association with modulation of C-fiber afferent activity in mice with spinal cord injury.

Spinal cord injury (SCI) above the sacral level causes bladder dysfunction and remodeling with fibrosis. This study examined the antifibrotic effects using nintedanib, an inhibitor of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptors, on detrusor overactivity (DO) and bladder fibrosis, as well as the modulation mechanisms of C-fiber afferent pathways. Thirty female C57BL/6 mice were divided into group A (spinal intact), group B (SCI with vehicle), and group C (SCI with nintedanib). At 2 weeks after SCI, vehicle or 50 mg/kg nintedanib was administered subcutaneously for 2 weeks. Then, cystometry was conducted, followed by RT-PCR measurements of fibrosis-related molecules, muscarinic, β-adrenergic, TRP and purinergic receptors in the bladder or L6-S1 dorsal root ganglia (DRG). Trichrome stain and Western blot analysis of transforming growth factor-beta and fibronectin were performed in the bladder. TRPV1 expression in L6 DRG was measured by immunohistochemistry. In cystometry, intercontraction intervals, nonvoiding contractions, voided volume, and voiding efficiency were significantly improved in group C versus group B. RT-PCR, Western blotting, and trichrome staining revealed the fibrotic changes in the bladder of group B, which was improved in group C. Increased messenger RNA levels of TRPV1, TRPA1, P2X2 , and P2X3 in DRG of group B were significantly decreased in group C. TRPV1 immunoreactivity in DRG was increased in group B, but decreased in group C. Nintedanib improves storage and voiding dysfunctions and bladder fibrosis in SCI mice. Also, nintedanib-induced improvement of DO is associated with reduced expression of C-fiber afferent markers, suggesting the modulation of bladder C-fiber afferent activity.

Acute stress induces visceral hypersensitivity via glucocorticoid receptor-mediated membrane insertion of TRPM8: Involvement of a non-receptor tyrosine kinase Pyk2.

Psychological stress is an important factor for the development and recurrence of irritable bowel syndrome (IBS). The mechanisms underlying stress-induced visceral hypersensitivity (VH), a key pathophysiological component in IBS, are still incompletely understood. We aimed to test whether transient receptor potential melastatin 8 (TRPM8) participates in acute stress-induced VH. Rats were subjected to 1-hour water avoidance stress (WAS). Visceral sensitivity was measured with visceromotor response to colorectal distension. Western blot and immunofluorescence were applied to evaluate the expression of GR and TRPM8 and activation of PKA, Akt, and PKC pathways. WAS-caused VH depended on glucocorticoid receptors (GRs) and TRPM8 channels. In a dorsal root ganglion (DRG)-derived cell line, corticosterone rapidly (within 30minutes) induced membrane expression of TRPM8. This effect was inhibited by GR antagonism and was mimicked by membrane-impermeable corticosterone. PKA, PI3K/Akt, and PKC pathways, which lied downstream of GR and acted in parallel to promote membrane expression of TRPM8, contributed to WAS-induced VH. The non-receptor tyrosine kinase Pyk2, which may serve as a convergence point for PKA, PI3K/Akt, and PKC pathways, facilitated membrane insertion of TRPM8 via tyrosine-phosphorylating TRPM8 in L6-S2 DRGs and participated in WAS-induced VH. Collectively, acute stress-induced VH could involve membrane-bound GR-dependent enhancement of TRPM8 function in nociceptive DRG neurons. Mechanistically, Pyk2 could act as a key mediator that coordinates multiple protein kinase signaling and triggers phosphorylation and membrane insertion of TRPM8.

PD24-09 POST-INJURY TIME COURSE OF LOWER URINARY TRACT DYSFUNCTION AFTER SPINAL CORD INJURY (SCI) IN THE MOUSE MODEL

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II (PD24)1 Apr 2020PD24-09 POST-INJURY TIME COURSE OF LOWER URINARY TRACT DYSFUNCTION AFTER SPINAL CORD INJURY (SCI) IN THE MOUSE MODEL Tetsuichi Saito*, Masahiro Kurobe, Daisuke Gotoh, Taro Igarashi, Osamu Ishizuka, and Naoki Yoshimura Tetsuichi Saito*Tetsuichi Saito* More articles by this author , Masahiro KurobeMasahiro Kurobe More articles by this author , Daisuke GotohDaisuke Gotoh More articles by this author , Taro IgarashiTaro Igarashi More articles by this author , Osamu IshizukaOsamu Ishizuka More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000881.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lower urinary tract dysfunction (LUTD) due to SCI causes detrusor overactivity (DO) and detrusor sphincter dyssynergia (DSD) leading to inefficient voiding with increased residual urine. Animal models of SCI have been used to study these conditions; however, the adequate timing of evaluation after SCI in mouse models has not been explored. Thus, this study evaluated the time-course changes of bladder and external urinary sphincter (EUS) activity as well as mechanosensitive channels in L6-S1 dorsal root ganglia (DRG). METHODS: Female C57BL/6N mice were used and SCI was induced by Th8/9 spinal cord transection. The mice were divided into 3 groups; A: 2 weeks post SCI, B: 4weeks post SCI and C: 6 weeks post SCI, and evaluated by single-filling cystometery and EUS-electromyography (EMG) under an awake condition. After evaluation, L6-S1 DRG were harvested to measure mRNA expression of TRP channels and mechanosensitive ion channels such as ASICs and Piezo. RESULTS: In SCI mice, non-voiding contractions (NVC) during bladder filling were confirmed at 2 weeks post-SCI and did not increase over time to 6 weeks (Fig. 1A). In EUS-EMG measurements, DSD was observed at 2 weeks, but periodic EMG reductions during bladder contraction, resulting in urination, were not observed in most 2-weeks SCI mice, thereby leading to urinary retention (Fig. 1A). At 4 weeks, SCI mice showed increases of EMG activity reduction time with increased voiding efficiency (VE). At 6 weeks, SCI mice exhibited further increases in bladder capacity, residual volume and EMG reduction time compared to groups A and B without significant changes in VE compared to 4 weeks (Fig. 1A). RT-PCR of L6-S1 DRG showed increased mRNA levels of TRPV1 and ASIC1-3 in SCI mice with a decrease of ASIC2-3 at 6 weeks compared to 4 weeks while Piezo2 showed a slow increase at 6 weeks (Fig. 1B). CONCLUSIONS: These results indicate that DO evident as NVC is established in the early phase (2weeks) whereas DSD is completed later at 4weeks with a slight improvement evident as increased EMG reduction time at 6 weeks post SCI, and that ASIC and Piezo2 mechanosensitive channels may be involved in the establishment of DSD in early (2-4 weeks) and late phases (4-6 weeks) of SCI, respectively. These data would help us to understand the progression mechanisms of SCI-induced LUTD. Source of Funding: DOD W81XWH-17-1-0403 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e535-e536 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tetsuichi Saito* More articles by this author Masahiro Kurobe More articles by this author Daisuke Gotoh More articles by this author Taro Igarashi More articles by this author Osamu Ishizuka More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

MP06-17 THE EFFECT OF INTRAVESICAL INSTILLATION OF LIPOSOME-CONJUGATED NGF ANTISENSE ON BLADDER HYPERSENSITIVITY AND NGF EXPRESSION IN A RAT MODEL OF PROSTATIC INFLAMMATION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-17 THE EFFECT OF INTRAVESICAL INSTILLATION OF LIPOSOME-CONJUGATED NGF ANTISENSE ON BLADDER HYPERSENSITIVITY AND NGF EXPRESSION IN A RAT MODEL OF PROSTATIC INFLAMMATION Taro Igarashi*, Pradeep Tyagi, Shinsuke Mizoguchi, Masahiro Kurobe, Daisuke Gotoh, Tetsuichi Saito, Zhou Wang, Akira Furuta, Yasuyuki Suzuki, Shin Egawa, and Naoki Yoshimura Taro Igarashi*Taro Igarashi* More articles by this author , Pradeep TyagiPradeep Tyagi More articles by this author , Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Masahiro KurobeMasahiro Kurobe More articles by this author , Daisuke GotohDaisuke Gotoh More articles by this author , Tetsuichi SaitoTetsuichi Saito More articles by this author , Zhou WangZhou Wang More articles by this author , Akira FurutaAkira Furuta More articles by this author , Yasuyuki SuzukiYasuyuki Suzuki More articles by this author , Shin EgawaShin Egawa More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Local upregulation of nerve growth factor (NGF) in the bladder has been implicated in lower urinary tract (LUT) dysfunction due to afferent sensitization. Also, male LUT symptoms are known to be associated with prostatic inflammation (PI) in human BPH tissues. Thus, we examined the effect of liposomes conjugated with NGF antisense oligonucleotide (OND) on bladder hypersensitivity and NGF expression in PI rats. METHODS: Male SD rats were divided into 4 groups: (1) Sham, (2) Sham-NS; with intravesical instillation of normal saline (NS, 0.5ml), (3) PI-NS; with PI and instillation of NS, (4) PI-OND; with PI and instillation of liposome-conjugated NGF antisense. PI was induced by intraprostatic formalin injection (5%, 50 μl per each ventral lobe). On day 14, phosphorothioated NGF antisense OND (0.5ml, 12μM) complexed with liposomes (PI-OND) or NS (Sham-NS and PI-NS) was instilled directly into the bladder with a 30G needle after draining urine, and kept for 60 minutes. On day 28, we evaluated awake cystometry (CMG) and harvested tissues to measure mRNA expressions of NGF in the bladder and C-fiber afferent markers (TRPV1 & TRPA1) in L6-S1 dorsal root ganglia (DRG). RESULTS: In CMG, PI-OND group had significantly longer intercontraction intervals (ICIs) vs. PI-NS group while ICIs were significantly reduced in PI-NS group vs. Sham group (Fig. 1A, 1B). mRNA expressions of NGF in bladder mucosa and TRPV1 & TRPA1 in DRG in PI-OND group was significantly lower vs. PI-NS group while there were no significant differences between PI-OND and Sham (Fig. 1C). CONCLUSIONS: These results demonstrate that bladder NGF is an important mediator to induce bladder overactivity following PI and that liposome-based, local NGF-targeting therapy could be effective for bladder hypersensitivity induced by prostate-to-bladder cross-sensitization after prostatic inflammation. Source of Funding: NIH U54DK112079 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e57-e58 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taro Igarashi* More articles by this author Pradeep Tyagi More articles by this author Shinsuke Mizoguchi More articles by this author Masahiro Kurobe More articles by this author Daisuke Gotoh More articles by this author Tetsuichi Saito More articles by this author Zhou Wang More articles by this author Akira Furuta More articles by this author Yasuyuki Suzuki More articles by this author Shin Egawa More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut-brain axis.

The major sensory nerve pathway between the colon and central nervous system (spinal cord and brain) that underlies the gut-brain axis, is via spinal afferent neurons, with cell bodies in dorsal root ganglia (DRG). Our aim was to identify the sensory nerve endings in the colon that arise from single colorectal-projecting DRG neurons. C57BL/6 mice were anesthetized and lumbosacral L6-S1 DRG injected with dextran biotin. Mice recovered for 7 days. The whole colon was then removed and stained to visualize single axons and nerve endings immunoreactive to calcitonin gene-related peptide (CGRP). Single axons arising from DRG were identified in the distal colon and their morphological features and CGRP immunoreactivity characterized. After entering the colon, single axons ramified rostrally or caudally along many rows of myenteric ganglia with little circumferential displacement, giving off varicose endings in multiple ganglia. Nerve endings arising from two classes of colorectal-projecting DRG neuron were identified. One class was peptidergic neurons that had nerve endings in circular muscle, myenteric ganglia, and submucosa. Another class of nonpeptidergic neurons innervated mucosal crypts, myenteric ganglia, and submucosa. Different morphological types of nerve endings which innervate different anatomical layers of colon can arise from the same axon and sensory neuron in DRG. These findings suggest single peptidergic and nonpeptidergic sensory neurons in DRG are potentially capable of detecting sensory stimuli from different anatomical layers of the colon, via different types of nerve endings.