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The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. Transient receptor potential vanilloid 1 (TRPV1) channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (P ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild-type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell-deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression, whereas control (no stress) mast cell-deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia (DRG) in WT mice exposed to RVS, but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction. NEW & NOTEWORTHY: Using pharmacological tools and transgenic mice in a repeated variate stress (RVS) model in female mice, we demonstrate that transient receptor potential vanilloid 1 (TRPV1) and mast cells contribute to the increased voiding frequency observed following RVS. TRPV1 and mast cells should continue to be considered as targets to improve bladder function in stress-induced bladder dysfunction. [ABSTRACT FROM AUTHOR]

Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one., Introduction The function of the micturition reflex is to store urine and then to completely empty urine (i.e., void) at socially appropriate times. Supralumbar spinal cord injury (SCI) results in [...]

The expression of acid-sensing ion channel (ASIC) isoforms, ASIC1, ASIC2a, and ASIC3, was examined in the urinary bladder after cyclophosphamide (CYP)-induced cystitis of varying duration (4 h, 48 h, and chronic). Immunohistochemical, Western blot, and quantitative PCR approaches were used to evaluate channel expression and effects of CYP-induced cystitis in whole urinary bladder and split-bladder preparations from control (no inflammation) and CYP-treated rats. Quantitative PCR demonstrated significant (P [less than or equal to] 0.01) increases in ASIC2a and ASIC3 transcripts with CYP-induced cystitis (48 h and chronic) in the urothelium but no changes (e.g., ASIC3) or modest changes (e.g., ASIC2a) in detrusor smooth muscle. ASIC1 mRNA expression in the urothelium or detrusor was not affected by CYP-induced cystitis. Immunohistochemistry for ASIC2a and ASIC3 protein expression revealed significant (P [less than or equal to] 0.01) increases in ASIC immunoreactivity in the urothelium and suburothelial plexus with CYP-induced cystitis at all time points examined. Western blotting for ASIC2a and ASIC3 protein expression was complementary and revealed significant (P [less than or equal to] 0.01) increases in ASIC immunoreactivity. For the first time, these studies demonstrate that CYP-induced cystitis alters ASIC2a and ASIC3 expression in the urinary bladder; ASIC1 transcript expression is not altered by CYP-induced cystitis. Future studies are necessary to determine ASIC isoform contributions to micturition reflexes in control and inflamed urinary bladder. urothelium; suburothelial nerve plexus; acid-sensing ion channel isoform 1; acid-sensing ion channel isoform 2a; acid-sensing ion channel isoform 3; quantitative polymerase chain reaction doi: 10.1152/ajprenal.00618.2009.

Chemokines, otherwise known as chemotactic cytokines, are proinflammatory mediators of the immune response and have been implicated in altered sensory processing, hyperalgesia, and central sensitization following tissue injury or inflammation. To address the role of CXCL12/CXCR4 signaling in normal micturition and inflammation-induced bladder hyperreflexia, bladder inflammation in adult female Wistar rats (175-250 g) was induced by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every 3rd day for 10 days) time points. CXCL12, and its receptor, CXCR4, were examined in the whole urinary bladder of control and CYP-treated rats using enzyme-linked immunosorbent assays (ELISAs), quantitative PCR (qRT-PCR), and immunostaining techniques. ELISAs, qRT-PCR, and immunostaining experiments revealed a significant (P [less than or equal to] 0.01) increase in CXCL12 and CXCR4 expression in the whole urinary bladder, and particularly in the urothelium, with CYP treatment. The functional role of CXCL12/CXCR4 signaling in micturition was evaluated using conscious cystometry with continuous instillation of saline and CXCR4 receptor antagonist (AMD-3100; 5 [micro]M) administration in control and CYP (48 h)-treated rats. Receptor blockade of CXCR4 using AMD-3100 increased bladder capacity in control (no CYP) rats and reduced CYP-induced bladder hyperexcitability as demonstrated by significant (P [less than or equal to] 0.01) increases in intercontraction interval, bladder capacity, and void volume. These results suggest a role for CXCL12/CXCR4 signaling in both normal micturition and with bladder hyperreflexia following bladder inflammation. urothelium; chemokines; cystometry; AMD-3100; bladder hyperreflexia doi:10.1152/ajprenal.00628.2009

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction. nerve growth factor; urothelium; transgenic mouse; bladder hypersensitivity; inflammation; afferent innervation doi:10.1152/ajpregu.00367.2009

Cytokines are upregulated in a variety of inflammatory conditions and cytokine/ receptor interactions can activate JAK-STAT signaling. Previous studies demonstrated upregulation of numerous cytokines in the urinary bladder following cyclophosphamide (CYP)-induced cystitis. The role of JAK-STAT signaling in urinary bladder inflammation and referred somatic sensitivity has not been addressed. The contribution of JAK-STAT signaling pathways in CYP-induced bladder hyperreflexia and referred somatic hypersensitivity was determined in CYP-treated rats using a JAK2 inhibitor, AG490. Acute (4 h; 150 mg/kg ip), intermediate (48 h; 150 mg/kg ip), or chronic (75 mg/kg ip, once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Phosphorylation status of STAT-3 was increased in urinary bladder after CYP-induced cystitis (4 h, 48 h, chronic). Blockade of JAK2 with AG490 (5-15 mg/kg ip or intravesical) significantly (P [less than or equal to] 0.05) reduced bladder hyperreflexia and hind paw sensitivity in CYP-treated rats. These studies demonstrate a potential role for JAK-STAT signaling pathways in bladder hyperreflexia and referred pain induced by CYP-induced bladder inflammation. somatic sensitivity; bladder hyperreflexia; cystometry; AG490 doi: 10.1152/ajprenal.00110.2009.

Previous studies demonstrated changes in urinary bladder neurotrophin content and upregulation of neurotrophin receptors, TrkA and the p75 neurotrophin receptor ([p75.sup.NTR]), in micturition reflex pathways after cyclophosphamide (CYP)-induced cystitis, [p75.sup.NTR] can bind nerve growth factor (NGF) and modulate NGF-TrkA binding and signaling. We examined [p75.sup.NTR] expression and the role of [p75.sup.NTR] in the micturition reflex in control and CYP-treated rats. [p75.sup.NTR] hnmunoreactivity was present throughout the urinary bladder. CYP-induced cystitis (4 h, 48 h, chronic) increased (P [less than or equal to] 0.05) [p75.sup.NTR] expression in whole urinary bladder as shown by Western blotting. The role of [p75.sup.NTR] in bladder function in control and CYP-treated rats was determined using conscious cystometry and immunoneutralization or PD90780, a compound known to specifically block NGF binding to [p75.sup.NTR]. An anti-[p75.sup.NTR] monoclonal antibody or PD90780 was infused intravesically and cystometric parameters were evaluated. Both methods of [p75.sup.NTR] blockade significantly (P [less than or equal to] 0.05) decreased the intercontraction interval and void volume in control and CYP-treated rats. Intravesical infusion of PD90780 also significantly (P [less than or equal to] 0.001) increased intravesical pressure and increased the number of nonvoiding contractions during the filling phase. Control intravesical infusions of isotype-matched IgG and vehicle were without effect. Intravesical instillation of PD90780 significantly (P [less than or equal to] 0.01) reduced the volume threshold to elicit a micturition contraction in control rats (no inflammation) and CYP-treated in a closed urinary bladder system. These studies demonstrate 1) ubiquitous [p75.sup.NTR] expression in urinary bladder and increased expression with CYP-induced cystitis and 2) [p75.sup.NTR] blockade at the level of the urinary bladder produces bladder hyperreflexia in control and CYP-treated rats. The overall activity of the urinary bladder reflects the balance of NGF-[p75.sup.NTR] and NGF-TrkA signaling. PD90780; conscious cystometry; Western blotting; bladder hyperreflexia; immunoneutralization

Regulation of the VEGF-VEGF receptor system was examined in the urinary bladder after acute (2-48 h) and chronic (10 days) cyclophosphamide (CYP)-induced cystitis. ELISAs demonstrated significant (P [less than or equal to] 0.01) upregulation of VEGF in whole urinary bladder with acute and chronic CYP-induced cystitis; however, the magnitude of increase was greater after acute (2-4 h) cystitis. Immunohistochemistry for VEGF immunoreactivity revealed a significant (P [less than or equal to] 0.05) increase in VEGF immunoreactivity in the urothelium, suburothelial vasculature, and detrusor smooth muscle with acute (4 and 48 h) CYP treatment. RT-PCR identified the isoform VEGF-164, the VEGF receptor VEGFR-2, and the VEGF co-receptors neuropilin (Npn)-1 and Npn-2 in the urinary bladder. Quantitative PCR demonstrated upregulation of VEGF-164 transcript with acute and chronic CYP-induced cystitis, but VEGFR-2, Npn-1, and Npn-2 transcripts were upregulated (P [less than or equal to] 0.01) in whole bladder only with chronic CYP-induced cystitis. Additional studies demonstrated regulation of VEGF transcript expression in the urinary bladder by nerve growth factor (NGF) in a novel line of NGF-overexpressing mice. These studies demonstrated that urinary bladder inflammation and NGF regulate the VEGF-VEGF receptor system in the urinary bladder. Functional role(s) for the VEGF-VEGF receptor system in urinary bladder inflammation remain to be determined. urothelium; quantitative polymerase chain reaction; immunohistochemistry; detrusor smooth muscle; vasculature; nerve growth factor

These studies examined the expression of cyclooxygenase-2 (COX-2) expression in the urothelium and suburothelial space and detrusor from rats treated with cyclophosphamide (CYP) to induce acute (4 h), intermediate (48 h), or chronic (10-day) cystitis. Western blot analysis and immunohistochemistry were used to demonstrate COX-2 expression. In whole mount preparations of urinary bladder, nerve fibers in the suburothelial plexus, and inflammatory cell infiltrates were characterized for COX-2 expression after CYP-induced cystitis. COX-2 expression significantly (P [less than or equal to] 0.01) increased in the urothelium + suburothelium and detrusor smooth muscle with acute, intermediate, and chronic (10-day) CYP-induced cystitis, but expression in urothelium + suburothelium was significantly greater. CYP-induced upregulation of COX-2 showed by immunostaining in the urothelium + suburothelium was similar to that observed with Western blot analysis and also demonstrated COX-2 inflammatory cell infiltrates (CD86+) and nerve fibers (PGP+) in the suburothelial plexus. Although COX-2 expression was significantly (P [less than or equal to] 0.01) increased in detrusor smooth muscle, immunohistochemistry failed to demonstrate an obvious change in COX-2-immunoreactivity (IR) in detrusor muscle, but COX-2 inflammatory infiltrates were present throughout the detrusor. COX-2-IR nerve fibers exhibited increased density in the suburothelial plexus with acute or chronic CYP-induced cystitis. COX-2-IR macrophages (CD86+) were present throughout the urinary bladder with acute and chronic CYP-induced cystitis. These studies demonstrate cellular targets in the urinary bladder where COX-2 inhibitors may act. urothelium; inflammatory infiltrates; detrusor; nerves

Phosphorylated ERK expression has been demonstrated in the central and peripheral nervous system after various stimuli, including visceral stimulation. Changes in the activation (i.e., phosphorylation) of extracellular signal-regulated kinases (pERK) were examined in the urinary bladder after 4 h (acute), 48 h (intermediate), or chronic (10 day) cyclophosphamide (CYP) treatment. CYP-induced cystitis significantly (P [less than or equal to] 0.01) increased pERK expression in the urinary bladder with intermediate (48 h) and chronic CYP treatment. Immunohistochemistry for pERK immunore-activity revealed little pERK-IR in control or acute (4 h) CYP-treated rat urinary bladders. However, pERK expression was significantly (P [less than or equal to] 0.01) upregulated in the urothelium after 48 h or chronic CYP treatment. Whole mount preparations of urothelium/lamina propria or detrusor smooth muscle from control (noninflamed) rats showed no pERK-IR in PGP9.5-labeled nerve fibers in the suburothelial plexus. However, with CYP-treatment (48 h, chronic), a few pERK-IR nerve fibers in the suburothelial plexus of whole mount preparations of bladder and at the serosal edge of urinary bladder sections were observed, pERK-IR cells expressing the CD86 antigen were also observed in urinary bladder from CYP-treated rats (48 h, chronic). Treatment with the upstream inhibitor of ERK phosphorylation, U0126, significantly (P [less than or equal to] 0.01) increased bladder capacity in CYP-treated rats (48 h). These studies suggest that therapies targeted at pERK pathways may improve urinary bladder function in CYP-treated rats. neurotrophins; mitogen-activated protein kinase; plasticity; inflammation; urothelium; U0126 doi:10.1152/ajpregu.00857.2006