The purpose of this study was to determine the role of cyclooxygenase-2 (COX-2) and its metabolites in lower urinary tract function after induction of acute (4 h), intermediate (48 h), or chronic (10 day) cyclophosphamide (CYP)-induced cystitis. Bladders were harvested from euthanized female rats for analyses. Conscious cystometry was used to assess the effects of a COX-2-specific inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl2(5H)-furanone (DFU, 5 mg/kg sc), a disubstituted furanone, in CYP-induced cystitis. COX-2 mRNA was increased in inflamed bladders after acute (12-fold) and chronic (9-fold) treatment. COX-2 protein expression in inflamed bladders paralleled COX-2 mRNA expression. Prostaglandin D2-methoxime expression in the bladder was significantly (P < or = 0.01) increased in acute (3-fold) and chronic (5.5-fold) cystitis. Prostaglandin E2 was significantly (P < or = 0.01) increased (2-fold) in the bladder with intermediate (1.7-fold) and chronic (2.6-fold) cystitis. COX-2-immunoreactive cell profiles were distributed throughout the inflamed bladder and coexpressed histamine immunoreactivity. Conscious cystometry in rats treated with CYP + DFU showed increased micturition intervals 4 and 48 h after CYP treatment and decreased intravesical pressures during filling and micturition compared with rats treated with CYP + vehicle. These studies suggest an involvement of urinary bladder COX-2 and its metabolites in altered micturition reflexes with CYP-induced cystitis.

Cyclophosphamide (CYP)-induced cystitis alters micturition function and produces reorganization of the micturition reflex. This reorganization may involve cytokine expression in the urinary bladder. These studies have determined candidate cytokines in the bladder that may contribute to the reorganization process. An RNase protection assay was used to measure changes in rat bladder cytokine mRNA [interferon-γ (IFN)-γ, interleukin-1α/β (IL-1α/β), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, and tumor necrosis factor-α/β (TNF-α/β)] after acute (4 h), intermediate (48 h), or chronic (10 day) cystitis. The correlation between bladder cytokine mRNA and protein expression was also determined by immunoassay. Although at each time point after cystitis significant changes in bladder cytokine mRNA were observed, the magnitude differed (acute > intermediate > chronic). Acute cystitis demonstrated the most robust changes ( P ≤ 0.005; IL-1β, 330-fold increase; IL-2, 20-fold increase; IL-4, 8-fold increase; IL-6, 80-fold increase) in cytokine mRNA expression and TNF-α or TNF-β mRNA were only increased (2–10-fold) after acute cystitis. More modest increases in cytokine mRNA expression were observed after 48-h or 10-day cystitis. Cytokine protein expression generally paralleled that of mRNA. Increased cytokine expression after CYP-induced cystitis, alone or in combination with other inflammatory mediators or growth factors, may contribute to altered lower urinary tract function after cystitis.

Cystitis decreases bladder NGF and BDNF expression, whereas MPG expression is increased. This change may reflect neurotrophin release at the bladder and retrograde transport to the MPG. TrkA-IR and TrkB-IR are increased in bladder postganglionic cells and bladders with cystitis. This increase may reflect a shift in Trk staining from urothelium to detrusor muscle and nerve fibers with cystitis. Neurotrophin/Trk interactions in the bladder and MPG may contribute to bladder overactivity with cystitis.

Cheppudira BP, Girard BM, Malley SE, Schutz KC, May V, Vizzard MA. Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis.

Merrill L, Malley S, Vizzard MA. Repeated variate stress in male rats induces increased voiding frequency, somatic sensitivity, and urinary bladder nerve growth factor expression. Am J Physiol Regul Integr Comp Physiol 305: R147-R156, 2013. First published May 8, 2013 doi:10.1152/ajpregu.00089.2013.-Stress exacerbates symptoms of functional lower urinary tract disorders including interstitial cystitis (IC)/bladder pain syndrome (BPS) and overactive bladder (OAB) in humans, but mechanisms contributing to symptom worsening are unknown. These studies address stress-induced changes in the structure and function of the micturition reflex using an animal model of stress in male rats. Rats were exposed to 7 days of repeated variate stress (RVS). Target organ (urinary bladder, thymus, adrenal gland) tissues were collected and weighed following RVS. Evans blue (EB) concentration and histamine, myeloperoxidase (MPO), nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), and CXCL12 protein content (ELISA) were measured in the urinary bladder, and somatic sensitivity of the hindpaw and pelvic regions was determined following RVS. Bladder function was evaluated using continuous, open outlet intravesical infusion of saline in conscious rats. Increases in body weight gain were significantly (P Յ 0.01) attenuated by day 5 of RVS, and adrenal weight was significantly (P Յ 0.05) increased. Histamine, MPO, NGF, and CXCL12 protein expression was significantly (P Յ 0.01) increased in the urinary bladder after RVS. Somatic sensitivity of the hindpaw and pelvic regions was significantly (P Յ 0.01) increased at all monofilament forces tested (0.1-4 g) after RVS. Intercontraction interval, infused volume, and void volume were significantly (P Յ 0.01) decreased after RVS. These studies demonstrate increased voiding frequency, histamine, MPO, NGF, and CXCL12 bladder content and somatic sensitivity after RVS suggesting an inflammatory component to stress-induced changes in bladder function and somatic sensitivity. micturition; stress; nerve growth factor; ELISA; somatic sensitivity STRESS CONTRIBUTES to symptom exacerbation in many disease states, including functional disorders of the urinary bladder such as overactive bladder (OAB) and interstitial cystitis (IC)/ bladder pain syndrome (BPS) (3,38,58,72). Urinary frequency is a common symptom among patients with OAB or IC/BPS, although the end result of frequent voiding may differ [reduce incontinence episodes (OAB) vs. reduce pain with bladder filling (IC/BPS)]. Patients with IC/BPS report symptom worsening during stress, as do patients with other disorders associated with IC/BPS including rheumatoid arthritis, psoriasis, and irritable bowel syndrome (47,72). Symptom worsening during stress may be due, in part, to disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Cortisol, through feedback on the HPA axis, normally acts to attenuate inflammation (47); however, abnormalities in the feedback may cause dysregulation of the inflammatory response. Therefore, patients...

Arms L, Girard BM, Malley SE, Vizzard MA. Expression and function of CCL2/CCR2 in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation. Am J Physiol Regul Integr Comp Physiol 305: F111-F122, 2013. First published April 17, 2013 doi:10.1152/ajprenal.00139.2013.-Chemokines are proinflammatory mediators of the immune response, and there is growing evidence for chemokine/receptor signaling involvement in pronociception. Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain syndrome characterized by pain, pressure, or discomfort perceived to be bladder-related with at least one urinary symptom. We have explored the expression and functional roles of CCL2 (monocyte chemoattractant protein-1) and its high-affinity receptor, CCR2, in micturition reflex function and somatic sensitivity in rats with urinary bladder inflammation induced by cyclophosphamide (CYP) treatment of varying duration (4 h, 48 h, chronic). Real-time quantitative RT-PCR, ELISAs, and immunohistochemistry demonstrated significant (P Յ 0.01) increases in CCL2 and CCR2 expression in the urothelium and in Fast Blue-labeled bladder afferent neurons in lumbosacral dorsal root ganglia with CYP-induced cystitis. Intravesical infusion of RS504393 (5 M), a specific CCR2 antagonist, reduced voiding frequency and increased bladder capacity and void volume in rats with CYP-induced cystitis (4 h), as determined with open outlet, conscious cystometry. In addition, CCR2 blockade, at the level of the urinary bladder, reduced referred somatic sensitivity of the hindpaw and pelvic region in rats with CYP treatment, as determined with von Frey filament testing. We provide evidence of functional roles for CCL2/CCR2 signaling at the level of the urinary bladder in reducing voiding frequency and somatic sensitivity following CYPinduced cystitis (4 h). These studies suggest that chemokines/receptors may be novel targets with therapeutic potential in the context of urinary bladder inflammation. chemokine/receptor signaling; conscious cystometry; von Frey filaments; urothelium BLADDER PAIN SYNDROME (BPS)/Interstitial Cystitis (IC) is a chronic pain syndrome characterized by pain, pressure, or discomfort perceived to be bladder-related with at least one urinary symptom (28,29). Although the etiology and pathogenesis of BPS/IC are unknown, numerous theories, including infection, inflammation, autoimmune disorder, toxic urinary agents, urothelial dysfunction and neurogenic causes have been proposed (22,34,50,54,55). We have hypothesized that pain associated with BPS/IC involves an alteration of visceral sensation/bladder sensory physiology. BPS/IC patients have a lower threshold for sensing bladder volume and often experience pain at normal bladder volumes, suggesting altered sensory processing within the urinary tract and/or its innervation (24, 48). The majority of biopsies from BPS/IC patients reveal some degree of inflammation; therefore; inflammatory mediators, including, but not limited to, chemokines, may contribute to inf...

ticotropin-releasing factor (CRF) is a prominent neuropeptide involved in micturition reflexes, and different roles in these reflexes have been suggested. These studies examined the expression of CRF in the urinary bladder and lumbosacral sacral parasympathetic nucleus (SPN) in response to cyclophosphamide (CYP)-induced cystitis (4 h, 48 h, or chronic) in rats. The expression of CRF receptors, CRF 1 and CRF2, was examined in urinary bladder from control and CYP-treated rats. Urinary bladder and lumbosacral spinal cord were harvested from rats killed by isoflurane (4%) and thoracotomy. CRF protein expression in whole urinary bladders significantly (P Յ 0.01) increased with 48 h or chronic CYP treatment. CRF immunoreactivity (IR) was increased significantly (P Յ 0.01) in the urothelium and SPN after CYP treatment. CRF IR nerve fibers increased in density in the suburothelial plexus and detrusor smooth muscle whole mounts with CYP-induced cystitis. CRF 2 receptor transcript was expressed in the urothelium or detrusor smooth muscle, and CRF2 receptor expression increased in whole bladder with CYP-treatment, whereas no CRF1 receptor transcript was expressed in either urothelium or detrusor. Immunohistochemical studies demonstrated CRF 2 IR in urinary bladder nerve fibers and urothelial cells from control animals, whereas no CRF 1 IR was observed. These studies demonstrated changes in the expression of CRF in urinary bladder and SPN region with CYPinduced cystitis and CRF receptor (CRF 2) expression in nerve fibers and urothelium in control rats. CRF may contribute to urinary bladder overactivity and altered sensory processing with CYP-induced cystitis. inflammation; sacral parasympathetic nucleus; urothelium; enzymelinked immunosorbet assay THE CHEMICALLY (cyclophosphamide, CYP) induced bladder inflammation model is associated with alterations in neurochemical (57, 61), electrophysiological (66), organizational (62), and functional properties (14) of micturition pathways. These changes may be mediated by chemical mediators (e.g., neurotrophins, cytokines, neuropeptides) produced in the bladder, spinal cord, or dorsal root ganglia with cystitis (5, 27, 57, 59, 61).Corticotropin-releasing factor (CRF) is of particular relevance in the rat, since it is present in descending projections from the pontine micturition center or more specifically from Barrington's nucleus to the sacral parasympathetic nucleus (SPN; see Refs. 30,31,49,56). Historically, Barrington's nucleus has been viewed as the supraspinal switching center that regulates storage and elimination of urine (25,33,41). Recent studies have led to the suggestion that Barrington's nucleus may contain neurons that control a broad range of pelvic organ functions (28,29,34,38,55). CRF is prominently expressed in the descending pathway from Barrington's nucleus to the SPN in the lumbosacral spinal cord, and prominent CRF immunoreactivity (IR) is expressed in the SPN of adult rats (37,53,54). Our recent studies have demonstrated an age-dependent upregulation of CRF IR ...

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting in the urinary bladder, produces increased voiding frequency, and results in increased referred somatic hypersensitivity. Additional NGF-mediated pleiotropic changes might contribute to the increased voiding frequency and pelvic hypersensitivity observed in these transgenic mice, such as modulation of other growth factor/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific uroplakin II promoter. In the present study, we examined NGF, brain-derived neurotrophic factor (BDNF), and associated receptor [p75(NTR), tyrosine kinase (Trk)A, TrkB] transcript and protein expression in urothelium and detrusor smooth muscle of NGF-overexpressing (OE) and littermate wild-type mice, using real-time quantitative reverse transcription-polymerase chain reaction, ELISAs, and semiquantitation of immunohistochemistry. We focused on these growth factor/receptors given the established roles of NGF/TrkA, NGF/p75(NTR), and BDNF/TrkB systems in bladder function. Increased voiding frequency in NGF-OE mice was confirmed by examining urination patterns. BDNF, TrkA, and TrkB protein expression was significantly (P ≤ 0.01) reduced and p75(NTR) protein expression was significantly (P ≤ 0.01) increased in urinary bladder of NGF-OE mice. The NGF-OE-induced changes in neurotrophic factor/receptor expression in urinary bladder may represent compensatory changes to reduce voiding frequency in the NGF-OE mouse.