Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury.

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.

Intravesical PAC1 Receptor Antagonist, PACAP(6-38), Reduces Urinary Bladder Frequency and Pelvic Sensitivity in NGF-OE Mice.

Chronic NGF overexpression (OE) in the urothelium, achieved through the use of a highly urothelium-specific uroplakin II promoter, stimulates neuronal sprouting in the urinary bladder, produces increased voiding frequency and non-voiding contractions, and referred somatic sensitivity. Additional NGF-mediated pleiotropic changes might contribute to increased voiding frequency and pelvic hypersensitivity in NGF-OE mice such as neuropeptide/receptor systems including PACAP(Adcyap1) and PAC1 receptor (Adcyap1r1). Given the presence of PAC1-immunoreactive fibers and the expression of PAC1 receptor expression in bladder tissues, and PACAP-facilitated detrusor contraction, whether PACAP/receptor signaling contributes to increased voiding frequency and somatic sensitivity was evaluated in NGF-OE mice. Intravesical administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased intercontraction interval (2.0-fold) and void volume (2.5-fold) in NGF-OE mice. Intravesical instillation of PACAP(6-38) also decreased baseline bladder pressure in NGF-OE mice. PACAP(6-38) had no effects on bladder function in WT mice. Intravesical administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in WT mice. PACAP/receptor signaling contributes to the increased voiding frequency and pelvic sensitivity observed in NGF-OE mice.

Expression and function of CCL2/CCR2 in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation.

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 ≤ 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 µM), 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 CYP-induced cystitis (4 h). These studies suggest that chemokines/receptors may be novel targets with therapeutic potential in the context of urinary bladder inflammation.

Increased expression of interleukin-6 family members and receptors in urinary bladder with cyclophosphamide-induced bladder inflammation in female rats.

Recent studies suggest that janus-activated kinases-signal transducer and activator of transcription signaling pathways contribute to increased voiding frequency and referred pain of cyclophosphamide (CYP)-induced cystitis in rats. Potential upstream chemical mediator(s) that may be activated by CYP-induced cystitis to stimulate JAK/STAT signaling are not known in detail. In these studies, members of the interleukin (IL)-6 family of cytokines including, leukemia inhibitory factor (LIF), IL-6, and ciliary neurotrophic factor (CNTF) and associated receptors, IL-6 receptor (R) α, LIFR, and gp130 were examined in the urinary bladder in control and CYP-treated rats. Cytokine and receptor transcript and protein expression and distribution were determined in urinary bladder after CYP-induced cystitis using quantitative, real-time polymerase chain reaction (Q-PCR), western blotting, and immunohistochemistry. Acute (4 h; 150 mg/kg; i.p.), intermediate (48 h; 150 mg/kg; i.p.), or chronic (75 mg/kg; i.p., once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Q-PCR analyses revealed significant (p ≤ 0.01) CYP duration- and tissue- (e.g., urothelium, detrusor) dependent increases in LIF, IL-6, IL-6Rα, LIFR, and gp130 mRNA expression. Western blotting demonstrated significant (p ≤ 0.01) increases in IL-6, LIF, and gp130 protein expression in whole urinary bladder with CYP treatment. CYP-induced cystitis significantly (p ≤ 0.01) increased LIF-immunoreactivity (IR) in urothelium, detrusor, and suburothelial plexus whereas increased gp130-IR was only observed in urothelium and detrusor. These studies suggest that IL-6 and LIF may be potential upstream chemical mediators that activate JAK/STAT signaling in urinary bladder pathways.

Neurotrophin/receptor expression in urinary bladder of mice with overexpression of NGF in urothelium.

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.

PACAP/VIP and receptor characterization in micturition pathways in mice with overexpression of NGF in urothelium.

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting or proliferation in the urinary bladder, produces urinary bladder hyperreflexia, and results in increased referred somatic hypersensitivity. Additional NGF-mediated changes might contribute to the urinary bladder hyperreflexia and pelvic hypersensitivity observed in these transgenic mice such as upregulation of neuropeptide/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 pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), and associated receptor (PAC1, VPAC1, VPAC2) transcripts or protein expression in urothelium and detrusor smooth muscle and lumbosacral dorsal root ganglia in NGF-overexpressing and littermate wildtype mice using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical approaches. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-immunoreactivity in urothelium of NGF-OE mice whereas PACAP transcript and PACAP-immunoreactivity were decreased in urothelium of NGF-OE mice. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice. VIP transcript expression and immunostaining was not altered in urinary bladder of NGF-OE mice. Changes in PACAP, VIP, and associated receptor transcripts and protein expression in micturition pathways resemble some, but not all, changes observed after induction of urinary bladder inflammation known to involve NGF production.

Involvement of JAK-STAT signaling/function after cyclophosphamide-induced bladder inflammation in female rats.

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 < or = 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.

Postnatal expression of corticotropin releasing factor (CRF) in rat urinary bladder.

Corticotropin releasing factor (CRF) is a neuropeptide expressed in micturition reflex circuitry and different roles in these reflexes have been suggested. These studies examined the expression of CRF/CRF receptors in the urinary bladder during postnatal development in the rat. Urinary bladder was harvested from rats (postnatal (P) day 0-adult) euthanized by isoflurane (4%) and thoracotomy. CRF protein expression significantly (p

Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis.

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 < or = 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 < or = 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 < or = 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.

Exaggerated expression of inflammatory mediators in vasoactive intestinal polypeptide knockout (VIP-/-) mice with cyclophosphamide (CYP)-induced cystitis.

Vasoactive intestinal polypeptide (VIP) is an immunomodulatory neuropeptide distributed in micturition pathways. VIP(-/-) mice exhibit altered bladder function and neurochemical properties in micturition pathways after cyclophosphamide (CYP)-induced cystitis. Given VIP's role as an anti-inflammatory mediator, we hypothesized that VIP(-/-) mice would exhibit enhanced inflammatory mediator expression after cystitis. A mouse inflammatory cytokine and receptor RT2 profiler array was used to determine regulated transcripts in the urinary bladder of wild type (WT) and VIP(-/-) mice with or without CYP-induced cystitis (150 mg/kg; i.p.; 48 h). Four binary comparisons were made: WT control versus CYP treatment (48 h), VIP(-/-) control versus CYP treatment (48 h), WT control versus VIP(-/-) control, and WT with CYP treatment (48 h) versus VIP(-/-) with CYP treatment (48 h). The genes presented represent (1) greater than 1.5-fold change in either direction and (2) the p value is less than 0.05 for the comparison being made. Several regulated genes were validated using enzyme-linked immunoassays including IL-1beta and CXCL1. CYP treatment significantly (p < or = 0.001) increased expression of CXCL1 and IL-1beta in the urinary bladder of WT and VIP(-/-) mice, but expression in VIP(-/-) mice with CYP treatment was significantly (p < or = 0.001) greater (4.2- to 13-fold increase) than that observed in WT urinary bladder (3.6- to 5-fold increase). The data suggest that in VIP(-/-) mice with bladder inflammation, inflammatory mediators are increased above that observed in WT with CYP. This shift in balance may contribute to increased bladder dysfunction in VIP(-/-) mice with bladder inflammation and altered neurochemical expression in micturition pathways.

Expression of fractalkine and fractalkine receptor in urinary bladder after cyclophosphamide (CYP)-induced cystitis.

Alterations in the expression of the chemokine, fractalkine (CX3CL1), were examined in the urinary bladder after cyclophosphamide (CYP)-induced cystitis of varying duration: acute (4 h or 48 h), or chronic (10 day). CYP-induced cystitis significantly (p

Expression of corticotropin-releasing factor and CRF receptors in micturition pathways after cyclophosphamide-induced cystitis.

Corticotropin-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 CRF(2), 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 < or = 0.01) increased with 48 h or chronic CYP treatment. CRF immunoreactivity (IR) was increased significantly (P < or = 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 CRF(2) receptor expression increased in whole bladder with CYP-treatment, whereas no CRF(1) 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 CYP-induced 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.

Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic ganglia and bladder.

PURPOSE: We examined neurotrophin and receptor tyrosine kinase (Trk) expression in the bladder and major pelvic ganglia (MPG) after cyclophosphamide induced cystitis in rats. MATERIALS AND METHODS: The bladder and MPG were used in immunohistochemical studies, enzyme-linked immunoassays and Western blots for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), TrkA and TrkB. Bladder postganglionic MPG cells were labeled by tracing techniques. RESULTS: NGF and BDNF expression was decreased in the bladder of all rats after cystitis (p < or =0.001). NGF and BDNF expression was increased in the MPG in male rats with cystitis (p < or =0.01). Cells expressing TrkA and TrkB immunoreactivity (IR) increased 78% to 81% in the MPG in male rats with cystitis. TrkA-IR or TrkB-IR bladder postganglionic cells increased 50% to 74% with cystitis. Cystitis increased TrkA-IR 5 to 10-fold and TrkB-IR 10 to 12-fold in detrusor muscle. TrkA-IR and TrkB-IR were prominent in control urothelium but decreased with cystitis. After cystitis TrkB-IR nerve fibers and TrkA-IR cellular infiltrates were more apparent compared to controls. CONCLUSIONS: 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.

Differential expression of bladder neurotrophic factor mRNA in male and female rats after bladder outflow obstruction.

PURPOSE: We validated a male rat model of bladder outflow obstruction and compared the expression of bladder neurotrophic factor mRNA in male and female rats 6 weeks after bladder outlet obstruction. MATERIALS AND METHODS: We examined the proximal urethra in male Wistar rats. Urethral lumen reducing ligatures were placed in 15 females and 19 males, while 10 male and 10 female controls underwent sham surgery. Awake cystometry was performed 6 weeks after surgery. Ribonuclease protection assay was used to measure changes in bladder neurotrophic factor mRNA expression in the 2 sexes. RESULTS: Average bladder capacity in rats with bladder outlet obstruction increased 3-fold in males and 4.4-fold in females compared with controls, while bladder weight increased 2.2 and 4.3-fold, respectively. Filling and threshold pressure increased significantly and nonvoiding bladder contractions were recorded in 100% of female and 80% of male rats with bladder outlet obstruction. An 8-fold increase in bladder brain derived neurotrophic factor mRNA was noted in each sex after obstruction. A 2-fold increase in bladder nerve growth factor mRNA after obstruction was only observed in females. CONCLUSIONS: This male rat model of bladder outlet obstruction was created by placing lumen reducing ligatures at the urethrovesical junction. The dramatic increase in bladder brain derived neurotrophic factor mRNA expression and differential expression of nerve growth factor mRNA in male and female rats with bladder outlet obstruction suggest that additional neurotrophic factors may contribute to the lower urinary tract neuroplasticity associated with bladder outlet obstruction and this contribution may be gender dependent.

Changes in urinary bladder cytokine mRNA and protein after cyclophosphamide-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-gamma (IFN)-gamma, interleukin-1alpha/beta (IL-1alpha/beta), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, and tumor necrosis factor-alpha/beta (TNF-alpha/beta)] 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