- Email: [email protected]
− Mice
Bladder Dysfunction and Altered Somatic Sensitivity in PACAPⴚ/ⴚ Mice Victor May and Margaret A. Vizzard* From the Departments of Anatomy and Neurobiology (VM) and Neurology, University of Vermont College of Medicine (MAV), Burlington, Vermont
Abbreviations and Acronyms AA ⫽ acetic acid BC ⫽ bladder capacity CYP ⫽ cyclophosphamide DCD ⫽ detrusor contraction duration DRG ⫽ dorsal root ganglia ICI ⫽ intercontraction interval LUT ⫽ lower urinary tract PAC ⫽ PACAP PACAP ⫽ pituitary adenylate cyclase activating polypeptide RV ⫽ post-void residual volume VIP ⫽ vasoactive intestinal polypeptide VV ⫽ voided volume Submitted for publication April 30, 2009. Study received institutional animal care and use committee approval. Supported by National Institutes of Health Grants DK051369, DK060481 and DK065989, and National Institutes of Health Grant P20 RR16435 from the COBRE Program of the National Center. * Correspondence: Department of Neurology, University of Vermont College of Medicine, D415A Given Research Building, Burlington, Vermont 05405 (telephone: 802-656-3209; FAX: 802656-8704; e-mail: [email protected]).
772
www.jurology.com
Purpose: PACAP and receptors are expressed in micturition pathways. Studies show that PACAP has a role in detrusor smooth muscle contraction to facilitate adenosine triphosphate release from urothelium and PACAP antagonism decreases cyclophosphamide induced bladder hyperreflexia. Materials and Methods: PACAP contributions to micturition and somatic sensation were studied in PACAP knockout (PACAP⫺/⫺), litter mate heterozygote (PACAP⫹/⫺) and WT mice by conscious cystometry with continuous intravesical saline or acetic acid (0.5%) instillation, urination patterns, somatic sensitivity testing of hind paw and pelvic regions with calibrated von Frey filaments, and morphological bladder assessments. Results: PACAP⫺/⫺ mice had an increased bladder mass with fewer but larger urine spots. In PACAP⫺/⫺ mice the lamina propria and detrusor smooth muscle were significantly thicker but the urothelium was unchanged. PACAP⫺/⫺ mice had increased bladder capacity, voided volume and intercontraction interval with significantly increased detrusor contraction duration and large residual volume. WT mice responded to acetic acid (0.5%) with a decrease in voided volume and intercontraction interval but PACAP⫹/⫺ and PACAP⫺/⫺ mice did not respond. PACAP⫺/⫺ mice were less responsive to somatic stimulation. PACAP⫹/⫺ mice also had bladder dysfunction, and somatic and visceral sensory abnormalities but to a lesser degree. Conclusions: PACAP gene disruption contributes to changes in bladder morphology and function, and somatic and visceral hypoalgesia. Key Words: urinary bladder, pituitary adenylate cyclase-activating polypeptide, reflex, sensation, mice THE neuropeptide PACAP is involved in LUT function.1–3 PACAP is a member of the VIP, secretin, glucagon family of hormones that shows about 68% homology to VIP.4 PACAP can act on 3 types of G-protein coupled receptors, including PAC1, VIP PAC1 and VIP PAC2 receptors.4 Dense PACAP immunoreactive sensory fibers are present in the bladder, including suburothelial plexus.2,5 PACAP/receptor expression in DRG and spinal cord afferent pro-
jections are up-regulated in CYP induced bladder inflammation. 1 We have noted specific PACAP receptor expression in bladder smooth muscle, urothelium, lumbosacral DRG and spinal cord, suggesting that PACAP and PAC1 receptor signaling participate at multiple levels in the LUT.2 Functionally PACAP peptides increase contractility in rat and mouse detrusor smooth muscle. 2,3 Bladder hyperreflexia induced by CYP is decreased by the PAC1 an-
0022-5347/10/1832-0772/0 THE JOURNAL OF UROLOGY® Copyright © 2010 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 183, 772-779, February 2010 Printed in U.S.A. DOI:10.1016/j.juro.2009.09.077
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
773
tagonist PACAP6-38.2 PACAP application evoked adenosine triphosphate release from rat cultured urothelium that was blocked by the PAC1 receptor selective antagonist M65.6 In addition to roles for PACAP/receptor signaling in LUT function, differential effects of PACAP were reported in peripheral nociception.7 We determined the effects of PACAP gene disruption in mice (PACAP⫺/⫺, PACAP⫹/⫺ and WT litter mates) on bladder function, somatic sensitivity, bladder morphology and somatic (pelvic and hind paw) sensitivity. These studies demonstrated that PACAP gene disruption contributes to bladder morphology and function changes, and somatic and visceral hypoalgesia.
the hair bent slightly. We used established criteria to determine positive responses to hind paw or pelvic region stimulation.10,12 Pelvic and hind paw testing was done in the same mice but somatic regions were evaluated in randomly assigned mice at different times. All somatic testing was done in blinded fashion and groups were decoded after data analysis.
METHODS
Bladder Function on Cystometry
Animals The PACAP⫺/⫺ mouse model was generated in a pathogen-free facility at UCLA and back crossed for at least 8 generations with C57Bl/6.8,9 PACAP⫺/⫺, litter mate PACAP⫹/⫺ and WT mice8,9 were bred at our institution and lack of the PACAP gene was confirmed by polymerase chain reaction genotyping.9 The University of Vermont institutional animal care and use committee approved animal use. Animal care was done under the supervision of the University of Vermont office of animal care management according to Association for Assessment and Accreditation of Laboratory Animal Care International and National Institutes of Health guidelines.
Urination Patterns PACAP⫺/⫺, PACAP⫹/⫺ and WT mice of each gender were placed individually in standard cages for 1 hour with bedding replaced with Whatman grade 3 filter paper. Food and water were provided freely. Urine spots were photographed under ultraviolet light,10 spot area in cm2 was determined, and large (0.2 to 10 cm2)10 and small (less than 0.2 cm2)11 spots were counted. Mice were placed in cages and data were analyzed by an individual blinded to mouse strain. Groups were decoded after data analysis.
Mechanical Sensitivity Testing Due to divergent roles for PACAP in somatic sensation7 and PACAP expression in sensory neurons1,14 tactile allodynia was tested using calibrated von Frey hairs with a force of 0.04, 0.16, 0.4, 1 and 4 gm to the abdomen and hind paw10,12 among genotypes. Mice were tested in individual Plexiglas® chambers with a stainless steel wire grid floor. Mice were acclimated to the chambers for 2 hours. Due to limited supply the same mice were evaluated for somatic sensitivity 1 to 2 weeks before intravesical catheter implantation, and 1 to 2 weeks before catheter implantation and somatic testing to determine urination patterns. von Frey hairs were applied in an up-down method for 1 to 3 seconds with an interstimulus interval of 15 seconds. Pelvic region stimulation was confined to the lower abdominal area overlying the bladder. The plantar region of the hind paw and the lower abdominal area were tested by perpendicular application of von Frey hairs until
Bladder Catheter Implantation A lower midline abdominal incision was made using general anesthesia with isoflurane (2.5% to 3.5%). Polyethylene-10 tubing with the end flared by heat was inserted into the bladder dome and secured in place with a 6-zero nylon purse-string suture.10 The distal end of the tubing was sealed, tunneled subcutaneously and externalized. Animals were maintained for 72 hour to ensure complete recovery. Postoperative analgesics were given for 48 hours. Animals were placed while conscious and unrestrained in recording cages with a balance and pan below for urine collection and measurement.2,10 Intravesical pressure changes were recorded using a Small Animal Cystometry System (Med Associates, St. Albans, Vermont). Saline at room temperature was infused (25 l per minute) to elicit repetitive bladder contractions. At least 6 reproducible micturition cycles were recorded after an initial stabilization period (25 to 30 minutes). BC was measured as the amount of saline infused in the bladder at the time when micturition commenced.11,13 Voided saline was collected to determine VV. After collection the infusion was stopped and RV was determined by withdrawing urine from the bladder catheter with a syringe. ICI, maximal voiding pressure, pressure threshold for voiding and baseline resting pressure were measured.14 DCD was determined by the width of the micturition contraction at its base. Nonvoiding bladder contractions, defined as greater than 5 cm H2O from baseline pressure without a release of fluid from the urethra, were quantified during the filling phase. AA (0.5%) was infused into the bladder in some litter mates after saline infusion to evaluate nociceptive bladder responses. Mice were removed from study when adverse events occurred, including a 20% or greater decrease in body weight postoperatively, a significant postoperative event, lethargy, pain or distress not relieved by our institutional animal care and use committee approved regimen of postoperative analgesics or hematuria in control rodents.10 No mice were excluded from analysis according to established criteria but 3 were removed because they chewed the exteriorized tubing. Behavioral movements, eg grooming or defecation, rendered bladder pressure recording unusable during these events. Experiments were done at similar times of the day.10 Mice were sacrificed with isoflurane (4%) and thoracotomy at the conclusion of study. Bladders were dissected and weighed.
Bladder Histological Analysis Histological analysis of bladder sections (15 m) from intravesical saline studies only was done using hematoxylin and eosin staining. Digital images of hematoxylin and eosin stained sections were captured using a MagnaFire™ camera and an Olympus® microscope using a 20⫻ objec-
774
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
tive. Bladder layer thickness was measured using MetaMorph®, version 4.5r4. Measurements were made of urothelium, lamina propria and muscular layer thickness. Section measurements were done in triplicate and averaged. We matched the bladder region being compared, ie bladder dome or trigone, among litter mates. Parameters were measured as the distance along a line drawn normal to the long axis of the specimen from the luminal edge to the serosal urothelium, lamina propria and detrusor edges.13
Statistical Analysis All values are shown as the mean ⫾ SEM. Data were compared with ANOVA using SAS® statistical software. When F ratios exceeded the critical value of p ⱕ0.05, the Newman-Keul post hoc test was used to compare means.
RESULTS WT, PACAPⴙ/ⴚ and PACAPⴚ/ⴚ Mice There were no differences in body mass among WT, PACAP⫹/⫺ and PACAP⫺/⫺ litter mates but the bladder-to-body weight ratio was significantly increased in PACAP⫺/⫺ mice due to the increased bladder mass (fig. 1). Fluid intake measured in 24 hours was similar among litter mates (see table). As quantified on filter paper in a 1-hour period, urine spots of PACAP⫺/⫺ mice were significantly fewer (p ⱕ0.01)
Urine spots, fluid intake and bladder pressure in 10 WT, PACAP⫺/⫺ and PACAP⫹/⫺ litter mates each
Fluid intake (ml/24 hrs) Urine spots (No./hr) Pressure (cm H2O): Max voiding Baseline Threshold
Mean ⫾ SEM WT
Mean ⫾ SEM PACAP⫺/⫺
Mean ⫾ SEM PACAP⫹/⫺
7.2 ⫾ 0.8 12.4 ⫾ 2.5
7.4 ⫾ 1.0 3.2 ⫾ 1.2
7.0 ⫾ 0.5 6.2 ⫾ 1.5
67.2 ⫾ 1.5 12.4 ⫾ 2.5 14.5 ⫾ 2.5
68.5 ⫾ 1.0 14.2 ⫾ 2.2 21.5 ⫾ 4.5
66.5 ⫾ 1.5 13.2 ⫾ 1.5 16.8 ⫾ 2.0
but greater in area than those of WT mice (8.2 ⫾ 3.5 vs 3.3 ⫾ 2.5 cm2, see table). PACAP⫹/⫺ mice also produced significantly fewer urine spots than WT mice (p ⱕ0.01). Large (0.2 to 10 cm2) and small (less than 0.2 cm2) diameter urine spots were observed for all genotypes but there were differences in the number of only large spots, likely representing actual voiding events, in PACAP⫺/⫺ and PACAP⫹/⫺ mice. No differences were noted between PACAP⫺/⫺ and PACAP⫹/⫺ mice (see table). Bladder Histology Histological analysis of the bladder at the dome and trigone regions revealed significantly increased lam-
Figure 1. Bladder histological analysis in PACAP⫺/⫺, PACAP⫹/⫺ and WT mice. Representative 15 m cryostat bladder sections of WT (A) and PACAP⫺/⫺ (B) mice reveal significantly increased lamina propria (LP) and detrusor smooth muscle (SM) thickness. U, urothelium. H & E, scale bar indicates 120 m. Histological results were supported by morphometric analysis (C). Asterisk indicates p ⱕ0.01. Body weight did not differ among mice (D) but bladder-to-body weight ratio was significantly increased in PACAP⫺/⫺ mice due to increased bladder mass (E). g, gm. Values represent mean ⫾ SEM in 7 to 10 mice per group (C to E).
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
775
Figure 2. Representative cystometrogram traces show infused volume (IF), bladder pressure (BP) and VV in conscious unrestrained PACAP⫺/⫺ mice with 25 l per minute continuous intravesical infusion of room temperature saline (A to C) and 0.5% AA (D to F). PACAP⫺/⫺ mice had large BC before micturition (A), long detrusor contraction duration (B) and large VV (C) with incomplete emptying compared to WT mice. PACAP⫺/⫺ mice did not respond to AA compared to WT mice (fig. 3).
ina propria and detrusor thickness in PACAP⫺/⫺ mice (p ⱕ0.01, fig. 1, A to C). There were no differences in bladder layer thickness in bladder dome vs trigone regions. No differences in urothelial thickness were observed in WT vs PACAP⫺/⫺ mice.
Bladder Function In response to continuous intravesical saline instillation PACAP⫺/⫺ mice showed significantly larger BC and VV, and longer ICI than WT and PACAP⫹/⫺ mice (p ⱕ0.01, figs. 2 to 4). RV in PACAP⫺/⫺ and
Figure 3. Representative cystometrogram traces show infused volume (IF), bladder pressure (BP) and VV in conscious, unrestrained WT mice with 25 l per minute continuous intravesical infusion of room temperature saline (A to C) and 0.5% AA (D to F). WT mice responded to AA instillation with increased voiding frequency (E) and decreased VV (F).
776
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
Figure 4. ICI in seconds (s) (A), BC with saline or AA (B), DCD (C) and RV (D) on cystometry in conscious, unrestrained WT, PACAP⫺/⫺ and PACAP⫹/⫺ mice with continuous infusion of saline or 0.5% AA. ICI was significantly longer in PACAP⫺/⫺ vs WT mice with each instillation (p ⱕ0.01) (A). No ICI changes were noted for AA vs saline in PACAP⫺/⫺ and PACAP⫹/⫺ mice. ICI was significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ mice with AA vs WT mice (p ⱕ0.01). BC with saline or AA was significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ vs WT mice (p ⱕ0.01) (B). For saline and AA VV was also significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ mice (p ⱕ0.01). Significant differences in BC and VV for saline and AA were noted in PACAP⫺/⫺ and PACAP⫹/⫺ mice (p ⱕ0.01). AA in PACAP⫺/⫺ and PACAP⫹/⫺ mice did not affect BC vs that in WT mice. DCD in PACAP⫺/⫺ mice was significantly longer than in WT and PACAP⫹/⫺ mice (p ⱕ0.01) (C). RV was significantly increased in PACAP⫹/⫺ and PACAP⫺/⫺ vs WT mice (p ⱕ0.01) (D). Values represent mean ⫾ SEM in 7 to 10 mice per group.
PACAP⫹/⫺ mice was also significantly greater than in WT mice (p ⱕ0.01, fig. 4, D). The DCD associated with voiding was significantly increased 1.9 to 4.1fold in PACAP⫺/⫺ mice compared to that in WT and PACAP⫹/⫺ mice (figs. 2, 3 and 4, C). In WT mice AA (0.5%) bladder instillation decreased ICI and VV (figs. 3 and 4, A and B). Intravesical AA instillation in PACAP⫺/⫺ and PACAP⫹/⫺ mice produced no significant change in VV or ICI (figs. 2 and 4, A and B). We observed nonvoiding bladder contractions during bladder filling with intravesical saline or AA instillation infrequently in any litter mate and they were not considered further. No significant differences in maximum voiding, micturition threshold or baseline filling pressure with intravesical saline instillation were observed among WT, PACAP⫺/⫺ and PACAP⫹/⫺ mice (see table). Somatic Sensitivity We evaluated baseline mechanical somatic sensitivity using a calibrated series of von Frey hairs on the plantar surface of the hind paw and pelvic region overlying the bladder in WT, PACAP⫺/⫺ and
PACAP⫹/⫺ mice.9,12 Somatic sensitivity in the pelvic region was significantly decreased in PACAP⫺/⫺ mice with von Frey hairs among the filament forces tested (0.1 to 4 gm) compared to that in WT and PACAP⫹/⫺ mice (p ⱕ0.01, fig. 5, A). Pelvic sensitivity was also significantly decreased in PACAP⫹/⫺ mice but the magnitude of change was less than in PACAP⫺/⫺ mice (p ⱕ0.01, fig. 5, A). Somatic sensitivity was significantly decreased in the hind paw in PACAP⫺/⫺ and PACAP⫹/⫺ vs WT mice (p ⱕ0.01, fig. 5, B). PACAP⫺/⫺ mice showed significantly less hind paw sensitivity than PACAP⫹/⫺ mice (p ⱕ0.01, fig. 5, B).
DISCUSSION To our knowledge this study reveals several novel roles of PACAP in the micturition reflex, and somatic and visceral sensation. PACAP⫺/⫺ mice produced fewer but larger urine spots. The bladder in PACAP⫺/⫺ mice had increased mass, correlating with significantly thicker lamina propria and detrusor smooth muscle layers, while the urothelium appeared unchanged. PACAP⫺/⫺ mice showed greater
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
777
Figure 5. Baseline pelvic (A) and hind paw (B) somatic sensitivity testing with von Frey hairs in WT (circles), PACAP⫺/⫺ (triangles) and PACAP⫹/⫺ (squares) mice. Pelvic region was tested at lower abdominal area over bladder. PACAP⫺/⫺ mice had significantly decreased pelvic response frequency with all hairs tested vs WT and PACAP⫹/⫺ mice. PACAP⫹/⫺ mice also had significantly decreased pelvic response frequency vs WT with some hairs tested. PACAP⫺/⫺ mice had significantly decreased hind paw response frequency with some hairs tested vs WT and PACAP⫹/⫺ mice. PACAP⫹/⫺ mice also had significantly decreased hind paw response frequency with some hairs tested vs WT mice. Values represent mean ⫾ SEM in 8 to 10 mice per group. Asterisk indicates PACAP⫺/⫺ vs WT p ⱕ0.01. Pound sign indicates PACAP⫺/⫺ vs PACAP⫹/⫺ p ⱕ0.01.
BC, VV and ICI. Also, PACAP⫺/⫺ mice had significantly increased DCD and greater RV. WT mice responded to AA (0.5%) intravesical instillation with decreased VV and ICI but PACAP⫹/⫺ and PACAP⫺/⫺ mice did not respond. PACAP⫺/⫺ mice were less responsive, ie hypoalgesic, to somatic stimulation of the hind paw or pelvic region with calibrated von Frey filaments. PACAP⫹/⫺ also showed bladder dysfunction, and somatic and visceral sensory abnormalities but to a lesser degree. These studies suggest that PACAP gene disruption contributes to changes in bladder morphology and function, and somatic and visceral hypoalgesia. PACAP and PAC1 signaling are prominent regulators of bladder physiology through actions on urothelium, detrusor smooth muscle and/or bladder afferent nerves.1–3 PACAP and VIP peptides regulate smooth muscle function in a tissue and species specific manner.15,16 We previously reported the direct effects of PACAP on bladder smooth muscle contractility.2 PACAP increased bladder smooth muscle tone and potentiated electric field stimulation induced contractions2 that were tetrodotoxin insensitive, suggesting direct smooth muscle effects.2 PACAP evoked adenosine triphosphate release from rat urothelial cell cultures, which was significantly blocked by the PAC1 receptor selective antagonist M65.6 PACAP/receptor expression is regulated by CYP induced cystitis.1 Administration of the PAC1 receptor antagonist PACAP6-38 significantly decreases CYP induced bladder hyperreflexia.2 Mice with PACAP gene deletion (PACAP⫺/⫺) or disruption (PACAP⫹/⫺) showed bladder dysfunction consistent with outlet obstruction. Conscious cystometry in PACAP⫺/⫺ mice revealed greater VV and
ICI. PACAP⫺/⫺ mice did not achieve complete bladder emptying, and had large RV and prolonged bladder emptying (increased DCD). An interpretation of these data is that PACAP gene deletion results in incomplete compensation by the detrusor muscle to overcome outlet obstruction. Despite evidence of detrusor smooth muscle hypertrophy increases in peak micturition pressure in PACAP⫺/⫺ mice were not observed, suggesting incomplete compensation. Given previous studies demonstrating PACAP stimulatory effects on detrusor function,2,3 PACAP⫺/⫺ mice may have decreased detrusor contractility. Among genotypes more detailed analyses of urethral outlet morphology and detrusor contractility in vitro may help clarify some of these issues. The current study in PACAP⫺/⫺ mice revealed some distinctions in bladder structure and function compared with a recent study in VIP⫺/⫺ mice.10 VIP⫺/⫺ and PACAP⫺/⫺ mice showed increased detrusor smooth muscle thickness, VV and ICI but in contrast to PACAP⫺/⫺ animals, VIP⫺/⫺ mice have low RV and no differences in baseline hind paw sensitivity, and responded as expected to intravesical AA instillation.10 Furthermore, only PACAP⫺/⫺ mice showed cystometric signs consistent with outlet obstruction, ie large RV and increased DCD. Previous series demonstrated that PACAP is found in capsaicin sensitive DRG populations,17 is co-expressed with transient receptor potential vanilloid receptor 1 immunoreactivity in bladder nerves of the suburothelial plexus (Vizzard et al, unpublished data) and is released from the spinal cord in response to capsaicin.18 Given that PACAP⫺/⫺ and PACAP⫹/⫺ mice showed decreased somatic (hind paw and pelvic region) sensitivity in response to
778
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
calibrated filament probing and did not respond to intravesical instillation of AA (0.5%), the changes in sensory function may also be a significant contributor to the differences in bladder/urethral defects between PACAP and VIP null animals. The sensory dysfunction observed using somatic testing techniques may also extend to sensory dysfunction of mechanosensitive bladder afferents. PACAP⫺/⫺ mice showed increased BC, VV and RV with a tendency toward an increased pressure threshold (see table), suggesting that bladder mechanosensitive afferents may be less sensitive to bladder distention after PACAP depletion. This is supported by decreased somatic mechanosensitivity in the pelvic area. Furthermore, increases in RV and DCD apart from sensory dysfunction may indicate detrusor dysfunction. Thus, sensory and detrusor dysfunction in PACAP null mice may contribute to the observed bladder dysfunction. Partial urethral obstruction and CYP induced cystitis are associated with an increased bladder mass and altered expression of bladder nerve growth factor.19,20 Studies of intestinal21 and bladder10 morphology in VIP⫺/⫺ mice revealed significant increases in smooth muscle thickness. Given the ability of VIP to inhibit airway smooth muscle proliferation,22 a regulatory role for VIP in smooth muscle proliferation was suggested.21 Our results suggest a similar regulatory role for PACAP in de-
trusor smooth muscle. The increased bladder mass in PACAP⫺/⫺ mice was consistent with cystometry and urine spot data demonstrating increased VV and fewer but significantly larger urine spots. PACAP⫺/⫺ mice also showed increased lamina propria thickness, as previously noted in animal models of outlet obstruction.23 Reasons underlying the increased lamina propria thickness in PACAP⫺/⫺ mice may include tissue remodeling of the afferent and efferent nerves24 in the suburothelial plexus and detrusor,25 myofibroblasts and/or vasculature,26 as described in animal models of outlet obstruction and potentially influenced by changes in bladder nerve growth factor content.24,27 Future studies will explore the relationship between bladder neurotrophic factor expression and bladder morphological changes in PACAP⫺/⫺ mice. In conclusion, these data suggest that PACAP gene disruption results in 1) broad effects on LUT function that may reflect underlying sensory and bladder dysfunction, and 2) novel effects on somatic sensation.
ACKNOWLEDGMENTS Susan Malley and Abbey Dattilio provided technical support. Dr. Bopaiah Cheppudira, Department of Pharmacology, University of Illinois at Chicago provided instruction in somatic testing techniques. Dr. Peter Zvara provided discussions on the manuscript.
REFERENCES 1. Vizzard MA: Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. J Comp Neurol 2000; 420: 335.
7. Sandor K, Bolcskei K, McDougall JJ et al: Divergent peripheral effects of pituitary adenylate cyclase-activating polypeptide-38 on nociception in rats and mice. Pain 2009; 141: 143.
2. Braas KM, May V, Zvara P et al: Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes. Am J Physiol Regul Integr Comp Physiol 2006; 290: R951.
8. Colwell CS, Michel S, Itri J et al: Selective deficits in the circadian light response in mice lacking PACAP. Am J Physiol Regul Integr Comp Physiol 2004; 287: R1194.
3. Herrera GM, Braas KM, May V et al: PACAP enhances mouse urinary bladder contractility and is upregulated in micturition reflex pathways after cystitis. Ann N Y Acad Sci 2006; 1070: 330.
9. Girard BA, Lelievre V, Braas KM et al: Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice. J Neurochem 2006; 99: 499.
4. Arimura A: Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 1992; 37: 287. 5. Fahrenkrug J and Hannibal J: Pituitary adenylate cyclase activating polypeptide immunoreactivity in capsaicin-sensitive nerve fibres supplying the rat urinary tract. Neuroscience 1998; 83: 1261. 6. Girard BM, Wolf-Johnston A, Braas KM et al: PACAP-mediated ATP release from rat urothelium and regulation of PACAP/VIP and receptor mRNA in micturition pathways after cyclophosphamide (CYP)-induced cystitis. J Mol Neurosci 2008; 36: 310.
10. Studeny S, Cheppudira BP, Meyers S et al: Urinary bladder function and somatic sensitivity in vasoactive intestinal polypeptide (VIP)-/- mice. J Mol Neurosci 2008; 36: 175. 11. Birder LA, Nakamura Y, Kiss S et al: Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nat Neurosci 2002; 5: 856. 12. Rudick CN, Chen MC, Mongiu AK et al: Organ cross talk modulates pelvic pain. Am J Physiol Regul Integr Comp Physiol 2007; 293: R1191.
13. Herrera GM, Pozo MJ, Zvara P et al: Urinary bladder instability induced by selective suppression of the murine small conductance calciumactivated potassium (SK3) channel. J Physiol 2003; 551: 893. 14. Maggi CA, Santicioli P and Meli A: The nonstop transvesical cystometrogram in urethane-anesthetized rats: a simple procedure for quantitative studies on the various phases of urinary bladder voiding cycle. J Pharmacol Methods 1986; 15: 157. 15. Mizumoto A, Fujimura M, Ohtawa M et al: Pituitary adenylate cyclase activating polypeptide stimulates gallbladder motility in conscious dogs. Regul Pept 1992; 42: 39. 16. Seebeck J, Lowe M, Kruse ML et al: The vasorelaxant effect of pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in isolated rat basilar arteries is partially mediated by activation of nitrergic neurons. Regul Pept 2002; 107: 115. 17. Zhang YZ, Hannibal J, Zhao Q et al: Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury. Neuroscience 1996; 74: 1099.
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
18. Zhang Y, Malmberg AB, Yaksh TL et al: Capsaicin-evoked release of pituitary adenylate cyclase activating peptide (PACAP) and calcitonin generelated peptide (CGRP) from rat spinal cord in vivo. Regul Pept 1997; 69: 83. 19. Steers WD, Kolbeck S, Creedon D et al: Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 1991; 88: 1709.
tion in the gene encoding vasoactive intestinal polypeptide: a model for the study of intestinal ileus and Hirschsprung’s disease. Peptides 2007; 28: 1688. 22. Maruno K, Absood A and Said SI: VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. Am J Physiol 1995; 268: L1047.
20. Vizzard MA: Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol 2000; 278: R1027.
23. Pampinella F, Roelofs M, Castellucci E et al: Proliferation of submesothelial mesenchymal cells during early phase of serosal thickening in the rabbit bladder is accompanied by transient keratin 18 expression. Exp Cell Res 1996; 223: 327.
21. Lelievre V, Favrais G, Abad C et al: Gastrointestinal dysfunction in mice with a targeted muta-
24. Steers WD, Ciambotti J, Etzel B et al: Alterations in afferent pathways from the urinary bladder of
779
the rat in response to partial urethral obstruction. J Comp Neurol 1991; 310: 1. 25. Andersson KE and Hedlund P: Pharmacologic perspective on the physiology of the lower urinary tract. Urology 2002; 60: 13. 26. Matsumoto S, Hanai T, Ohnishi N et al: Bladder smooth muscle cell phenotypic changes and implication of expression of contractile proteins (especially caldesmon) in rats after partial outlet obstruction. Int J Urol 2003; 10: 339. 27. Steers WD, Creedon DJ and Tuttle JB: Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol 1996; 155: 379.
Abbreviations and Acronyms AA ⫽ acetic acid BC ⫽ bladder capacity CYP ⫽ cyclophosphamide DCD ⫽ detrusor contraction duration DRG ⫽ dorsal root ganglia ICI ⫽ intercontraction interval LUT ⫽ lower urinary tract PAC ⫽ PACAP PACAP ⫽ pituitary adenylate cyclase activating polypeptide RV ⫽ post-void residual volume VIP ⫽ vasoactive intestinal polypeptide VV ⫽ voided volume Submitted for publication April 30, 2009. Study received institutional animal care and use committee approval. Supported by National Institutes of Health Grants DK051369, DK060481 and DK065989, and National Institutes of Health Grant P20 RR16435 from the COBRE Program of the National Center. * Correspondence: Department of Neurology, University of Vermont College of Medicine, D415A Given Research Building, Burlington, Vermont 05405 (telephone: 802-656-3209; FAX: 802656-8704; e-mail: [email protected]).
772
www.jurology.com
Purpose: PACAP and receptors are expressed in micturition pathways. Studies show that PACAP has a role in detrusor smooth muscle contraction to facilitate adenosine triphosphate release from urothelium and PACAP antagonism decreases cyclophosphamide induced bladder hyperreflexia. Materials and Methods: PACAP contributions to micturition and somatic sensation were studied in PACAP knockout (PACAP⫺/⫺), litter mate heterozygote (PACAP⫹/⫺) and WT mice by conscious cystometry with continuous intravesical saline or acetic acid (0.5%) instillation, urination patterns, somatic sensitivity testing of hind paw and pelvic regions with calibrated von Frey filaments, and morphological bladder assessments. Results: PACAP⫺/⫺ mice had an increased bladder mass with fewer but larger urine spots. In PACAP⫺/⫺ mice the lamina propria and detrusor smooth muscle were significantly thicker but the urothelium was unchanged. PACAP⫺/⫺ mice had increased bladder capacity, voided volume and intercontraction interval with significantly increased detrusor contraction duration and large residual volume. WT mice responded to acetic acid (0.5%) with a decrease in voided volume and intercontraction interval but PACAP⫹/⫺ and PACAP⫺/⫺ mice did not respond. PACAP⫺/⫺ mice were less responsive to somatic stimulation. PACAP⫹/⫺ mice also had bladder dysfunction, and somatic and visceral sensory abnormalities but to a lesser degree. Conclusions: PACAP gene disruption contributes to changes in bladder morphology and function, and somatic and visceral hypoalgesia. Key Words: urinary bladder, pituitary adenylate cyclase-activating polypeptide, reflex, sensation, mice THE neuropeptide PACAP is involved in LUT function.1–3 PACAP is a member of the VIP, secretin, glucagon family of hormones that shows about 68% homology to VIP.4 PACAP can act on 3 types of G-protein coupled receptors, including PAC1, VIP PAC1 and VIP PAC2 receptors.4 Dense PACAP immunoreactive sensory fibers are present in the bladder, including suburothelial plexus.2,5 PACAP/receptor expression in DRG and spinal cord afferent pro-
jections are up-regulated in CYP induced bladder inflammation. 1 We have noted specific PACAP receptor expression in bladder smooth muscle, urothelium, lumbosacral DRG and spinal cord, suggesting that PACAP and PAC1 receptor signaling participate at multiple levels in the LUT.2 Functionally PACAP peptides increase contractility in rat and mouse detrusor smooth muscle. 2,3 Bladder hyperreflexia induced by CYP is decreased by the PAC1 an-
0022-5347/10/1832-0772/0 THE JOURNAL OF UROLOGY® Copyright © 2010 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 183, 772-779, February 2010 Printed in U.S.A. DOI:10.1016/j.juro.2009.09.077
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
773
tagonist PACAP6-38.2 PACAP application evoked adenosine triphosphate release from rat cultured urothelium that was blocked by the PAC1 receptor selective antagonist M65.6 In addition to roles for PACAP/receptor signaling in LUT function, differential effects of PACAP were reported in peripheral nociception.7 We determined the effects of PACAP gene disruption in mice (PACAP⫺/⫺, PACAP⫹/⫺ and WT litter mates) on bladder function, somatic sensitivity, bladder morphology and somatic (pelvic and hind paw) sensitivity. These studies demonstrated that PACAP gene disruption contributes to bladder morphology and function changes, and somatic and visceral hypoalgesia.
the hair bent slightly. We used established criteria to determine positive responses to hind paw or pelvic region stimulation.10,12 Pelvic and hind paw testing was done in the same mice but somatic regions were evaluated in randomly assigned mice at different times. All somatic testing was done in blinded fashion and groups were decoded after data analysis.
METHODS
Bladder Function on Cystometry
Animals The PACAP⫺/⫺ mouse model was generated in a pathogen-free facility at UCLA and back crossed for at least 8 generations with C57Bl/6.8,9 PACAP⫺/⫺, litter mate PACAP⫹/⫺ and WT mice8,9 were bred at our institution and lack of the PACAP gene was confirmed by polymerase chain reaction genotyping.9 The University of Vermont institutional animal care and use committee approved animal use. Animal care was done under the supervision of the University of Vermont office of animal care management according to Association for Assessment and Accreditation of Laboratory Animal Care International and National Institutes of Health guidelines.
Urination Patterns PACAP⫺/⫺, PACAP⫹/⫺ and WT mice of each gender were placed individually in standard cages for 1 hour with bedding replaced with Whatman grade 3 filter paper. Food and water were provided freely. Urine spots were photographed under ultraviolet light,10 spot area in cm2 was determined, and large (0.2 to 10 cm2)10 and small (less than 0.2 cm2)11 spots were counted. Mice were placed in cages and data were analyzed by an individual blinded to mouse strain. Groups were decoded after data analysis.
Mechanical Sensitivity Testing Due to divergent roles for PACAP in somatic sensation7 and PACAP expression in sensory neurons1,14 tactile allodynia was tested using calibrated von Frey hairs with a force of 0.04, 0.16, 0.4, 1 and 4 gm to the abdomen and hind paw10,12 among genotypes. Mice were tested in individual Plexiglas® chambers with a stainless steel wire grid floor. Mice were acclimated to the chambers for 2 hours. Due to limited supply the same mice were evaluated for somatic sensitivity 1 to 2 weeks before intravesical catheter implantation, and 1 to 2 weeks before catheter implantation and somatic testing to determine urination patterns. von Frey hairs were applied in an up-down method for 1 to 3 seconds with an interstimulus interval of 15 seconds. Pelvic region stimulation was confined to the lower abdominal area overlying the bladder. The plantar region of the hind paw and the lower abdominal area were tested by perpendicular application of von Frey hairs until
Bladder Catheter Implantation A lower midline abdominal incision was made using general anesthesia with isoflurane (2.5% to 3.5%). Polyethylene-10 tubing with the end flared by heat was inserted into the bladder dome and secured in place with a 6-zero nylon purse-string suture.10 The distal end of the tubing was sealed, tunneled subcutaneously and externalized. Animals were maintained for 72 hour to ensure complete recovery. Postoperative analgesics were given for 48 hours. Animals were placed while conscious and unrestrained in recording cages with a balance and pan below for urine collection and measurement.2,10 Intravesical pressure changes were recorded using a Small Animal Cystometry System (Med Associates, St. Albans, Vermont). Saline at room temperature was infused (25 l per minute) to elicit repetitive bladder contractions. At least 6 reproducible micturition cycles were recorded after an initial stabilization period (25 to 30 minutes). BC was measured as the amount of saline infused in the bladder at the time when micturition commenced.11,13 Voided saline was collected to determine VV. After collection the infusion was stopped and RV was determined by withdrawing urine from the bladder catheter with a syringe. ICI, maximal voiding pressure, pressure threshold for voiding and baseline resting pressure were measured.14 DCD was determined by the width of the micturition contraction at its base. Nonvoiding bladder contractions, defined as greater than 5 cm H2O from baseline pressure without a release of fluid from the urethra, were quantified during the filling phase. AA (0.5%) was infused into the bladder in some litter mates after saline infusion to evaluate nociceptive bladder responses. Mice were removed from study when adverse events occurred, including a 20% or greater decrease in body weight postoperatively, a significant postoperative event, lethargy, pain or distress not relieved by our institutional animal care and use committee approved regimen of postoperative analgesics or hematuria in control rodents.10 No mice were excluded from analysis according to established criteria but 3 were removed because they chewed the exteriorized tubing. Behavioral movements, eg grooming or defecation, rendered bladder pressure recording unusable during these events. Experiments were done at similar times of the day.10 Mice were sacrificed with isoflurane (4%) and thoracotomy at the conclusion of study. Bladders were dissected and weighed.
Bladder Histological Analysis Histological analysis of bladder sections (15 m) from intravesical saline studies only was done using hematoxylin and eosin staining. Digital images of hematoxylin and eosin stained sections were captured using a MagnaFire™ camera and an Olympus® microscope using a 20⫻ objec-
774
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
tive. Bladder layer thickness was measured using MetaMorph®, version 4.5r4. Measurements were made of urothelium, lamina propria and muscular layer thickness. Section measurements were done in triplicate and averaged. We matched the bladder region being compared, ie bladder dome or trigone, among litter mates. Parameters were measured as the distance along a line drawn normal to the long axis of the specimen from the luminal edge to the serosal urothelium, lamina propria and detrusor edges.13
Statistical Analysis All values are shown as the mean ⫾ SEM. Data were compared with ANOVA using SAS® statistical software. When F ratios exceeded the critical value of p ⱕ0.05, the Newman-Keul post hoc test was used to compare means.
RESULTS WT, PACAPⴙ/ⴚ and PACAPⴚ/ⴚ Mice There were no differences in body mass among WT, PACAP⫹/⫺ and PACAP⫺/⫺ litter mates but the bladder-to-body weight ratio was significantly increased in PACAP⫺/⫺ mice due to the increased bladder mass (fig. 1). Fluid intake measured in 24 hours was similar among litter mates (see table). As quantified on filter paper in a 1-hour period, urine spots of PACAP⫺/⫺ mice were significantly fewer (p ⱕ0.01)
Urine spots, fluid intake and bladder pressure in 10 WT, PACAP⫺/⫺ and PACAP⫹/⫺ litter mates each
Fluid intake (ml/24 hrs) Urine spots (No./hr) Pressure (cm H2O): Max voiding Baseline Threshold
Mean ⫾ SEM WT
Mean ⫾ SEM PACAP⫺/⫺
Mean ⫾ SEM PACAP⫹/⫺
7.2 ⫾ 0.8 12.4 ⫾ 2.5
7.4 ⫾ 1.0 3.2 ⫾ 1.2
7.0 ⫾ 0.5 6.2 ⫾ 1.5
67.2 ⫾ 1.5 12.4 ⫾ 2.5 14.5 ⫾ 2.5
68.5 ⫾ 1.0 14.2 ⫾ 2.2 21.5 ⫾ 4.5
66.5 ⫾ 1.5 13.2 ⫾ 1.5 16.8 ⫾ 2.0
but greater in area than those of WT mice (8.2 ⫾ 3.5 vs 3.3 ⫾ 2.5 cm2, see table). PACAP⫹/⫺ mice also produced significantly fewer urine spots than WT mice (p ⱕ0.01). Large (0.2 to 10 cm2) and small (less than 0.2 cm2) diameter urine spots were observed for all genotypes but there were differences in the number of only large spots, likely representing actual voiding events, in PACAP⫺/⫺ and PACAP⫹/⫺ mice. No differences were noted between PACAP⫺/⫺ and PACAP⫹/⫺ mice (see table). Bladder Histology Histological analysis of the bladder at the dome and trigone regions revealed significantly increased lam-
Figure 1. Bladder histological analysis in PACAP⫺/⫺, PACAP⫹/⫺ and WT mice. Representative 15 m cryostat bladder sections of WT (A) and PACAP⫺/⫺ (B) mice reveal significantly increased lamina propria (LP) and detrusor smooth muscle (SM) thickness. U, urothelium. H & E, scale bar indicates 120 m. Histological results were supported by morphometric analysis (C). Asterisk indicates p ⱕ0.01. Body weight did not differ among mice (D) but bladder-to-body weight ratio was significantly increased in PACAP⫺/⫺ mice due to increased bladder mass (E). g, gm. Values represent mean ⫾ SEM in 7 to 10 mice per group (C to E).
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
775
Figure 2. Representative cystometrogram traces show infused volume (IF), bladder pressure (BP) and VV in conscious unrestrained PACAP⫺/⫺ mice with 25 l per minute continuous intravesical infusion of room temperature saline (A to C) and 0.5% AA (D to F). PACAP⫺/⫺ mice had large BC before micturition (A), long detrusor contraction duration (B) and large VV (C) with incomplete emptying compared to WT mice. PACAP⫺/⫺ mice did not respond to AA compared to WT mice (fig. 3).
ina propria and detrusor thickness in PACAP⫺/⫺ mice (p ⱕ0.01, fig. 1, A to C). There were no differences in bladder layer thickness in bladder dome vs trigone regions. No differences in urothelial thickness were observed in WT vs PACAP⫺/⫺ mice.
Bladder Function In response to continuous intravesical saline instillation PACAP⫺/⫺ mice showed significantly larger BC and VV, and longer ICI than WT and PACAP⫹/⫺ mice (p ⱕ0.01, figs. 2 to 4). RV in PACAP⫺/⫺ and
Figure 3. Representative cystometrogram traces show infused volume (IF), bladder pressure (BP) and VV in conscious, unrestrained WT mice with 25 l per minute continuous intravesical infusion of room temperature saline (A to C) and 0.5% AA (D to F). WT mice responded to AA instillation with increased voiding frequency (E) and decreased VV (F).
776
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
Figure 4. ICI in seconds (s) (A), BC with saline or AA (B), DCD (C) and RV (D) on cystometry in conscious, unrestrained WT, PACAP⫺/⫺ and PACAP⫹/⫺ mice with continuous infusion of saline or 0.5% AA. ICI was significantly longer in PACAP⫺/⫺ vs WT mice with each instillation (p ⱕ0.01) (A). No ICI changes were noted for AA vs saline in PACAP⫺/⫺ and PACAP⫹/⫺ mice. ICI was significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ mice with AA vs WT mice (p ⱕ0.01). BC with saline or AA was significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ vs WT mice (p ⱕ0.01) (B). For saline and AA VV was also significantly greater in PACAP⫺/⫺ and PACAP⫹/⫺ mice (p ⱕ0.01). Significant differences in BC and VV for saline and AA were noted in PACAP⫺/⫺ and PACAP⫹/⫺ mice (p ⱕ0.01). AA in PACAP⫺/⫺ and PACAP⫹/⫺ mice did not affect BC vs that in WT mice. DCD in PACAP⫺/⫺ mice was significantly longer than in WT and PACAP⫹/⫺ mice (p ⱕ0.01) (C). RV was significantly increased in PACAP⫹/⫺ and PACAP⫺/⫺ vs WT mice (p ⱕ0.01) (D). Values represent mean ⫾ SEM in 7 to 10 mice per group.
PACAP⫹/⫺ mice was also significantly greater than in WT mice (p ⱕ0.01, fig. 4, D). The DCD associated with voiding was significantly increased 1.9 to 4.1fold in PACAP⫺/⫺ mice compared to that in WT and PACAP⫹/⫺ mice (figs. 2, 3 and 4, C). In WT mice AA (0.5%) bladder instillation decreased ICI and VV (figs. 3 and 4, A and B). Intravesical AA instillation in PACAP⫺/⫺ and PACAP⫹/⫺ mice produced no significant change in VV or ICI (figs. 2 and 4, A and B). We observed nonvoiding bladder contractions during bladder filling with intravesical saline or AA instillation infrequently in any litter mate and they were not considered further. No significant differences in maximum voiding, micturition threshold or baseline filling pressure with intravesical saline instillation were observed among WT, PACAP⫺/⫺ and PACAP⫹/⫺ mice (see table). Somatic Sensitivity We evaluated baseline mechanical somatic sensitivity using a calibrated series of von Frey hairs on the plantar surface of the hind paw and pelvic region overlying the bladder in WT, PACAP⫺/⫺ and
PACAP⫹/⫺ mice.9,12 Somatic sensitivity in the pelvic region was significantly decreased in PACAP⫺/⫺ mice with von Frey hairs among the filament forces tested (0.1 to 4 gm) compared to that in WT and PACAP⫹/⫺ mice (p ⱕ0.01, fig. 5, A). Pelvic sensitivity was also significantly decreased in PACAP⫹/⫺ mice but the magnitude of change was less than in PACAP⫺/⫺ mice (p ⱕ0.01, fig. 5, A). Somatic sensitivity was significantly decreased in the hind paw in PACAP⫺/⫺ and PACAP⫹/⫺ vs WT mice (p ⱕ0.01, fig. 5, B). PACAP⫺/⫺ mice showed significantly less hind paw sensitivity than PACAP⫹/⫺ mice (p ⱕ0.01, fig. 5, B).
DISCUSSION To our knowledge this study reveals several novel roles of PACAP in the micturition reflex, and somatic and visceral sensation. PACAP⫺/⫺ mice produced fewer but larger urine spots. The bladder in PACAP⫺/⫺ mice had increased mass, correlating with significantly thicker lamina propria and detrusor smooth muscle layers, while the urothelium appeared unchanged. PACAP⫺/⫺ mice showed greater
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
777
Figure 5. Baseline pelvic (A) and hind paw (B) somatic sensitivity testing with von Frey hairs in WT (circles), PACAP⫺/⫺ (triangles) and PACAP⫹/⫺ (squares) mice. Pelvic region was tested at lower abdominal area over bladder. PACAP⫺/⫺ mice had significantly decreased pelvic response frequency with all hairs tested vs WT and PACAP⫹/⫺ mice. PACAP⫹/⫺ mice also had significantly decreased pelvic response frequency vs WT with some hairs tested. PACAP⫺/⫺ mice had significantly decreased hind paw response frequency with some hairs tested vs WT and PACAP⫹/⫺ mice. PACAP⫹/⫺ mice also had significantly decreased hind paw response frequency with some hairs tested vs WT mice. Values represent mean ⫾ SEM in 8 to 10 mice per group. Asterisk indicates PACAP⫺/⫺ vs WT p ⱕ0.01. Pound sign indicates PACAP⫺/⫺ vs PACAP⫹/⫺ p ⱕ0.01.
BC, VV and ICI. Also, PACAP⫺/⫺ mice had significantly increased DCD and greater RV. WT mice responded to AA (0.5%) intravesical instillation with decreased VV and ICI but PACAP⫹/⫺ and PACAP⫺/⫺ mice did not respond. PACAP⫺/⫺ mice were less responsive, ie hypoalgesic, to somatic stimulation of the hind paw or pelvic region with calibrated von Frey filaments. PACAP⫹/⫺ also showed bladder dysfunction, and somatic and visceral sensory abnormalities but to a lesser degree. These studies suggest that PACAP gene disruption contributes to changes in bladder morphology and function, and somatic and visceral hypoalgesia. PACAP and PAC1 signaling are prominent regulators of bladder physiology through actions on urothelium, detrusor smooth muscle and/or bladder afferent nerves.1–3 PACAP and VIP peptides regulate smooth muscle function in a tissue and species specific manner.15,16 We previously reported the direct effects of PACAP on bladder smooth muscle contractility.2 PACAP increased bladder smooth muscle tone and potentiated electric field stimulation induced contractions2 that were tetrodotoxin insensitive, suggesting direct smooth muscle effects.2 PACAP evoked adenosine triphosphate release from rat urothelial cell cultures, which was significantly blocked by the PAC1 receptor selective antagonist M65.6 PACAP/receptor expression is regulated by CYP induced cystitis.1 Administration of the PAC1 receptor antagonist PACAP6-38 significantly decreases CYP induced bladder hyperreflexia.2 Mice with PACAP gene deletion (PACAP⫺/⫺) or disruption (PACAP⫹/⫺) showed bladder dysfunction consistent with outlet obstruction. Conscious cystometry in PACAP⫺/⫺ mice revealed greater VV and
ICI. PACAP⫺/⫺ mice did not achieve complete bladder emptying, and had large RV and prolonged bladder emptying (increased DCD). An interpretation of these data is that PACAP gene deletion results in incomplete compensation by the detrusor muscle to overcome outlet obstruction. Despite evidence of detrusor smooth muscle hypertrophy increases in peak micturition pressure in PACAP⫺/⫺ mice were not observed, suggesting incomplete compensation. Given previous studies demonstrating PACAP stimulatory effects on detrusor function,2,3 PACAP⫺/⫺ mice may have decreased detrusor contractility. Among genotypes more detailed analyses of urethral outlet morphology and detrusor contractility in vitro may help clarify some of these issues. The current study in PACAP⫺/⫺ mice revealed some distinctions in bladder structure and function compared with a recent study in VIP⫺/⫺ mice.10 VIP⫺/⫺ and PACAP⫺/⫺ mice showed increased detrusor smooth muscle thickness, VV and ICI but in contrast to PACAP⫺/⫺ animals, VIP⫺/⫺ mice have low RV and no differences in baseline hind paw sensitivity, and responded as expected to intravesical AA instillation.10 Furthermore, only PACAP⫺/⫺ mice showed cystometric signs consistent with outlet obstruction, ie large RV and increased DCD. Previous series demonstrated that PACAP is found in capsaicin sensitive DRG populations,17 is co-expressed with transient receptor potential vanilloid receptor 1 immunoreactivity in bladder nerves of the suburothelial plexus (Vizzard et al, unpublished data) and is released from the spinal cord in response to capsaicin.18 Given that PACAP⫺/⫺ and PACAP⫹/⫺ mice showed decreased somatic (hind paw and pelvic region) sensitivity in response to
778
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
calibrated filament probing and did not respond to intravesical instillation of AA (0.5%), the changes in sensory function may also be a significant contributor to the differences in bladder/urethral defects between PACAP and VIP null animals. The sensory dysfunction observed using somatic testing techniques may also extend to sensory dysfunction of mechanosensitive bladder afferents. PACAP⫺/⫺ mice showed increased BC, VV and RV with a tendency toward an increased pressure threshold (see table), suggesting that bladder mechanosensitive afferents may be less sensitive to bladder distention after PACAP depletion. This is supported by decreased somatic mechanosensitivity in the pelvic area. Furthermore, increases in RV and DCD apart from sensory dysfunction may indicate detrusor dysfunction. Thus, sensory and detrusor dysfunction in PACAP null mice may contribute to the observed bladder dysfunction. Partial urethral obstruction and CYP induced cystitis are associated with an increased bladder mass and altered expression of bladder nerve growth factor.19,20 Studies of intestinal21 and bladder10 morphology in VIP⫺/⫺ mice revealed significant increases in smooth muscle thickness. Given the ability of VIP to inhibit airway smooth muscle proliferation,22 a regulatory role for VIP in smooth muscle proliferation was suggested.21 Our results suggest a similar regulatory role for PACAP in de-
trusor smooth muscle. The increased bladder mass in PACAP⫺/⫺ mice was consistent with cystometry and urine spot data demonstrating increased VV and fewer but significantly larger urine spots. PACAP⫺/⫺ mice also showed increased lamina propria thickness, as previously noted in animal models of outlet obstruction.23 Reasons underlying the increased lamina propria thickness in PACAP⫺/⫺ mice may include tissue remodeling of the afferent and efferent nerves24 in the suburothelial plexus and detrusor,25 myofibroblasts and/or vasculature,26 as described in animal models of outlet obstruction and potentially influenced by changes in bladder nerve growth factor content.24,27 Future studies will explore the relationship between bladder neurotrophic factor expression and bladder morphological changes in PACAP⫺/⫺ mice. In conclusion, these data suggest that PACAP gene disruption results in 1) broad effects on LUT function that may reflect underlying sensory and bladder dysfunction, and 2) novel effects on somatic sensation.
ACKNOWLEDGMENTS Susan Malley and Abbey Dattilio provided technical support. Dr. Bopaiah Cheppudira, Department of Pharmacology, University of Illinois at Chicago provided instruction in somatic testing techniques. Dr. Peter Zvara provided discussions on the manuscript.
REFERENCES 1. Vizzard MA: Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. J Comp Neurol 2000; 420: 335.
7. Sandor K, Bolcskei K, McDougall JJ et al: Divergent peripheral effects of pituitary adenylate cyclase-activating polypeptide-38 on nociception in rats and mice. Pain 2009; 141: 143.
2. Braas KM, May V, Zvara P et al: Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes. Am J Physiol Regul Integr Comp Physiol 2006; 290: R951.
8. Colwell CS, Michel S, Itri J et al: Selective deficits in the circadian light response in mice lacking PACAP. Am J Physiol Regul Integr Comp Physiol 2004; 287: R1194.
3. Herrera GM, Braas KM, May V et al: PACAP enhances mouse urinary bladder contractility and is upregulated in micturition reflex pathways after cystitis. Ann N Y Acad Sci 2006; 1070: 330.
9. Girard BA, Lelievre V, Braas KM et al: Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice. J Neurochem 2006; 99: 499.
4. Arimura A: Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 1992; 37: 287. 5. Fahrenkrug J and Hannibal J: Pituitary adenylate cyclase activating polypeptide immunoreactivity in capsaicin-sensitive nerve fibres supplying the rat urinary tract. Neuroscience 1998; 83: 1261. 6. Girard BM, Wolf-Johnston A, Braas KM et al: PACAP-mediated ATP release from rat urothelium and regulation of PACAP/VIP and receptor mRNA in micturition pathways after cyclophosphamide (CYP)-induced cystitis. J Mol Neurosci 2008; 36: 310.
10. Studeny S, Cheppudira BP, Meyers S et al: Urinary bladder function and somatic sensitivity in vasoactive intestinal polypeptide (VIP)-/- mice. J Mol Neurosci 2008; 36: 175. 11. Birder LA, Nakamura Y, Kiss S et al: Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1. Nat Neurosci 2002; 5: 856. 12. Rudick CN, Chen MC, Mongiu AK et al: Organ cross talk modulates pelvic pain. Am J Physiol Regul Integr Comp Physiol 2007; 293: R1191.
13. Herrera GM, Pozo MJ, Zvara P et al: Urinary bladder instability induced by selective suppression of the murine small conductance calciumactivated potassium (SK3) channel. J Physiol 2003; 551: 893. 14. Maggi CA, Santicioli P and Meli A: The nonstop transvesical cystometrogram in urethane-anesthetized rats: a simple procedure for quantitative studies on the various phases of urinary bladder voiding cycle. J Pharmacol Methods 1986; 15: 157. 15. Mizumoto A, Fujimura M, Ohtawa M et al: Pituitary adenylate cyclase activating polypeptide stimulates gallbladder motility in conscious dogs. Regul Pept 1992; 42: 39. 16. Seebeck J, Lowe M, Kruse ML et al: The vasorelaxant effect of pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in isolated rat basilar arteries is partially mediated by activation of nitrergic neurons. Regul Pept 2002; 107: 115. 17. Zhang YZ, Hannibal J, Zhao Q et al: Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury. Neuroscience 1996; 74: 1099.
BLADDER DYSFUNCTION AND ALTERED SOMATIC SENSITIVITY IN PACAP⫺/⫺ MICE
18. Zhang Y, Malmberg AB, Yaksh TL et al: Capsaicin-evoked release of pituitary adenylate cyclase activating peptide (PACAP) and calcitonin generelated peptide (CGRP) from rat spinal cord in vivo. Regul Pept 1997; 69: 83. 19. Steers WD, Kolbeck S, Creedon D et al: Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 1991; 88: 1709.
tion in the gene encoding vasoactive intestinal polypeptide: a model for the study of intestinal ileus and Hirschsprung’s disease. Peptides 2007; 28: 1688. 22. Maruno K, Absood A and Said SI: VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. Am J Physiol 1995; 268: L1047.
20. Vizzard MA: Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol 2000; 278: R1027.
23. Pampinella F, Roelofs M, Castellucci E et al: Proliferation of submesothelial mesenchymal cells during early phase of serosal thickening in the rabbit bladder is accompanied by transient keratin 18 expression. Exp Cell Res 1996; 223: 327.
21. Lelievre V, Favrais G, Abad C et al: Gastrointestinal dysfunction in mice with a targeted muta-
24. Steers WD, Ciambotti J, Etzel B et al: Alterations in afferent pathways from the urinary bladder of
779
the rat in response to partial urethral obstruction. J Comp Neurol 1991; 310: 1. 25. Andersson KE and Hedlund P: Pharmacologic perspective on the physiology of the lower urinary tract. Urology 2002; 60: 13. 26. Matsumoto S, Hanai T, Ohnishi N et al: Bladder smooth muscle cell phenotypic changes and implication of expression of contractile proteins (especially caldesmon) in rats after partial outlet obstruction. Int J Urol 2003; 10: 339. 27. Steers WD, Creedon DJ and Tuttle JB: Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol 1996; 155: 379.