PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Mol Neurosci. Author manuscript; available in PMC 2009 November 1.
Published in final edited form as:
PMCID: PMC2707851
NIHMSID: NIHMS110582

Expression of Phosphorylated cAMP Response Element Binding Protein (pCREB) in Bladder Afferent Pathways in VIP−/− Mice with Cyclophosphamide (CYP)-induced Cystitis

Abstract

The expression of phosphorylated cAMP response element binding protein (pCREB) in dorsal root ganglia (DRG) with and without CYP-induced cystitis (150 mg/kg, i.p; 48 hr) was determined in VIP−/− and wildtype (WT) mice. p-CREB-immunoreactivity (IR) was determined in bladder (Fastblue) afferent cells. Nerve growth factor (NGF) bladder content was determined by ELISAs. Basal expression of pCREB-IR in DRG of VIP−/− mice was (p ≤ 0.01) greater in L1, L2, L5-S1 DRG compared to WT mice. CYP treatment in WT mice increased (p ≤ 0.05) pCREB-IR in L1, L2, L5-S1 DRG. CYP treatment in VIP−/− mice (p ≤ 0.01) increased (p ≤ 0.01) p-CREB-IR in L6-S1 DRG compared to WT with CYP. In WT mice, bladder afferent cells (20–38%) in DRG expressed pCREB-IR under basal conditions. With CYP, pCREB-IR increased in bladder afferent cells (60–65%; L6-S1 DRG) in WT mice. In VIP−/− mice, bladder afferent cells (12–58%) expressed pCREB-IR under basal conditions and CYP increased pCREB expression (78–84%) in L6-S1 DRG. Urinary bladder NGF expression in VIP−/− mice under basal conditions or after cystitis was significantly greater than WT. Detrusor smooth muscle thickness was significantly increased in VIP−/− mice. Bladder NGF expression may contribute to differences in pCREB expression.

Index entries: inflammation, urinary bladder, afferent neurons, growth factors, cytokines

Introduction

Patients with interstitial cystitis (IC) or painful bladder syndrome (PBS), a painful, chronic urinary bladder inflammation syndrome, exhibit urinary frequency, urgency and suprapubic and pelvic pain and pain at low to moderate bladder pressure (Petrone et al., 1995). Although the etiology and pathogenesis of IC/PBS are unknown, numerous theories including; infection, autoimmune disorder, toxic urinary agents, deficiency in bladder wall lining and neurogenic causes have been proposed (Petrone et al., 1995; Ho et al., 1997; Johansson et al., 1997; Driscoll and Teichman, 2001; Sant and Hanno, 2001). We have hypothesized that pain associated with IC/PBS involves an alteration of visceral sensation/bladder sensory physiology. Altered visceral sensations from the urinary bladder (i.e., pain at low or moderate bladder filling) that accompany IC/PBS (Petrone et al., 1995; Ho et al., 1997; Johansson et al., 1997; Driscoll and Teichman, 2001; Sant and Hanno, 2001) may be mediated by many factors including changes in the properties of peripheral bladder afferent pathways such that bladder afferent neurons respond in an exaggerated manner to normally innocuous stimuli (allodynia). These changes may be mediated, in part, by inflammatory changes in the urinary bladder.

Neuropeptides are potential mediators or modulators of inflammation and are found in human micturition pathways (Chapple et al., 1992; Lasanen et al., 1992; Smet et al., 1997; Morgan et al., 1999; Uckert et al., 2002). Changes in the expression of neuropeptides have been observed with bladder overactivity (Chapple et al., 1992; Lasanen et al., 1992; Smet et al., 1997) and in animal models of bladder inflammation (Vizzard, 2000d, 2001; Zvarova and Vizzard, 2006). In this study, we have examined the contribution of vasoactive intestinal polypeptide (VIP) in afferent pathways to the urinary bladder by using wildtype and VIP−/− mice under control conditions or after induction of acute bladder inflammation. VIP is a 28 amino-acid peptide, belonging to the glucagon/secretin superfamily of hormones (Dickinson et al., 1999) and acts through two high affinity receptors, the VPAC1 and VPAC2 receptors (Harmar et al., 1998). VIP may exert excitatory or inhibitory actions in neural pathways controlling micturition and these functions may be altered with neural injury, disease or inflammation. Previous studies (Girard et al., 2006) have demonstrated that the closely related neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), does not compensate for VIP in VIP−/− mice. Thus, the VIP and PACAP systems appear distinct.

Numerous studies involving a chemically (cyclophosphamide; CYP)-induced bladder inflammation (Cox, 1979; Maggi et al., 1992; Lantéri-Minet et al., 1995; Vizzard, 2000a; Vizzard, 2000d; Zvarova and Vizzard, 2006) model have demonstrated alterations in neurochemical (Vizzard, 2000a; Vizzard, 2000d, 2001; Zvarova and Vizzard, 2006), electrophysiological (Jennings and Vizzard, 1999; Yoshimura and de Groat, 1999), organizational (Vizzard and Boyle, 1999; Vizzard, 2000b) and functional properties of bladder afferent neurons in dorsal root ganglia (DRG) and in central reflex micturition pathways as well as changes in the urinary bladder (xx). Neurotrophins, including nerve growth factor (NGF) have been implicated in mediating some of these changes. In vitro studies demonstrate that activation of downstream intracellular signaling molecules, especially transcription factors CREB (camp-response element binding protein), c-Jun and Elk-1 are important steps in neurotrophin-signaling cascades (Hawley et al., 1992; Gold et al., 1993; Riccio et al., 1999; Avelino et al., 2002; Arthur et al., 2004). Previous studies have demonstrated that CYP-induced cystitis in the rat induces the expression of phosphorylated cAMP response element binding protein (p-CREB) in bladder afferent cells in the lumbosacral DRG (Qiao and Vizzard, 2004). In the present study, we: (1) examined the contribution of VIP to the expression of p-CREB in micturition afferent pathways under control or inflamed conditions by using antibodies that specifically recognize the phosphorylated form of CREB (p-CREB) in VIP−/− and wildtype mice; (2) determined whether p-CREB expression is associated with bladder afferent cells in the DRG by retrograde labeling of bladder afferent neurons with a conventional tracing dye, Fast blue (FB) and (3) determined NGF bladder content in VIP−/− and WT mice under basal (no inflammation) conditions and after CYP-induced cystitis.

Materials and Methods

Animals

VIP−/− mice (Dr. James Waschek, University of California, LA, USA) (Colwell et al., 2003) were used in these experiments. The VIP−/− mouse model was prepared using a VIP gene disruption strategy with confirmation of targeted mutation in mice and subsequent backcrossing to the C57BL/6 strain for at least six generations (Colwell et al., 2003). Mice used were bred locally at The University of Vermont and lack of the VIP gene was confirmed by PCR genotyping of tailsnips. Mice were housed (12-hr light/dark cycle) in groups (5) in the UVM animal vivarium with water and food provided ad libitum. VIP−/− and wildtype (WT) controls from the same litter were analyzed in most cases. When a WT littermate was unavailable, an age-matched control mouse from another C57BL/6 litter was used. The Institutional Animal Care and Use Committee at The University of Vermont approved all experiments.

Retrograde labelling of bladder afferents

To label dorsal root ganglion (DRG) cell bodies with an axonal projection to the bladder, the fluorescent dye Fast Blue (FB) (EMS-Chemie, Germany, 4% in water), was injected into the bladder wall of anesthetized mice using a slight modification of a published procedure (Qiao and Vizzard, 2002a; Qiao and Vizzard, 2002b; Qiao and Vizzard, 2004). Briefly, a total volume 20 µl, divided into 5–10 injections, was injected into the entire surface of the bladder wall using a Hamilton syringe with particular care to avoid injections into the bladder lumen, major blood vessels, or overlying fascial layers. At each injection site, the needle (28 gauge) was kept in place for several seconds after injection, and the site was washed with saline to minimize contamination of adjacent organs with FB. FB was injected 5–6 days before CYP treatment as described below.

Acute cyclophosphamide (CYP)-induced cystitis

To produce an acute inflammation of the bladder, the cytostatic drug, cyclophosphamide (CYP; Sigma, 40 mg/ml in saline), was injected (150 mg/kg; i.p.) (Qiao and Vizzard, 2002a, 2004; Zvarova and Vizzard, 2006). After CYP-treatment, animal health status was observed daily. The animals were euthanized 48 hr after treatment.

Euthanasia, tissue harvesting and preparation

Mice were anesthetized with isoflurane (0.4%), euthanized by thoracotomy and urinary bladders harvested. Bladder weight and animal weights were recorded. Dorsal root ganglia (DRG) were identified, harvested and immersion-fixed in paraformaldehyde solution (4%) overnight at 4 °C. Spinal cord segments were identified based upon at least two criteria: (1) the T13 DRG was present after the last rib, and (2) the L6 vertebra was the last moveable vertebra followed by the fused sacral vertebrae. Another less precise criterion is the observation that the L6 DRG are the smallest ganglia following the largest DRG, L5. Tissue was then placed overnight in sucrose (30%) in 0.1 M PBS for cryoprotection at 4 °C. DRG (L1, L2, L5–S1) were sectioned parasagittally at a thickness of 20 µm on a cryostat. Every third section (1:2 series) of DRG was thaw-mounted on gelled (0.5%) slides. Some DRG (L1, L2, L6, S1) were specifically chosen for analysis based upon the previously determined segmental representation of urinary bladder circuitry (Donovan et al., 1983; Keast and de Groat, 1992; Nadelhaft and Vera, 1995). Bladder afferents are not distributed within the L4–L5 DRG (Donovan et al., 1983; Keast and de Groat, 1992) that contain only somatic afferents nor are neurons that are involved in urinary bladder function observed in the L4–L5 spinal segments (Donovan et al., 1983; Keast and de Groat, 1992; Nadelhaft and Vera, 1995). Thus, the L5 DRG served as an internal control for these studies.

Immunostaining for phosphorylated cAMP response element binding protein (p-CREB)

DRG sections for WT and VIP−/− mice were processed for pCREB-immunoreactivity (IR) using an on–slide processing technique (Vizzard, 1997). Groups of control (no inflammation) and CYP-treated WT and VIP−/− mice were processed simultaneously to decrease the possible incidence of variation in staining and background between tissues and between animals. DRG sections were incubated overnight at room temperature with rabbit anti–pCREB antibody (Cell Signaling Technology, MA, USA, 1: 1000) in 1% goat serum and 0.1 M KPBS (Phosphate Buffer Solution with potassium), and then washed (3 × 15 min) with 0.1 M KPBS, pH 7.4. Tissue was then incubated with Cy3-conjugated goat anti–rabbit IgG (1:500; Jackson ImmunoResearch) for 2 hr at room temperature. After several rinses with 0.1 M KPBS, tissues were mounted with Citifluor (Citifluor, London, UK) on slides and coverslipped. Control tissues incubated in the absence of primary or secondary antibody were also processed and evaluated for specificity or background staining levels. In the absence of primary antibody, no positive immunostaining was observed.

Hematoxylin and Eosin Staining of Urinary Bladder

Sections of urinary bladder (25 µm) from VIP−/− and WT mice were stained with hematoxylin/eosin (H/E). Urinary bladders were harvested from animals euthanized as described but not subjected to any other protocol. Bladders were blotted dry and the weights recorded. Subsequently, the tissue was postfixed in 4% paraformaldehyde, placed in ascending concentrations of sucrose (10–30%) in 0.1MPBS for cryoprotection, sectioned and counterstained using the Mayer’s technique for hematoxylin and eosin (Bancroft and Stevens, 1990). Bladder sections (1:2 series) were thaw-mounted on gelled (0.5%) microscope slides.

Assessment of positive staining in lumbosacral DRG

Staining observed in experimental tissue was compared with that observed from experiment-matched negative controls. Tissues exhibiting immunoreactivity that was greater than the background level observed in experiment-matched negative controls were considered positively stained.

Data analysis of pCREB and FB-labeled DRG

Tissues were examined under an Olympus fluorescence photomicroscope for visualization of Cy3 and FB. Cy3 was visualized with a filter with an excitation range of 560–596 nm and an emission range of 610–655 nm. In DRG from control and CYP-treated rats, pCREB–immunoreactive (IR) cell profiles were counted in 5–8 sections of each selected DRG (L1, L2, L5–S1). Only cell profiles with a nucleus were quantified and only cells with nuclear expression of pCREB-IR were quantified. DRG sections with FB-labeled cells were viewed with a filter with an excitation wavelength of 340–380 nm and an emission wavelength of 420 nm. Cells colabeled with FB and pCREB –IR were similarly counted. Numbers of pCREB-IR cell profiles per DRG section are presented (mean ± SEM). The percentage of presumptive bladder afferent cells (FB-labeled) expressing pCREB–IR in each DRG examined is also presented (mean ± SEM). The results are not corrected for double-counting. pCREB-IR and FB-labeled cells were quantified by two individuals. Comparisons between control and CYP-treated groups were made by using analysis of variance (ANOVA). Percentage data were arcsin-transformed to meet the requirements of this statistical test. Animals, processed and analyzed on the same day, were tested as a block in the ANOVA. Thus, day was treated as a blocking effect in the model. Two variables were tested in the analysis: (1) inflammation versus no inflammation (control), and (2) mouse strain (WT versus VIP−/−). When F ratios exceeded the critical value (p ≤ 0.05), the Newman–Keuls test was used for multiple comparisons among means.

Enzyme-linked immunoassays for NGF in urinary bladder

In separate groups of VIP−/− and WT mice with and without CYP-induced cystitis (48 hr), mice were euthanized as described above and the urinary bladder was dissected and weighed. Individual bladders were solubilized in Tissue Protein Extraction Reagent (Pierce Biotechnology, Woburn, MA) with protease inhibitors (Roche Diagnostics GmbH, Germany). As previously described (Vizzard, 2000c; Murray et al., 2004), tissue was homogenized and centrifuged (10,000 rpm for 5 min); supernatants were used for NGF quantification. Total protein was determined (Pierce). The NGF E-max immunoassay system (Promega Corp., Madison, WI) demonstrates very low cross-reactivity with structurally related growth factors. The NGF standard generated a linear standard curve from 7.8–500 pg/ml (R2 = 0.996, p ≤ 0.001). Absorbance values of standards and samples were corrected by subtraction of background value. Samples were diluted to bring the absorbance values onto the linear portion of the standard curve. Curve fitting of standards and evaluation of NGF content of samples was performed using a least squares fit. Comparisons of NGF bladder content were made using ANOVA. When F ratios exceeded the critical value (p ≤ 0.05), the Newman-Keuls test was used to compare means. All values are expressed as mean ± S.E.M.

Detrusor smooth muscle thickness determination

Grayscale images acquired in tiff format were imported into Meta Morph image analysis software (version 4.5r4; Universal Imaging, Downingtown, PA). The opened image was first calibrated for pixel size by applying a previously created calibration file. In VIP−/− and WT mice, 4–5 bladder sections for each mouse (n=5–7) were used for smooth muscle measurements. Using the free hand drawing tool, a tangent was dropped perpendicular to the serosal surface of the bladder section. Measurements were performed using an established calibration. This process was repeated 4–5 times obtaining thickness measurements throughout an individual bladder section. Comparisons of detrusor smooth muscle thickness were made using ANOVA. When F ratios exceeded the critical value (p ≤ 0.05), the Dunnett’s test was used to compare means. All values are expressed as mean ± S.E.M.

Figure Preparation

Digital images were obtained using a CCD camera (MagnaFire SP; Optronics; Optical Analysis Corp., Nashua, NH) and LG-3 frame grabber attached to an Olympus microscope (Optical Analysis Corp.). Exposure times were held constant when acquiring images from VIP−/− and WT mice processed and analyzed on the same day. Images were imported into Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA) where groups of images were assembled and labeled.

Results

Basal p-CREB immunoreactivity in lumbosacral DRG is increased in VIP−/− mice

Basal expression of p-CREB-IR in lumbosacral DRG in VIP−/− mice was significantly (p ≤ 0.01) greater than that observed in DRG from WT mice (Fig.1 A, C). Significantly increased basal pCREB expression in VIP−/− mice was observed in all lumbosacral DRG (L1, L2, L5-S1) examined (Fig. 2). There was no difference in the number of p-CREB-IR DRG cells observed among different DRG levels (range 23.2–28.4 cells/section) in VIP−/− mice (Fig. 3). Numbers of pCREB-IR cells in WT mice under basal condition were similar among all DRG levels examined (range 10.3–15.4 cells/section) (Fig. 3).

Figure 1
p-CREB-IR in S1 dorsal root ganglia (DRG) from wildtype (WT; A, B) or VIP−/− (C, D) mice
Figure 2
Expression of pCREB-IR in lumbosacral DRG with and without CYP treatment in VIP−/− and wildtype (WT) mice
Figure 3
p-CREB-IR in L6 dorsal root ganglia (DRG) from wildtype (WT; A, B) or VIP−/− (C, D) mice

Increased p-CREB immunoreactivity in lumbosacral DRG in WT and VIP−/− mice with CYP-induced cystitis

Acute CYP-induced cystitis resulted in a significant (p ≤ 0.01) increase in p-CREB-IR in DRG levels (L1, L2, L5-S1) examined in WT mice (Fig. 1Fig. 3). The magnitude of change in pCREB expression in WT mice with CYP-induced cystitis was greatest in the L2 (2.5-fold), L6 (2.1-fold) and S1 (3.7-fold) DRG (Fig. 2, ,3).3). In contrast, CYP-induced cystitis significantly increased pCREB-IR only in L6 and S1 DRG in VIP −/− mice (Fig. 1Fig. 3). The magnitude of change in pCREB expression in VIP−/− mice with CYP-induced cystitis was less than that (L6, 1.9-fold; S1, 1.8-fold) observed for WT mice (Fig. 2). No changes in pCREB-IR in L1, L2 or L5 DRG were observed in VIP−/− mice with CYP-induced cystitis (Fig. 2).

p-CREB immunoreactivity in bladder afferent cells in the DRG

To determine if pCREB-IR was present in bladder afferent cells in the lumbosacral DRG under basal conditions or after CYP-induced cystitis, bladder afferent cells were identified by retrograde transport of Fastblue after injection into the detrusor smooth muscle (Fig. 4).

Figure 4
Bladder afferent cells (Fastblue, FB-labeled) exhibit p-CREB-IR in WT and VIP−/− mice under basal conditions and after CYP-induced cystitis

Basal expression

Under basal conditions in WT mice, p-CREB-IR was observed in 20–38% of FB labelled bladder afferents in L1, L2, L6 and S1 DRG (Fig. 4C). A significantly (p ≤ 0.05) greater percentage of bladder afferents cells exhibiting p-CREB-IR in L6, S1 DRG compared to L1, L2 DRG was observed in WT mice (Fig. 4B, C). In VIP −/− mice, 12–58% of bladder afferent cells expressed p-CREB-IR with L6-S1 DRG exhibiting a significantly (p ≤ 0.05) greater percentage than that observed in L1-L2 DRG (Fig. 4C). Under basal conditions, the percentage of bladder afferent cells exhibiting pCREB-IR in L6, S1 DRG in VIP−/− mice was significantly (p ≤ 0.01) greater than that observed in WT L6, S1 DRG (Fig. 4C).

CYP-induced cystitis

CYP treatment in WT or VIP−/− mice did not affect the percentage of bladder afferent cells exhibiting pCREB-IR in the L1 or L2 DRG (Fig. 4C). In contrast, acute CYP-treatment in both WT and VIP−/− mice significantly increased the percentage of bladder afferent cells expressing pCREB-IR in L6 and S1 DRG (Fig. 4A–C). The magnitude of change in the L6 DRG induced by CYP treatment was similar in WT (1.8-fold) and VIP−/− (1.4-fold) mice (Fig. 4C). In contrast, in the S1 DRG, the magnitude of change in WT mice (2-fold) was greater than that observed in VIP−/− mice (1.3-fold)(Fig. 4C). Not all p-CREB-IR cells in the DRG were bladder afferent cells (Fig. 4A, B).

NGF Bladder Content in VIP−/− and WT mice with or without CYP-induced cystitis

Basal expression of NGF in whole urinary bladder was significantly (p ≤ 0.001) greater in VIP−/− mice compared to WT (Fig. 5). Both VIP−/− (2.3-fold increase) and WT (1.94-fold increase) mice exhibited a significant (p ≤ 0.001) increase in NGF bladder content with CYP-induced cystitis (48 hr)(Fig. 5); however, the magnitude of change (2.1-fold) observed was significantly greater in VIP−/− mice (Fig. 5).

Figure 5
Nerve growth factor (NGF) bladder content is significantly increased in VIP−/− mice under basal conditions and after induction of CYP-induced cystitis (48 hr) compared to wildtype (WT) mice

Increased detrusor smooth muscle thickness in VIP−/− mice

In VIP−/− mice, the urinary bladder/body mass ratio (Fig. 6A) and the thickness of the detrusor smooth muscle (Fig. 6B–D) were significantly (p ≤ 0.01) increased compared to littermate WT mice.

Figure 6
Increased bladder mass and detrusor muscle thickness in VIP−/− mice

Discussion

These studies demonstrate several novel findings with respect to pCREB expression in lumbosacral DRG and specifically in bladder afferent pathways and NGF expression in the urinary bladder of VIP−/− mice. First, pCREB expression in lumbosacral DRG was greater in VIP−/− mice under basal conditions and after bladder inflammation induced by cyclophosphamide (CYP). However, the magnitude of change in pCREB expression induced by CYP-induced cystitis was greater in WT mice. A similar finding was demonstrated when bladder afferent cells labeled with the retrograde tracing agent, Fastblue, were examined for pCREB expression in VIP−/− and WT mice with and without cystitis. To begin to understand what factors may contribute to altered pCREB expression in VIP−/− mice, we examined NGF expression in the urinary bladder because previous studies (Vizzard, 2000c; Murray et al., 2004) have implicated NGF in contributing to micturition reflex plasticity following inflammation. Previously, studies from our laboratory have demonstrated that pCREB expression and not c-jun expression is increased in bladder afferent cells after CYP-induced cystitis in rats (Qiao and Vizzard, 2004). Urinary bladder from VIP−/− mice exhibited greater mass, increased thickness of detrusor smooth muscle and increased NGF content compared to WT mice.

VIP−/− mice exhibit higher basal level of p-CREB-IR in DRG

A significantly higher basal level of p-CREB expression in lumbosacral DRG was observed in VIP−/− mice. This may be accounted for by a number of factors including, changes in (1) urinary bladder activity; (2) urinary bladder afferent activity or (3) expression in bladder-derived neurotrophic factors, including NGF. Greater basal activity in the primary bladder afferents in the DRG or in the target organ itself (i.e., urinary bladder) could result in increased pCREB expression in the absence of inflammation by increasing Ca++ and/or cAMP in VIP−/− mice. Primary afferent cells in the DRG express VIP (Morgan et al., 1999; Hernandez et al., 2006) and VIP receptors (Yashpal et al., 1991; Braas et al., 2006). Thus, absence of VIP in the VIP−/− mice could produce a direct effect on primary afferent cells. However, VIP acting through VIP receptors activates adenylate cyclase (Ishihara et al., 1992), increasing cAMP and inducing phosphorylation of CREB by cAMP dependent protein kinase A (Delgado et al., 1999; Dickinson et al., 1999; Delgado et al., 2000; Mayr and Montminy, 2001). Thus, in VIP−/− mice, one might have expected a decrease in pCREB expression in DRG cells if VIP at the level of the DRG directly mediated changes in pCREB expression. Therefore an effect of VIP deletion on the target organ (i.e., urinary bladder) may be more likely. This may also explain the regional differences in DRG expression of pCREB observed in the present study with DRG levels innervating the urinary bladder being specifically affected.

VIP functions in the lower urinary tract

A number of diverse and conflicting roles for VIP have been demonstrated in urinary bladder from numerous species. VIP has been shown to relax urinary bladder from human (Uckert et al., 2002) or pig (Hernandez et al., 2006), and contract or produce no effects on urinary bladder from the rat (Igawa et al., 1993; Erol et al., 1992). These contradictory findings might be attributable to species differences (Uckert et al., 2002). The majority of VIP in the lower urinary tract is located in postganglionic efferent neurons of the pelvic ganglia (Chapple et al., 1992; Smet et al., 1997; Wanigasekara et al., 2003). Although the functional effects of VIP on the bladder seem diverse, our results may be attributed to absence of VIP-mediated relaxation in VIP−/− mice. Changes in basal tone or contractility of the urinary bladder may increase pCREB expression in the lumbosacral DRG.

VIP−/− mice show increased p-CREB-IR in DRG in response to CYP-induced cystitis but magnitude of change is less than that in WT

CYP-induced cystitis increased pCREB expression in both VIP−/− and WT mice and although the absolute increase in numbers of pCREB-IR cells in DRG or percentages of bladder afferent cells expressing pCREB were greater in VIP−/− mice the magnitude of this change from basal conditions was less than that observed in WT mice. This could reflect saturation at the level of the DRG. In the future, it would be of interest to determine if pCREB is expressed in both A-δ and C-fiber bladder afferents or in only one population of bladder afferent cell. This could account for a maximum percentage of bladder afferent cells expressing pCREB. pCREB expression was also observed to increase in L5 DRG with CYP-induced cystitis. This likely represents viscerosomatic convergence or cross-talk among somatic and autonomic pathways ( Amir and Devor, 1996; Jaggar et al., 1999).

Increased NGF expression in urinary bladder of VIP−/− mice with and without inflammation compared to WT

Both neurotrophins and cytokines may affect pCREB expression (Hawley et al., 1992; Singh et al., 1996; Riccio et al., 1999; Arthur et al., 2004). This possibility has been suggested in studies addressing pCREB involvement with peripheral nerve injury (Gold et al., 1993), somatic (Ji and Rupp, 1997; Messersmith et al., 1998), and visceral inflammation (Qiao and Vizzard, 2004) and sympathetic neuron survival (Riccio et al., 1999). The influence of target organ-neuron interactions in the adult animal has been extensively demonstrated (Steers et al., 1991; Vizzard, 2000c; Qiao and Vizzard, 2002a; Murray et al., 2004; Steers and Tuttle, 2006). Under basal and inflamed conditions, VIP−/− bladders expressed greater NGF content compared to WT bladders. Altered urinary bladder NGF expression in VIP−/− mice may contribute to altered pCREB expression in DRG under both basal and inflamed bladder conditions. Regulation of neuropeptide expression by NGF in DRG has been demonstrated (Verge et al., 1995). NGF suppressed VIP as well as neuropeptide Y, galanin, and cholecystokinin expression in rat DRG. Future studies will determine neuropeptide expression (i.e., PACAP, galanin) in DRG from VIP−/− mice and the potential contribution to altered pCREB expression in DRG.

Hypertrophy of the urinary bladder in VIP−/− mice

Hypertrophy of the urinary bladder smooth muscle is associated with increased NGF expression. Partial urethral obstruction, spinal cord injury and CYP-induced cystitis are associated with increases in bladder mass, altered expression of urinary bladder NGF (Steers and de Groat, 1988; Steers et al., 1991; Tuttle et al., 1994; Steers et al., 1996; Vizzard, 2000c) and the NGF receptor, TrkA (Qiao and Vizzard, 2002b; Qiao and Vizzard, 2004). In the present study, VIP−/− mice exhibited increased basal and inflammation-induced expression of NGF, increased bladder/body weight ratio and increased detrusor muscle thickness under basal conditions. A recent study (Lelievre et al., 2007) of intestinal morphology and function in VIP−/− mice has also demonstrated a significant increase in intestinal smooth muscle thickness. A regulatory role for VIP in intestinal smooth muscle proliferation has been suggested (Lelievre et al., 2007) given the ability of VIP to inhibit airway smooth muscle proliferation (Maruno et al., 1995). A similar regulatory role for VIP in detrusor smooth muscle may be possible.

Anti-inflammatory role of VIP in bladder reflex pathways

In addition to roles as neurotransmitter or neuromodulator in autonomic nervous system pathways, VIP exhibits considerable anti-inflammatory properties (Said, 1991; Voice et al., 2002; Szema et al., 2006), mediated through VPAC1 receptors on inflammatory cells (Delgado et al., 2000). It has been shown that VIP inhibits the production of pro-inflammatory compounds including, TNFα, iNOS, IL-1 and IL-12 (Karin et al., 1997; Chorny et al., 2006). Conversely, VIP upregulates production of the anti-inflammatory cytokine, IL-10 (Delgado et al., 1999). In diverse experimental models of inflammation, VIP has shown to improve symptoms and survival (Delgado et al., 1999) (Abad et al., 2003; Bik et al., 2004; Newman et al., 2005; Juarranz et al., 2005; Martinez et al., 2005; Gonzalez-Rey and Delgado, 2006). We have recently demonstrated (Vizzard et al., 2007b) that production of pro-inflammatory compounds including IL-1β, IL-6 and monocyte chemoattract protein (MCP-1; CCL2) is increased in urinary bladder of VIP−/− mice treated with CYP compared to WT. Thus, changes in expression of inflammatory compounds as well as NGF expression in VIP−/− urinary bladder may contribute to altered pCREB expression with inflammation. With respect to bladder function in VIP−/− mice, we have recently demonstrated (Vizzard et al., 2007a) increased urinary bladder hyperreflexia (smaller void volumes and increased voiding frequency) in VIP−/− mice treated with CYP.

In summary, these studies demonstrate increased basal expression of pCREB in unidentified lumbosacral DRG cells as well as bladder afferent cells in DRG in VIP−/− mice. Under basal conditions, NGF expression was significantly increased in urinary bladder of VIP−/− mice and VIP−/− exhibited increased bladder/body weight ratio. With inflammation, NGF bladder content further increased in the urinary bladder compared to WT with inflammation. Although bladder inflammation increased the absolute number of pCREB-IR DRG cells in VIP−/− mice, the magnitude of change was less than that observed in WT mice. pCREB expression under basal or inflamed conditions in VIP−/− mice in DRG cells may be related to changes in bladder-derived factors, including NGF.

Acknowledgements

The authors acknowledge the technical support of Susan E. Malley. This work was funded by NIH grants DK051369, DK060481, and DK065989. D.G. Jensen was supported by Drug Research Academy at Copenhagen University and Ferring Pharmaceutical, Copenhagen.

References

  • Abad C, Martinez C, Juarranz MG, et al. Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease. Gastroenterology. 2003;124:961–971. [PubMed]
  • Amir R, Devor M. Chemically mediated cross-excitation in rat dorsal root ganglia. J Neurosci. 1996;16:4733–4741. [PubMed]
  • Arthur JS, Fong AL, Dwyer JM, et al. Mitogen-and stress-activated protein kinase 1 mediates cAMP response element-binding protein phosphorylation and activation by neurotrophins. J Neurosci. 2004;24:4324–4332. [PubMed]
  • Avelino A, Cruz C, Cruz F. Nerve growth factor regulates galanin and c-jun overexpression occurring in dorsal root ganglion cells after intravesical resiniferatoxin application. Brain Res. 2002;951:264–269. [PubMed]
  • Bancroft JD, Stevens A. Theory and Practice of Histological Techniques. New York: Churchill Livingstone; 1990.
  • Bik W, Wolinska-Witort E, Chmielowska M, Baranowska-Bik A, Rusiecka-Kuczalek E, Baranowska B. Vasoactive intestinal peptide can modulate immune and endocrine responses during lipopolysaccharide-induced acute inflammation. Neuroimmunomodulation. 2004;11:358–364. [PubMed]
  • 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–R962. [PMC free article] [PubMed]
  • Chapple CR, Milner P, Moss HE, Burnstock G. Loss of sensory neuropeptides in the obstructed human bladder. Br J Urol. 1992;70:373–381. [PubMed]
  • Chorny A, Gonzalez-Rey E, Varela N, Robledo G, Delgado M. Signaling mechanisms of vasoactive intestinal peptide in inflammatory conditions. Regul Pept. 2006;137:67–74. [PubMed]
  • Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Liu X, Waschek JA. Disrupted circadian rhythms in VIP-and PHI-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2003;285:R939–R949. [PubMed]
  • Cox PJ. Cyclophosphamide cystitis--identification of acrolein as the causative agent. Biochem Pharmacol. 1979;28:2045. [PubMed]
  • Delgado M, Gomariz RP, Martinez C, Abad C, Leceta J. Anti-inflammatory properties of the type 1 and type 2 vasoactive intestinal peptide receptors: role in lethal endotoxic shock. Eur J Immunol. 2000;30:3236–3246. [PubMed]
  • Delgado M, Munoz-Elias EJ, Gomariz RP, Ganea D. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide enhance IL-10 production by murine macrophages: in vitro and in vivo studies. J Immunol. 1999;162:1707–1716. [PubMed]
  • Dickinson T, Mitchell R, Robberecht P, Fleetwood-Walker SM. The role of VIP/PACAP receptor subtypes in spinal somatosensory processing in rats with experimental peripheral mononeuropathy. Neuropharmacology. 1999;38:167–180. [PubMed]
  • Dmitrieva N, Shelton D, Rice ASC, McMahon SB. The role of nerve growth factor in a model of visceral inflammation. Neuroscience. 1997;78:449–459. [PubMed]
  • Donovan MK, Winternitz SR, Wyss JM. An analysis of the sensory innervation of the urinary system of the rat. Brain Res Bull. 1983;11:321–324. [PubMed]
  • Driscoll A, Teichman JMH. How do patients with interstitial cystitis present? J Urol. 2001;166:2118–2120. [PubMed]
  • Erol K, Ulak G, Donmez T, Cingi MI, Alpan RS, Ozdemir M. Effects of vasoactive intestinal polypeptide on isolated rat urinary bladder smooth muscle. Urol Int. 1992;49:151–153. [PubMed]
  • 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;99:499–513. [PubMed]
  • Gold BG, Storm-Dickerson T, Austin DR. Regulation of the transcription factor c-JUN by nerve growth factor in adult sensory neurons. Neurosci Lett. 1993;154:129–133. [PubMed]
  • Gonzalez-Rey E, Delgado M. Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide. Gastroenterology. 2006;131:1799–1811. [PubMed]
  • Harmar AJ, Arimura A, Gozes I, et al. International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacol Rev. 1998;50:265–270. [PubMed]
  • Hawley RJ, Scheibe RJ, Wagner JA. NGF induces the expression of the VGF gene through a cAMP response element. J Neurosci. 1992;12:2573–2581. [PubMed]
  • Hernandez M, Barahona MV, Recio P, et al. Neuronal and smooth muscle receptors involved in the PACAP-and VIP-induced relaxations of the pig urinary bladder neck. Br J Pharmacol. 2006;149:100–109. [PubMed]
  • Ho N, Koziol JA, Parsons CL. Epidemiology of interstitial cystitis. In: Sant Gr., editor. Interstitial Cystitis. Philadelphia: Lippincott-Raven Publishers; 1997. pp. 9–16.
  • Hu VY, Zvara P, Dattilio A, et al. Decrease in bladder overactivity with REN1820 in rats with cyclophosphamide induced cystitis. J Urol. 2005;173:1016–1021. [PubMed]
  • Hunt SP, Pini A, Evans G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature. 1987;328:632–634. [PubMed]
  • Igawa Y, Persson K, Andersson KE, Uvelius B, Mattiasson A. Facilitatory effect of vasoactive intestinal polypeptide on spinal and peripheral micturition reflex pathways in conscious rats with and without detrusor instability. J Urol. 1993;149:884–889. [PubMed]
  • Ishihara T, Shigemoto R, Mori K, Takahashi K, Nagata S. Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron. 1992;8:811–819. [PubMed]
  • Jaggar SI, Scott HC, Rice AS. Inflammation of the rat urinary bladder is associated with a referred thermal hyperalgesia which is nerve growth factor dependent. Br J Anaesth. 1999;83:442–448. [PubMed]
  • Jennings LJ, Vizzard MA. Cyclophosphamide-induced inflammation of the urinary bladder alters electrical properties of small diameter afferent neurons from dorsal root ganglia. FASEB Journal. 1999;13:A57.
  • Ji RR, Brenner GJ, Schmoll R, Baba H, Woolf CJ. Phosphorylation of extracellular signal-regulated protein kinase and cAMP-responsive element binding protein in nociceptive neurons after noxious stimulation. Proc 9th World Congress on Pain. 2000:191–198.
  • Ji RR, Rupp F. Phosphorylation of transcription factor CREB in rat spinal cord after formalin-induced hyperalgesia: relationship to c-fos induction. J. Neurosci. 1997;17:1776–1785. [PubMed]
  • Johansson SL, Ogawa K, Fall M. The pathology of interstitial cystitis. In: Sant Gr., editor. Interstitial Cystitis. Philadelphia: Lippincott-Raven Publishers; 1997. pp. 143–152.
  • Juarranz Y, Abad C, Martinez C, et al. Protective effect of vasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis Res Ther. 2005;7:R1034–R1045. [PMC free article] [PubMed]
  • Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997;9:240–246. [PubMed]
  • Keast JR, de Groat WC. Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol. 1992;319:615–623. [PubMed]
  • Lantéri-Minet M, Bon K, de Pommery J, Michiels JF, Menétrey D. Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involves as revealed by the expression of c-Fos and Krox-24 proteins. Exp Brain Res. 1995;105:220–232. [PubMed]
  • Lasanen LT, Tammela TL, Liesi P, Waris T, Polak JM. The effect of acute distension on vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) immunoreactive nerves in the female rat urinary bladder. Urol Res. 1992;20:259–263. [PubMed]
  • Lelievre V, Favrais G, Abad C, et al. Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: A model for the study of intestinal ileus and Hirschsprung's disease. Peptides. 1997;28:1688–1699. [PMC free article] [PubMed]
  • Maggi CA, Lecci A, Santiciolo P, Del Biance E, Giuliani S. Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents. J Auton Nerv Syst. 1992;38:201–208. [PubMed]
  • Martinez C, Juarranz Y, Abad C, et al. Analysis of the role of the PAC1 receptor in neutrophil recruitment, acute-phase response, and nitric oxide production in septic shock. J Leukoc Biol. 2005;77:729–738. [PubMed]
  • Maruno K, Absood A, Said SI. VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells. Am J Physiol. 1995;268:L1047–L1051. [PubMed]
  • Mayr B, Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol. 2001;2:599–609. [PubMed]
  • Messersmith DJ, Kim DJ, Iadarola MJ. Transcription factor regulation of prodynorphin gene expression following rat hindpaw inflammation. Mol Brain Res. 1998;53:259–269. [PubMed]
  • Morgan CW, Ohara PT, Scott DE. Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat. J Comp Neurol. 1999;407:381–394. [PubMed]
  • Murray E, Malley SE, Qiao LY, Hu VY, Vizzard MA. Cyclophosphamide induced cystitis alters neurotrophin and receptor tyrosine kinase expression in pelvic Ganglia and bladder. J Urol. 2004;172:2434–2439. [PubMed]
  • Nadelhaft I, Vera PL. Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve. J Comp Neurol. 1995;359:443–456. [PubMed]
  • Newman R, Cuan N, Hampartzoumian T, Connor SJ, Lloyd AR, Grimm MC. Vasoactive intestinal peptide impairs leucocyte migration but fails to modify experimental murine colitis. Clin Exp Immunol. 2005;139:411–420. [PubMed]
  • Petrone RL, Agha AH, Roy JB, Hurst RE. Urodynamic findings in patients with interstitial cystitis. J Urol. 1995;153:290A.
  • Qiao LY, Vizzard MA. Cystitis-induced upregulation of tyrosine kinase (TrkA, TrkB) receptor expression and phosphorylation in rat micturition pathways. J Comp Neurol. 2002a;454:200–211. [PubMed]
  • Qiao LY, Vizzard MA. Up-regulation of tyrosine kinase (Trka, Trkb) receptor expression and phosphorylation in lumbosacral dorsal root ganglia after chronic spinal cord (TS-T10) injury. J Comp Neurol. 2002b;449:217–230. [PubMed]
  • Qiao LY, Vizzard MA. Up-regulation of phosphorylated CREB but not c-Jun in bladder afferent neurons in dorsal root ganglia after cystitis. J Comp Neurol. 2004;469:262–274. [PubMed]
  • Riccio A, Ahn S, Davenport CM, Blendy JA, Ginty DD. Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science. 1999;286:2358–2361. [PubMed]
  • Said SI. Vasoactive intestinal polypeptide (VIP) in asthma. Ann N Y Acad Sci. 1991;629:305–318. [PubMed]
  • Sant G, Hanno PM. Interstitial cystitis: current issues and controversies in diagnosis. Urology. 2001;57:82. [PubMed]
  • Singh S, Natarajan K, Aggarwal BB. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a potent inhibitor of nuclear transcription factor-kappa B activation by diverse agents. J Immunol. 1996;157:4412–4420. [PubMed]
  • Smet PJ, Moore KH, Jonavicius J. Distribution and colocalization of calcitonin gene-related peptide, tachykinins, and vasoactive intestinal peptide in normal and idiopathic unstable human urinary bladder. Lab Invest. 1997;77:37–49. [PubMed]
  • Steers WD, Creedon DJ, Tuttle JB. Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol. 1996;155:379–385. [PubMed]
  • Steers WD, de Groat WC. Effect of bladder outlet obstruction on micturition reflex pathways in the rat. J Urol. 1988;140:864–871. [PubMed]
  • Steers WD, Kolbeck S, Creedon D, Tuttle JB. Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest. 1991;88:1709–1715. [PMC free article] [PubMed]
  • Steers WD, Tuttle JB. Mechanisms of Disease: the role of nerve growth factor in the pathophysiology of bladder disorders. Nat Clin Pract Urol. 2006;3:101–110. [PubMed]
  • Szema AM, Hamidi SA, Lyubsky S, et al. Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP. Am J Physiol Lung Cell Mol Physiol. 2006;291:L880–L886. [PubMed]
  • Tuttle JB, Steers WD, Albo M, Nataluk E. Neural input regulates tissue NGF and growth of the adult rat urinary bladder. J Auton Nerv Syst. 1994;49:147–158. [PubMed]
  • Uckert S, Stief CG, Lietz B, Burmester M, Jonas U, Machtens SA. Possible role of bioactive peptides in the regulation of human detrusor smooth muscle-functional effects in vitro and immunohistochemical presence. World J Urol. 2002;20:244–249. [PubMed]
  • Verge VMK, Richardson PM, Wiesenfeld-Hallin Z, Hokfelt T. Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J. Neurosci. 1995;15(3):2081–2096. [PubMed]
  • Vizzard MA. Increased expression of neuronal nitric oxide synthase in bladder afferent and spinal neurons following spinal cord injury. Develop Neurosci. 1997;19:232–246. [PubMed]
  • Vizzard MA. Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol. 2000a;278:R1027–R1039. [PubMed]
  • Vizzard MA. Alterations in spinal Fos protein expression induced by bladder stimulation following cystitis. Am J Physiol Regul Integr Comp Physiol. 2000b;278:R1027–R1039. [PubMed]
  • Vizzard MA. Changes in urinary bladder neurotrophic factor mRNA and NGF protein following urinary bladder dysfunction. Exp Neurol. 2000c;161:273–284. [PubMed]
  • Vizzard MA. Up-regulation of pituitary adenylate cyclase-activating polypeptide in urinary bladder pathways after chronic cystitis. J Comp Neurol. 2000d;420:335–348. [PubMed]
  • Vizzard MA. Alterations in neuropeptide expression in lumbosacral bladder pathways following chronic cystitis. J Chem Neuroanat. 2001;21:125–138. [PubMed]
  • Vizzard MA, Boyle MM. Increased expression of growth-associated protein (GAP-43) in lower urinary tract pathways following cyclophosphamide (CYP)-induced cystitis. Brain Res. 1999;844:174–187. [PubMed]
  • Vizzard MA, Braas KM, Studeny S, et al. Vasoactive intestinal polypeptide knockout (VIP−/−) mice exhibit altered bladder function and somatic sensitivity with cyclophosphamide (CYP)-induced cystitis. J Mol Neurosci. 2007a;33:311.
  • Vizzard MA, Malley S, Braas KM, Waschek JA, May V. Exaggerated cytokine and chemokine expression in vasoactive intestinal polypeptide knockout (VIP−/−) mice with cyclophosphamide (CYP)-induced cystitis. J Mol Neurosci. 2007b;33:338–339.
  • Voice JK, Dorsam G, Chan RC, Grinninger C, Kong Y, Goetzl EJ. Immunoeffector and immunoregulatory activities of vasoactive intestinal peptide. Regul Pept. 2002;109:199–208. [PubMed]
  • Wanigasekara Y, Kepper ME, Keast JR. Immunohistochemical characterisation of pelvic autonomic ganglia in male mice. Cell Tissue Res. 2003;311:175–185. [PubMed]
  • Yashpal K, Sarrieau A, Quirion R. [125I]vasoactive intestinal polypeptide binding sites: quantitative autoradiographic distribution in the rat spinal cord. J Chem Neuroanat. 1991;4:439–446. [PubMed]
  • Yoshimura N, de Groat WC. Increased excitability of afferent neurons innervating rat urinary bladder after chronic bladder inflammation. J Neurosci. 1999;19:4644–4653. [PubMed]
  • Zvara P, Vizzard MA. Exogenous overexpression of nerve growth factor in the urinary bladder produces bladder overactivity and altered micturition circuitry in the lumbosacral spinal cord. BMC Physiol. 2007;7:9. [PMC free article] [PubMed]
  • Zvarova K, Vizzard MA. Changes in galanin-immunoreactivity in rat micturition reflex pathways after cyclophosphamide (CYP)-induced cystitis. Cell Tissue Res. 2006;324:213–224. [PubMed]