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

Animals

Adult female Wistar rats (150–250 g), purchased from Charles River Canada (St. Constant, PQ, Canada), were housed two per cage and maintained in standard laboratory conditions with free access to food and water. All experimental protocols involving animal use were approved by the University of Vermont Institutional Animal Care and Use Committee (IACUC no. 08-085). Animal care was under the supervision of the University of Vermont's Office of Animal Care Management, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress, or distress.

CYP-Induced Cystitis in Female Rats

Rats were anesthetized with isoflurane (2%) and received intraperitoneal injection(s) of CYP (Sigma Aldrich, St. Louis, MO) to produce urinary bladder inflammation. To induce chronic bladder inflammation, CYP was injected (75 mg/kg ip) every third day for 8 days with euthanasia occurring on the 8th day (5, 19, 38). To induce acute bladder inflammation, CYP was injected (150 mg/kg ip) with euthanasia occurring 4 or 48 h after injection (5, 19, 38). Control rats received no treatment. For conscious cystometry studies, rats received CYP, as described with bladder function testing occurring 4 h after injection.

CCL2 and CCR2 Transcript Expression in Urinary Bladder: qRT-PCR

Bladders (n = 5 or 6 rats per group) were harvested, and the urothelium was separated from the detrusor. The bladder was cut open along the midline and pinned to a silicon-coated plate (Sylgard, Dow Corning, Midland, MI), the urothelium was removed with the aid of fine forceps and a dissecting microscope, and all tissues were snap-frozen on dry ice before processing, as previously described. Thus dissected, the urothelium also includes suburothelial structures; the term “urothelium” in this article refers to both urothelial and suburothelial structures. Total RNA was extracted using the STAT-60 total RNA/mRNA isolation reagent (Tel-Test “B”; Friendswood, TX), as previously described (5, 26). One to 2 μg of RNA per sample were used to synthesize cDNA using SuperScript II reverse transcriptase and random hexamer primers with the SuperScript II Preamplification System (Invitrogen, Carlsbad, CA) in a 20-μl final reaction volume. Amplification of cDNA was performed using oligonucleotide primers specific for CCL2, CCR2, or 18S (Table 1).

The qRT-PCR standards for all transcripts were prepared with the amplified CCL2, CCR2, and 18S cDNA products ligated directly into pCR2.1 TOPO vector using the TOPO TA cloning kit (Invitrogen). The nucleotide sequences of the inserts were verified by automated fluorescent dideoxy dye terminator sequencing (Vermont Cancer Center DNA Analysis Facility). To estimate the relative expression of the receptor transcripts, 10-fold serial dilutions of stock plasmids were prepared as quantitative standards. The range of standard concentrations was determined empirically.

Real-time qRT-PCR was performed using SYBR Green I detection (5, 26). cDNA templates, diluted 10-fold to minimize the inhibitory effects of the reverse transcription reaction components, were assayed using HotStart-IT SYBR Green qPCR Master Mix (USB, Cleveland, OH) containing 5 mM MgCl₂, 0.4 mM dATP, dGTP, dCTP, and dTTP, HotStart-IT Taq DNA polymerase, and 300 nM of each primer in a final 25-μl reaction volume. The real-time qRT-PCR was performed on an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA) (5, 26) using the following standard conditions: 1) 94°C for 2 min; and 2) amplification over 40 cycles at 94°C for 15 s and 58–60°C depending on primer set for 30 s. The amplified product from these amplification parameters was subjected to SYBR Green I melting analysis by ramping the temperature of the reaction samples from 60 to 95°C. A single DNA melting profile was observed under these dissociation assay conditions, demonstrating amplification of a single unique product free of primer dimers or other anomalous products.

For data analyses, a standard curve was constructed by amplification of serially diluted plasmids containing the target sequence. Data were analyzed at the termination of each assay using the Sequence Detection Software version 1.3.1 (Applied Biosystems, Norwalk, CT). In standard assays, default baseline settings were selected. The increase in SYBR Green I fluorescence intensity (ΔRn) was plotted as a function of cycle number, and the threshold cycle was determined by the software as the amplification cycle at which the ΔRn first intersects the established baseline. All data are expressed as the relative quantity of the gene of interest normalized to the relative quantity of reference gene (18S). Control samples were set equal to 100%.

Preparation of Tissue Samples for ELISAs

Rats from all experimental groups (control, 4 h, 48 h, and chronic; n = 6–8) were euthanized with isoflurane (4%), a thoracotomy was performed, and the urinary bladder was harvested. Individual bladders were immediately weighed and solubilized in tissue protein extraction agent (T-PER; Roche, Indianapolis, IN), a mild zwitterionic dialyzable detergent in 25 mM bicine, 150 mM sodium chloride (pH 7.6), supplemented with a protease inhibitor mix (16 μg/ml benzamidine, 2 μg/ml leupeptin, 50 μg/ml lima bean trypsin inhibitor, and 2 μg/ml pepstatin A, Sigma-Aldrich), and aliquots were removed for protein assays (5, 79). Tissue was homogenized using a Polytron homogenizer, centrifuged (10,000 rpm for 10 min), and the resulting supernatant was used for CCL2 protein quantification. Total protein was determined using the Coomassie Plus protein assay reagent kit (Thermo Fischer Scientific, Rockford, IL). CCL2 was quantified using standard 96-well ELISA plates (R&D Systems, Minneapolis, MN), according to the manufacturer's recommendations.

ELISAs for CCL2 in Urinary Bladder

Microtiter plates (R&D Systems) were coated with anti-CCL2 antibody. Sample and standard solutions were run in duplicate. Horseradish peroxidase-streptavidin conjugate was used to detect the antibody complex. Tetramethylbenzidine was the substrate, and the enzyme activity was measured by the change in optical density. The standards provided with these protocols generated a linear standard curve from 3.9–250 pg/ml (R² = 0.998, P ≤ 0.0001) for samples. The absorbance values of standards and samples were corrected by the subtraction of the background value (absorbance resulting from nonspecific binding). Samples were not diluted, and no samples fell below the detection limits of the assays.

Immunohistochemical Localization of CCR2 in Cryostat Sections of Urinary Bladder from Control and CYP-Treated Rats

The bladders were rapidly dissected from control and CYP-treated rats (n = 16; 4 each group), placed in 4% paraformaldehyde followed by overnight incubation in 30% sucrose in 0.1 M PBS for cryoprotection. Tissue was frozen in optimal cutting temperature compound, sectioned (20 μm) on a freezing cryostat, and mounted directly onto gelled (0.5%) microscope slides (5, 19). Sections were incubated overnight at room temperature in rabbit anti-CCR2 (1:2,000; Aviva Systems Biology, San Diego, CA) diluted in 1% goat serum and 0.1 M phosphate buffer. After overnight incubation, sections were washed (3 × 10 min) with 0.1 M PBS (pH 7.4). Sections were then incubated with goat anti-rabbit Cy3-conjugated secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature followed by washes (3 × 10 min) with PBS and coverslipping with antifade medium (Citifluor, Fisher Scientific, Pittsburgh, PA). Immunohistochemical controls included incubation of tissue sections with 1% goat serum and 0.1 M phosphate buffer alone (no primary antibody) or rabbit isotype control (1:500; Cell Signaling Technology, Danvers, MA), followed by normal washing and incubation with secondary antibodies to evaluate background staining levels. In the absence of primary antibody or in the presence of the isotype control, no positive immunostaining above background levels was observed. Control tissues that were incubated in the absence of secondary antibody were also processed and evaluated for specificity or background staining levels. In the absence of secondary antibody, no positive immunostaining was observed. Finally, immunoabsorptions with CCR2 peptide (5 μg/ml; Aviva Systems Biology) and antisera in urinary bladder sections resulted in no staining above background (data not shown).

Visualization and Semiquantitative Analyses of CCR2 in Urothelium and Detrusor Smooth Muscle

CCR2-immunoreactivity (IR) in bladder sections was visualized, and images were captured using an Olympus fluorescence photomicroscope (Optical Analysis, Nashua, NH). The filter was set to an excitation range of 560–569 nm and an emission range of 610–655 nm for visualization of Cy3. Images were captured, acquired in tagged image file format (TIFF), and imported into image analysis software (MetaMorph, version 4.5r4; University Imaging, Downingtown, PA) (5, 19). The free-hand drawing tool was used to select the urothelium, and the urothelium was measured in total pixels area (5, 19). To evaluate staining in the detrusor smooth muscle, being careful to avoid nondetrusor structures (e.g., blood vessels, inflammatory cells), a rectangle of fixed dimension (125 × 125 pixels) was placed on the section according to random x and y coordinates. This process was repeated 5 times for each image without overlap (6, 40). A threshold encompassing an intensity range of 100–250 grayscale values was applied to the region of interest in the least brightly stained condition first. The threshold was adjusted for each experimental series using concomitantly processed negative controls as a guide for setting background fluorescence. The same threshold was subsequently used for all images. Immunoreactivity was considered to be positive only when the staining for CCR2 exceeded the established threshold. Percent CCR2-IR above threshold in the total area selected was calculated. For the detrusor smooth muscle, the percent CCR2-IR above threshold was calculated by averaging the data collected from all rectangles placed on the image (6, 40).

Assessment of Immunohistochemical Staining in Urinary Bladder Regions

Immunohistochemistry and subsequent evaluation of CCR2-IR in bladder sections or whole mount preparations were performed on control and experimental tissues simultaneously to reduce the incidence of staining variation that can occur between tissues processed on different days. Staining in experimental tissue was compared with that in experiment-matched negative controls. Urinary bladder sections or whole mounts exhibiting immunoreactivity that was greater than background level in experiment-matched negative controls were considered positively stained.

Immunohistochemical Localization of CCR2 in Suburothelial Nerve Plexus in Urinary Bladder Whole Mounts

The urinary bladder was dissected rapidly and placed into oxygenated (95% O₂ and 5% CO₂) physiological saline solution (119.0 mmol NaCl, 4.7 mmol KCl, 24.0 mmol NaHCO₃, 1.2 mmol KH₂PO₄, 1.2 mmol MgSO₄·7H₂O, 11.0 mmol glucose) (38, 79). Starting at the urethra, a midline incision was made through the bladder, and then it was pinned flat onto a silicon-coated plate (Sylgard, Dow Corning, Midland, MI), maximally stretched, and then fixed in 2% paraformaldehyde + 0.2% picric acid for 1.5 h. After fixation, the urothelium was separated from the detrusor layer using fine-tip forceps, iris scissors, and a dissecting microscope, as previously described (38, 79). Notches were made in the region of the bladder neck to track orientation and assess regional immunoreactivity of the bladder. Urothelium and bladder musculature were processed for CCR2-IR, as described above. In some whole mount preparations processed for CCR2-IR, nerve fibers in the suburothelial nerve plexus were also stained with the pan-neuronal marker protein gene product (PGP9.5, 1: 3,000; AbD Serotec, Raleigh, NC) to determine potential expression of CCR2 in suburothelial nerve fibers and visualized with Cy2-conjugated species-specific secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Visualization of CCR2-IR in Suburothelial Plexus in Bladder Whole Mounts

Whole mount tissues from control and experimental groups (n = 4 for control and CYP treatment groups) were examined using an Olympus fluorescence photomicroscope (Optical Analysis) with a multiband filter set for simultaneous visualization of the Cy3 and Cy2 fluorophores. Cy2 was viewed using a filter with an excitation range of 447–501 nm and an emission range from 510 to 540 nm.

Retrograde Labeling of Bladder Afferent Neurons in Lumbosacral DRG

Five to seven days prior to CYP injection or no treatment, Fast Blue (FB; 4%, wt/vol; Polyol, Gross-Umstadt, Germany) was injected into the bladder to retrogradely label bladder afferent neurons in lumbosacral (L1, L2, L6, S1) DRG in control (n = 5) and 4 h CYP-treated (n = 7) rats. As previously described (18, 39), a total volume of 40 μl divided into six to eight injections was injected into the dorsal surface of the bladder wall with particular care to avoid injections into the bladder lumen, major blood vessels, or overlying fascial layers. At each injection site, the needle was kept in place for several seconds after injection, and the site was washed with saline to minimize contamination of adjacent organs with FB. The 4 h CYP treatment group was selected for analysis of CCR2 expression in FB-labeled (presumptive bladder) afferent neurons and to determine the bladder function effects of pharmacological blockade of CCR2 in cystometry experiments (described below) because CCR2 transcript and protein was significantly increased in whole urinary bladder with acute (4 h) bladder inflammation. Furthermore, longer duration of CYP treatment did not result in additional increases in expression.

Immunohistochemical Localization of CCR2 in Lumbosacral Dorsal Root Ganglia

In control situations and after CYP treatment (4 h) (n = 5–7), animals were deeply anesthetized with isoflurane (3–4%) and then euthanized via thoracotomy. The lumbosacral DRG were quickly removed and postfixed in 4% paraformaldehyde for 12 h. Tissue was then placed overnight in sucrose (30%) in 0.1 M PBS for cryoprotection. DRG were identified on the basis of at least two criteria: 1) the T13 DRG are present after the last rib and 2) the L6 vertebra was the last moveable vertebra followed by the fused sacral vertebrae (18, 39). Another less precise criterion is the observation that the L6 DRG are the smallest ganglia following the largest DRG, L5 (18, 39). DRG (L1, L2, L5-S1) were sectioned parasagittally at a thickness of 20 μm on a cryostat. Some DRG (L1, L2, L6, S1) were specifically chosen for analysis based upon the previously determined segmental representation of urinary bladder circuitry. The L5 DRG were used as internal controls because bladder afferents are not distributed within the L4-L5 DRG that contain only somatic afferents. Tissues from control animals (no CYP treatment) were handled in an identical manner to that described.

CCR2-Immunoreactivity in DRG

DRG sections from both control and 4 h CYP-treated rats were processed for CCR2-immunoreactivity (IR) using an on-slide processing technique using methodology described above with some minor modifications. Tissues from control and experimental animals 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 polyclonal anti-CCR2 antibody (1:1,000; Aviva Systems Biology) 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 h at room temperature. After several rinses with 0.1 M KPBS, tissues were mounted on slides and coverslipped with Citifluor (Citifluor, London, UK). Methodological and procedural controls, as well as immunoabsorptions involving DRG tissues, were performed as described above. In the absence of primary antibody or in the presence of the isotype control, no positive immunostaining that was above background levels was observed. Immunoabsorptions with CCR2 peptide (5 μg/ml; Aviva Systems Biology) and antisera in DRG sections resulted in no staining above background (data not shown). Repeated attempts to localize CCL2-IR in cryostat bladder and DRG sections and in urinary bladder whole mount preparations with several different antibodies and substantial trouble-shooting were not successful. Thus, data are not presented for immunohistochemical localization of CCL2-IR in urinary bladder or DRG.

Data Analysis of CCR2- 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 4 h CYP-treated rats, CCR2-immunoreactive cell profiles were counted in 6–8 sections of each selected DRG (L1, L2, L5-S1) (18, 39). Only cell profiles with a nucleus were quantified in a blinded fashion (18, 39). 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 labeled with FB and expressing CCR2-IR were similarly counted. Numbers of CCR2-immunoreactive cell profiles per DRG section are presented (means ± SE). The percentage of presumptive bladder afferent cells (FB-labeled) expressing CCR2-IR in each DRG examined is also presented (means ± SE). The results are not corrected for double counting.

Intravesical Catheter Implant

A lower midline abdominal incision was performed under general anesthesia with 2–3% isoflurane using aseptic surgical techniques (5, 6, 15, 31, 40). The end of polyethylene tubing (PE-50; Clay Adams, Parsippany, NJ) was flared with heat, inserted into the dome of the bladder, and secured in place with a 6–0 nylon purse-string suture (5, 6, 15, 31, 40). The distal end of the tubing was sealed and tunneled subcutaneously to the back of the neck where it was externalized, out of the animal's reach (5, 6, 15, 31, 40). Rats received buprenorphine (0.05 mg/kg sc) starting at the time of surgery and then every 8–12 h postoperatively for a total of four doses. Animals were maintained for 96 h after surgery before conscious cystometry was initiated to ensure complete recovery and clearance of postoperative analgesics.

Open Voiding, Conscious Cystometry With Continuous Intravesical Infusion of Saline and CCR2 Antagonist

The effects of CCR2 receptor blockade on bladder function in control (no inflammation; n = 6) and CYP-treated rats (4 h; n = 6) were evaluated with intravesical infusion of RS504393 (5 μM; Tocris, Bristol, United Kingdom), a highly specific CCR2 receptor antagonist. Unrestrained and conscious rats were placed in a recording cage over a scale and pan to collect and measure voided urine. To elicit repetitive bladder contractions using conscious cystometry, continuous intravesical infusion of room temperature saline was infused at a constant rate (10 ml/h). At least six reproducible micturition cycles were recorded after an initial stabilization period (15–30 min). Intravesical pressure changes were recorded using a Small Animal Cystometry System (Med Associates, St. Albans, VT) (5, 6, 15, 19, 40). Filling pressure, pressure threshold for voiding, maximal voiding pressure, and intercontraction interval were evaluated. Nonvoiding bladder contractions (NVCs), defined as rhythmic intravesical pressure increases 7 cm H₂O above baseline without the release of fluid from the urethra, were also determined per voiding cycle. Bladder capacity was measured as the amount of saline infused into the bladder at the time when micturition commenced (12, 30).

To evaluate the effects of CCR2 blockade on bladder function, rats were anesthetized (1–2% isoflurane) and saline or RS504393 (5 μM), a highly specific CCR2 receptor antagonist (7, 37, 81), was intravesically infused for 30 min. Prior to intravesical drug infusion, the bladder was manually emptied using the Credé maneuver. Bladders were then infused with ∼300 μl-1 ml (a volume less than bladder capacity to not elicit a bladder contraction and expulsion of instillate) of vehicle (1% DMSO in saline; Sigma-Aldrich), or RS504393 (5 μM), according to prior published studies (5, 6, 15, 40). Rats remained anesthetized (1–2% isoflurane) during infusion (30 min) to subdue the micturition reflex and prevent expulsion of drug from the bladder. To avoid potential variation resulting from circadian rhythms, experiments were conducted at similar times of the day (21). At the conclusion of the study, rats were euthanized as described above. Micturition function before and after vehicle or RS504393 intravesical instillation was evaluated in the same rats (control and 4-h CYP-induced cystitis). A variety of routes of administration and concentrations of RS504393 have been used in diverse applications (7, 37, 81). Thus, the concentration used in the present study was based on published studies (7, 37, 81) but was also determined empirically in pilot studies. In pilot studies, a range of RS504393 concentrations (1, 5, and 10 μM) were evaluated. The lowest concentration evaluated was without effect; 5 μM produced effects described in the current study, and 10 μM RS504393 produced results comparable to but not greater than those observed with 5 μM. Thus, the effects of 5 μM RS504393 on bladder function and somatic sensitivity are presented in this study.

Mechanical Sensitivity Testing

Mechanical sensitivity testing was performed in separate groups (n = 8–10 each; hindpaw, pelvic region) of rats not used for bladder function determination. Referred (secondary) hyperalgesia and tactile allodynia were tested using calibrated von Frey hairs with forces of 0.1–4 g applied to the hindpaw or pelvic region. Rats were tested in individual Plexiglas chambers with a stainless-steel wire grid floor. Rats were acclimated to the chambers for a period of 2 h (15, 57). The von Frey hairs were applied in an up-down method for 1–3 s with an inter-stimulus interval of 15 s. For pelvic region stimulation, stimulation was confined to the lower abdominal area overlying the urinary bladder. Testing of the plantar region of the hindpaw and lower abdominal area was performed by perpendicular application of von Frey hairs to the indicated areas until the hair bent slightly. The following behaviors were considered positive responses to pelvic region stimulation: sharp retraction of the abdomen, jumping, or immediate licking or scratching of the pelvic area (15, 57). A positive response to hindpaw stimulation was sharp withdrawal of the paw or licking of the tested hindpaw (15, 57). Somatic sensitivity before and after vehicle or RS504393 intravesical instillation was evaluated in the same rats (control and 4 h CYP-induced cystitis). All somatic testing was performed in a blinded manner with respect to treatment. The groups were decoded after data analysis.

Exclusion Criteria

Rats were removed from the study when adverse events occurred that included: 20% reduction in body weight postsurgery, a significant postoperative event, lethargy, pain, or distress not relieved by our IACUC-approved regimen of postoperative analgesics or hematuria in control rodents (5, 6, 15, 19, 40). In the present study, no rats were excluded from the study or from analysis due to any of these exclusion criteria. In addition, behavioral movements, such as grooming, standing, walking, and defecation rendered bladder pressure recordings unusable during these events.

Materials

RS504393 was prepared as a stock solution in DMSO, aliquoted and stored at −20°C until use. Aliquots were diluted with saline to achieve final concentration.

Figure Preparation

Digital images were obtained using a charge-coupled device camera (MagnaFire SP, Optronics; Optical Analysis, Nashua, NH) and LG-3 frame grabber attached to an Olympus fluorescence photomicroscope (Optical Analysis). Exposure times were held constant when acquiring images from control and CYP-treated groups processed and analyzed on the same day. Images were imported into a graphics-editing program (Adobe Photoshop, version 8.0, Adobe Systems, San Jose, CA) where groups of images were assembled and labeled.

Statistical Analyses

All values represent means ± SE. Data were compared with ANOVA. Percentage data were arcsine transformed to meet the requirements of this statistical test. Cystometry data were compared using repeated-measures ANOVA, where each animal served as its own control. Animals, processed and analyzed on the same day, were tested as a block in the ANOVA. When F ratios exceeded the critical value (P ≤ 0.05), the Newman-Keul's or Dunnett's post hoc tests were used to compare group means.

CCL2 Protein Expression in the Whole Urinary Bladder with CYP-Induced Cystitis

CCL2 protein expression in the whole urinary bladder increased significantly (P ≤ 0.01) compared with control urinary bladders (no CYP) with all durations of CYP treatment evaluated as determined with ELISAs (Fig. 1). CCL2 expression in the urinary bladder was greatest (9.5-fold) with 4 h of CYP treatment. With 4 h of CYP treatment, CCL2 expression in urinary bladder was significantly (P ≤ 0.01) greater than that with 48 h or chronic CYP treatment. CCL2 protein expression with 48 h of CYP treatment was significantly (P ≤ 0.01) greater than protein expression with chronic CYP treatment (Fig. 1).

CCL2 and CCR2 mRNA Transcript Levels With and Without CYP-Induced Cystitis

Real-time qRT-PCR experiments demonstrated significant increases (P ≤ 0.01) in CCL2 mRNA transcript expression in both the urothelium and detrusor smooth muscle with acute (4 h) and intermediate (48 h) CYP treatment, but not chronic CYP-induced bladder inflammation (Fig. 2A). Increases in CCL2 mRNA levels in urothelium and detrusor were most robust with 4 h of CYP treatment, and these levels were significantly (P ≤ 0.01) higher than those detected with 48 h of CYP treatment (Fig. 2A). CCL2 mRNA expression in urothelium and detrusor returned to control expression with chronic CYP treatment.

CCR2 mRNA transcript expression also significantly (P < 0.01) increased in the urothelium and detrusor with acute (4 h) and intermediate (48 h) CYP treatment. CCR2 mRNA expression increased significantly (P ≤ 0.01) in the urothelium with 4 h of CYP treatment only, whereas CCR2 mRNA levels increased significantly (P ≤ 0.01) in the detrusor with both 4 h and 48 h CYP treatment (Fig. 2B). CCR2 mRNA expression in urothelium and detrusor returned to control levels with chronic CYP treatment.

CCR2- IR in Urinary Bladder With Acute (4 h) CYP-Induced Cystitis

Low expression of CCR2-IR was present in the urothelium of control bladders (no CYP treatment) (Fig. 3, A and C). CCR2-IR increased significantly (P ≤ 0.01) in the urothelium in all layers (basal, intermediate, apical) with 4 h of CYP treatment in both cryostat urinary bladder sections (Fig. 3, B, E, and F) and whole mount preparations (Fig. 3D). Low expression of CCR2-IR was also present in the detrusor smooth muscle of control rats, but expression was not altered with CYP treatment of any duration examined (4 h, 48 h, chronic) (data not shown). CCR2-IR was not observed in whole mount preparations of the suburothelial nerve plexus in control or CYP-treated rats (data not shown).

CCR2-Immunoreactivity (IR) Is Increased in Lumbosacral DRG Cells With 4-h CYP-Induced Cystitis

CCR2 expression was observed in the cytoplasm of DRG cells in all levels examined (L1-L2; L5-S1) and the number of DRG cells exhibiting CCR2-IR was similar (8–15 cells/section) across DRG levels examined (Fig. 4A). The number of cells that expressed CCR2-IR (Fig. 4A) increased (17–45 cells/section) significantly (P ≤ 0.01) in L1, L2, L6, and S1 DRG examined after 4 h CYP. CCR2 expression was significantly (P ≤ 0.01) greater in L6 DRG with 4 h CYP treatment compared with L1 or L2 DRG with 4 h CYP treatment (Fig. 4A). CCR2-IR was observed primarily in small-diameter DRG cells (19.6 ± 2.4 μm), but some larger DRG cells (24.3 ± 3.2 μm) also exhibited CCR2-IR. No changes in CCR2-IR were observed in L5 DRG (Fig. 4A).

CCR2-IR in Fast Blue-Labeled Bladder Afferent Cells

To determine whether CCR2-IR was expressed in bladder afferent cells in DRG from control rats or from those treated with CYP (4 h), FB was injected into the urinary bladder to retrogradely label bladder afferent cells in the L1, L2, L6, and S1 DRG (Fig. 4, C and D). In control animals (Fig. 4B), 25.2–27.4% of bladder afferent cells expressed CCR2-IR in L1, L2, L6 and S1 DRG. After 4 h CYP (Fig. 4, B–D), there was a significant (P ≤ 0.01) increase in the percentage of bladder afferent cells that expressed CCR2-IR in L1 (70.2% ± 4.0) (Fig. 4C), L2 (69.2% ± 3.9), L6 (78.3% ± 12.2) (Fig. 4D), and S1 (75.3% ± 7.8) DRG. No differences were observed in the number of CCR2-IR cells in DRG examined between control and control with FB injection or between CYP-treated and CYP-treated with FB injection (data not shown).

CCR2 Receptor Blockade With Intravesical Infusion of RS504393 Using Conscious Cystometry in Rats With or Without CYP-Induced Cystitis (4 h)

Hindpaw and Pelvic Somatic Sensitivity With CYP-Induced Cystitis and Effects of CCR2 Antagonist, RS504393

Somatic sensitivity in the hindpaw was significantly (P ≤ 0.01) increased with the monofilament forces tested (0.1 to 4 g) following 4 h of CYP treatment compared with control rats (Fig. 7A), as previously described (15). Similarly, pelvic somatic sensitivity was also significantly (P ≤ 0.01) increased following 4-h CYP treatment at all monofilament forces evaluated (0.1 to 4 g) (Fig. 7B). Intravesical infusion of RS504393 (5 μM) significantly (P ≤ 0.01) decreased the somatic sensitivity in the hindpaw (Fig. 7A) and pelvic region (Fig. 7B) in rats with CYP-induced cystitis (4 h). Intravesical infusion of RS504393 in control rats (no CYP treatment) produced no change in somatic sensitivity in the hindpaw or pelvic region (data not shown).