Transgenic Mice Expressing MCP-1 by the Urothelium Demonstrate Bladder Hypersensitivity, Pelvic Pain and Voiding Dysfunction: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network Animal Model Study

<div><p>Monocyte chemoattractant protein-1 (MCP-1) is one of the key chemokines that play important roles in diverse inflammatory and chronic pain conditions. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction. To facilitate IC/BPS research, we used transgenic technology to develop a novel urothelial MCP-1 secretion mouse model (URO-MCP-1). A transgene consisting of the uroplakin II gene promoter and the mouse MCP-1 coding sequence with a secretory element was constructed and microinjected. URO-MCP-1 mice were found to express MCP-1 mRNA in the bladder epithelium and MCP-1 protein in the urine, and developed bladder inflammation 24 hours after intravesical administration of a single sub-noxious dose of lipopolysaccharide (LPS). The inflamed bladders of URO-MCP-1 mice exhibited elevated mRNAs for interleukin (IL)-1ß, IL-6, substance P precursor, and nerve growth factor as well as increased macrophage infiltration. In parallel with these phenotypic changes, URO-MCP-1 mice manifested significant functional changes at days 1 and 3 after cystitis induction. These functional changes included pelvic pain as measured by von Frey filament stimulation and voiding dysfunction (increased urinary frequency, reduced average volume voided per micturition, and reduced maximum volume voided per micturition) as measured by micturition cages. Micturition changes remained evident at day 7 after cystitis induction, although these changes were not statistically significant. Control wild-type C57BL/6 mice manifested no clear changes in histological, biochemical and behavioral features after similar cystitis induction with LPS. Taken together, our results indicate that URO-MCP-1 mice are hypersensitive to bladder irritants such as LPS and develop pelvic pain and voiding dysfunction upon cystitis induction, providing a novel model for IC/BPS research.</p></div

Evidence for the Role of Mast Cells in Cystitis-Associated Lower Urinary Tract Dysfunction: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network Animal Model Study

<div><p>Bladder inflammation frequently causes cystitis pain and lower urinary tract dysfunction (LUTD) such as urinary frequency and urgency. Although mast cells have been identified to play a critical role in bladder inflammation and pain, the role of mast cells in cystitis-associated LUTD has not been demonstrated. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and LUTD. In this study we investigated the role of mast cells in LUTD using a transgenic autoimmune cystitis model (URO-OVA) that reproduces many clinical correlates of IC/BPS. URO-OVA mice express the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the urothelium and develop bladder inflammation upon introduction of OVA-specific T cells. To investigate the role of mast cells, we crossed URO-OVA mice with mast cell-deficient <i>Kit</i>^<i>W-sh</i> mice to generate URO-OVA/<i>Kit</i>^<i>W-sh</i> mice that retained urothelial OVA expression but lacked endogenous mast cells. We compared URO-OVA mice with URO-OVA/<i>Kit</i>^<i>W-sh</i> mice with and without mast cell reconstitution in response to cystitis induction. URO-OVA mice developed profound bladder inflammation with increased mast cell counts and LUTD, including increased total number of voids, decreased mean volume voided per micturition, and decreased maximum volume voided per micturition, after cystitis induction. In contrast, similarly cystitis-induced URO-OVA/<i>Kit</i>^<i>W-sh</i> mice developed reduced bladder inflammation with no mast cells and LUTD detected. However, after mast cell reconstitution URO-OVA/<i>Kit</i>^<i>W-sh</i> mice restored the ability to develop bladder inflammation and LUTD following cystitis induction. We further treated URO-OVA mice with cromolyn, a mast cell membrane stabilizer, and found that cromolyn treatment reversed bladder inflammation and LUTD in the animal model. Our results provide direct evidence for the role of mast cells in cystitis-associated LUTD, supporting the use of mast cell inhibitors for treatment of certain forms of IC/BPS.</p></div

Mast cell-deficient URO-OVA/<i>Kit</i>^<i>W-sh</i> mice exhibited no significant changes in voiding habits after cystitis induction.

<p>At day 6 after cystitis induction URO-OVA mice (B), URO-OVA/<i>Kit</i>^<i>W-sh</i> mice (D), and mast cell-reconstituted URO-OVA/<i>Kit</i>^<i>W-sh</i> mice (F) were placed in micturition cages for 24-hour micturition recording (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168772#pone.0168772.t002" target="_blank">Table 2</a>). The baseline voiding habits of URO-OVA (A), URO-OVA/<i>Kit</i>^<i>W-sh</i> (C), and mast cell-reconstituted URO-OVA/<i>Kit</i>^<i>W-sh</i> mice (E) were included for comparison. Data are shown as the amount (gram) of urine collected in 2-minite intervals during the 24-hour period. The results are representative of 5 mice for each of the three groups. The dark period is indicated by red lines.</p

Cromolyn treatment reverses voiding dysfunction in URO-OVA mice after cystitis induction.

<p>Cromolyn treatment reverses voiding dysfunction in URO-OVA mice after cystitis induction.</p

Bladder inflammation is associated with pelvic pain in URO-MCP-1 mice.

<p>(A) Both wild-type C57BL/6 (<i>top panels</i>) and URO-MCP-1 mice (<i>bottom panels</i>) were treated intravesically with 100 μl PBS or 1 μg of LPS in 100 μl PBS and evaluated for pelvic response to von Frey filament stimulation at 1, 3 and 7 days after intravesical treatment. The baseline pelvic responses were included for comparison. Data are shown as mean ± SEM percent of response frequency. <i>*p</i><0.05 as compared to baselines. The results are representative of 8 mice in each group. (B) The same C57BL/6 and URO-MCP-1 mice exhibited no significant changes in tactile sensitivity (50% threshold) of the plantar region of the hind paw at 1, 3 and 7 days after intravesical PBS or LPS treatment as compared to baselines.</p

Cromolyn treatment reverses bladder inflammation in URO-OVA mice after cystitis induction.

<p>Mice were treated with saline or cromolyn daily beginning one day before cystitis induction up to day 13. The bladders were collected at day 14 and processed for histological H&E staining (A), flow cytometric analysis of bladder infiltrating CD8⁺ T cells and CD8⁺Vα2⁺ T cells (B), and RT-PCR analysis of mRNAs for inflammatory factors IFN-γ, IL-6, TNF-α, NGF and substance P precursor (pre-SP) (C). GAPDH was used as an internal control. M, a 100 bp DNA marker. *<i>p</i><0.05. The images are representative of 8 bladders per group.</p

Changes in voiding habits after cystitis induction.

<p>Changes in voiding habits after cystitis induction.</p

Changes in voiding habits after a single sub-noxious dose of intravesical LPS in URO-MCP-1 mice.

<p>Changes in voiding habits after a single sub-noxious dose of intravesical LPS in URO-MCP-1 mice.</p

Transgenic MCP-1 gene and expression of MCP-1 in URO-MCP-1 mice.

<p>(A) Schematic illustration of the transgenic MCP-1 DNA construct. The mouse MCP-1 coding sequence with a secretory element (0.52Kb) was placed downstream to the UPII gene promoter (UPII Pro; ~3.6 Kb). An intron sequence was placed between the UPII Pro and the MCP-1 coding sequence. The targeting regions of the primer pair used for PCR genotyping and RT-PCR are indicated by arrows. ATG, a start codon; TAG, a stop codon; poly(A), an additional poly(A) site. (B) URO-MCP-1 mice express MCP-1 mRNA in the bladder. RT-PCR was performed on various tissues from an URO-MCP-1 mouse. MCP-1 mRNA product is indicated by a red arrow. GAPDH was used as an internal control. pUPII-MCP-1, a plasmid containing the transgenic MCP-1 DNA sequence (a control for MCP-1 genomic DNA). C57BL/6 bladder, a C57BL/6 mouse bladder (a negative control). (C) URO-MCP-1 mice express MCP-1 mRNA in the bladder epithelium. The bladder epithelium from an URO-MCP-1 mouse was processed for RT-PCR. MCP-1 mRNA product is indicated by a red arrow. GAPDH was used as an internal control. pUPII-MCP-1 served as a control for MCP-1 genomic DNA. C57BL/6 bladder epithelium served as a negative control. (D) URO-MCP-1 mice express MCP-1 protein in the urine. Urine was collected from both C57BL/6 (n = 5) and URO-MCP-1 mice (n = 12) and analyzed for MCP-1 by ELISA. Data are shown as mean ± s.d. <i>*p</i><0.001 as compared to the urine of C57BL/6 mice.</p

Mast cell-deficient URO-OVA/<i>Kit</i>^<i>W-sh</i> mice developed reduced bladder inflammation after cystitis induction.

<p>(A) At day 7 after cystitis induction the bladders of URO-OVA and URO-OVA/<i>Kit</i>^<i>W-sh</i> mice (both with and without mast cell reconstitution) were collected, sectioned, and analyzed by histological H&E staining. The normal bladders of URO-OVA and URO-OVA/<i>Kit</i>^<i>W-sh</i> mice were included for comparison. Magnifications: X40 and X200. The images are representative of 6 bladders per group. (B) The bladder sections were stained with toluidine blue solution and mast cells were counted in 10 consecutive sections for each bladder. *<i>p</i><0.05 and **<i>p</i><0.01. MC, mast cells.</p