The pathophysiology of BPS/IC has not been clearly revealed; however, there is histological evidence showing urothelial thinning and denudation including disruption of glycosaminoglycan (GAG) layer, or infiltration of inflammatory cells into the lamina propria in the bladder tissue, especially in the bladder-centric phenotype of BPS/IC such as Hunner-type IC. Thus, increased permeability of the damaged bladder urothelium has been proposed as an important pathophysiological basis of BPS/IC, leading to enhanced penetration of urinary substances into the bladder wall to enhance bladder pain sensitivity. Although various animal models including those with chemical cystitis have been used for studying the bladder-centric etiological factors of BPS/IC, most of them are induced by acute inflammation, and animal models of chronic urothelial dysfunction are lacking. Therefore, this study was designed to establish a longer-lasting animal model of urothelial disruption associated with bladder inflammation using intravesical instillation of a mixture of hyaluronidase, which can degrade the GAG layer, and potassium chloride (KCl), which can directly sensitize afferent fibers following penetration through the disrupted urothelium.

Twelve female 8 weeks old C57BL/6 mice were used and divided into 3 groups; 1) Sham group (N=4), 2) One week group at 1 week after 3-times weekly instillation of hyaluronidase/KCl (N=4) and 3) Three weeks group at 3 weeks after 3-times, weekly instillation of hyaluronidase/KCl (N=4). For weekly intravesical instillation, the 0.5 ml solution containing 5 mg/ml of hyaluronidase and 75 mg/ml of KCl was infused through a 24-gauge angiocatheter and retained in the bladder for 1 hour.  Sham group was treated in the same manner, but using the 0.9% NaCl solution instead of hyaluronidase/KCl solution. Then, at 1 or 3 weeks after the 3rd treatment of hyaluronidase/KCl or NaCl, pelvic pain, bladder activity by cystometry, histology by H&E staining, expression levels of molecules related to urothelial tight junction and inflammatory responses by RT-PCR were evaluated.  Pelvic pain sensation was assessed by applying a von Frey monofilament to the suprapubic region five times with 3 minutes intervals.  Fifty percent (50%) thresholds were measured when the mice showed the responses such as sharp escaping, licking and/or jumping behaviors over 3 of 5 times trials.  For cystometric measurements, a PE50 catheter was inserted into the bladder through the dome under urethane anesthesia. Thereafter, intercontraction intervals (ICI), the number of non-voiding contractions (NVCs), maximum voiding bladder contraction pressure (MCP), intravesical baseline pressure (IVBP), voided volume, residual volume, bladder capacity and voiding efficiency were measured.To estimate the histological changes, H&E staining was performed and compared among three groups. In addition, RT-PCR was conducted to examine changes in mRNA expression levels of ZO-1, a urothelial tight junction molecule, and IL-1β, IL-6, CCL2, VEGFα, which represent inflammatory responses and are reportedly upregulated in the bladders of BPS/IC patients [1].

In pelvic pain assessment, 50% thresholds were decreased significantly in both 1 week (0.014 ± 0.007 g) and 3 weeks groups (0.016 ± 0.016 g) compared with Sham group (0.078 ± 0.028 g) (Fig. 1A). In cytometric analyses, NVCs (number/min) in both 1 week and 3 weeks groups (0.93 ± 0.2 and 0.82 ± 0.05, respectively, vs. 0.21± 0.06) were increased significantly, and voided volume (0.03 ± 0.01 vs. 0.05 ± 0.05 ml) and bladder capacity (0.05 ± 0.01 vs. 0.06 ± 0.01 ml) were significantly decreased in the 3 weeks group compared with Sham group (Fig. 1B). 
In histologic findings, mucosal thinning and denudation in the urothelium were seen in both 1 week and 3 weeks groups. Inflammatory responses were found focally in the bladder wall in association with infiltration of inflammatory cells restricted to the lamina propria. In molecular analyses, mRNA expression of ZO-1 was reduced in both 1 week and 3 weeks groups, and IL-1β, IL-6, CCL2, and VEGFα mRNA levels were up-regulated in both 1 week and 3 weeks groups compared with Sham group (Fig. 2).

Previous acute cystitis models with urothelial damage induced by drugs such as cyclophosphamide often exhibit severe and extensive injury of the bladder urothelium; therefore, these models do not seems to be adequate for studying the chronic disease process of BPS/IC. Our new model of hyaluronidase-induced urothelial damage with KCl-induced afferent sensitization showed persistent tactile allodynia in the pelvic region evident as enhanced sensitivity in the von Frey test, which lasted up to 3 weeks after repeated intravesical instillation.  In addition, the enhanced pain sensitivity in our hyaluronidase/KCl model was associated with the urothelial disruption shown by reduced ZO-1 expression and inflammatory responses evident as inflammatory cell infiltration in the lamina propria and increased cytokine production, similarly found in the bladder of BPS/IC patients [1,2].

Our new model of urothelial disruption and pelvic pain induced by repeated intravesical hyaluronidase and KCl instillation is thought to be adequate as a longer-lasting, chronic model for the study of bladder-centric pathophysiology of BPS/IC.

Jhang JF, Kuo HC. Pathomechanism of Interstitial Cystitis/Bladder Pain Syndrome and Mapping the Heterogeneity of Disease. Int Neurourol J 2016;20 Suppl 2:S95-104.
Kim HJ. Update on the Pathology and Diagnosis of Interstitial Cystitis/Bladder Pain Syndrome: A Review. Int Neurourol J 2016;20(1):13-17.