Bladder pain syndrome/Interstitial cystitis (BPS/IC) is a chronic disease that causes pelvic pain and lower urinary tract symptoms such as urinary frequency, urgency, and nocturia. Although the etiology of BPS/IC is thought to be multifactorial, increased permeability of the 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 induce afferent sensitization and pain sensation. However, reliable methods to evaluate the permeability of the urothelium are lacking.  Recent studies have shown that a novel magnetic resonance imaging (MRI) method can directly and quantitatively evaluate the bladder wall permeability in rats and BPS/IC humans (ref:1,2). Furthermore, recent clinical studies have reported that psychological stress plays an important role in inducing or worsening BPS/IC symptoms (ref:3). Accordingly, an animal model of psychological stress induced by water avoidance stress (WAS), which exhibits pelvic pain and urinary frequency, has been used to study the BPS/IC pathophysiology.  Previous studies have also shown that nerve growth factor (NGF) that is known to cause sensitization of nociceptive afferent pathways is increased in the bladder of BPS/IC patients and that extracellular matrix volume is increased in WAS rats.  Thus, in this study on WAS rats, we examined the bladder wall permeability by MRI, as well as bladder activity and bladder pain behavior induced after intravesical stimulation with low-dose protamine sulfate (LD-PS) (10mg/mL), concentration of PS which fails to produce significant changes in bladder activity in normal rats, and expressions of NGF, junction molecules and TRP channels in the bladder mucosa of WAS rats.

Female Wistar rats weighing 230-250gm were divided into; (1) the WAS group, which underwent 1-hour WAS for 10 consecutive days and (2) the control group not exposed to WAS.  Rats in the WAS group were placed on a glass platform (8×8cm) in the middle of a plastic container with 90cm in diameter and 50cm in height, which was filled with water to 1cm below the height of the platform. Rats in the control group were put in the same platform and container without water. 
Ten rats (WAS; N=5, Control; N=5) were used for MRI permeability evaluation. Under isoflurane anesthesia, MRI was performed using a 9.4T/30cm Brucker AVANCE III HD scanner with an 86mm transit coil and a 4-channel receive array coil positioned over the bladder. Bladder wall T1 resting time was measured using a variable TR sequence (6 TRs ranging from 400-10000ms) with a 0.7mm slice thickness and 0.1mm in-plane resolution before and after transurethral instillation of 0.3ml of a novel contrast agent mixture containing Gadobutrol and Feraheme (ref:2). T1-weighted imaging was performed using standard turbospin echo sequences at 9.4 Tesla, before and after transurethral instillation of 0.3ml of novel contrast mixture of paramagnetic gadolinium- and supraparamagnetic iron oxide-based contrast agents. In each rat, 10 pixels of the anterior bladder wall were evaluated. The T1 enhancement of the urothelium and lamina propria layers was calculated as follows: T1(enhancement)=[T1(precontrast)–T1(postcontrastI)]/(Tprecontrast).
Additional rats were divided into 4 groups; control (N=5), control + LD-PS (N=5), WAS (N=5), and WAS + LD-PS (N=5).  All groups underwent awake cystometry using the method as follows: a PE50 tube was inserted into the bladder through the dome under isoflurane anesthesia.  After the recovery from anesthesia, rats in the groups with LD-PS underwent cystometry after 1 hour of intravesical infusion of LD-PS whereas rats in the groups without LD-PS received saline infusion. After 1 hour of LD-PS or saline infusion, the bladder was emptied, and the catheter was connected via a three-way stopcock to a pressure transducer and to a syringe pump. Physiological saline was then continuously infused into the bladder at a rate of 0.04mL/min. Cytometric parameters such as residual volume, voiding efficiency, basal pressure, micturition pressure, inter-contraction intervals were examined.
A separate group of WAS and control rats (N=5 each) were evaluated with bladder pain behavior after intravesical infusion of LD-PS.  A PE10 catheter was transurethrally inserted into the bladder to drain urine. After 300μL of LD-PS was intra-vesically infused and kept for 1 min, the rats were placed into metabolic cages. Thereafter, the number of licking and freezing, which respectively represent urethral and bladder-related pain behaviors, was counted for a 15min period.
After the experiments, bladder tissues were harvested for RT-PCR and ELISA analyses. Bladder sections were also evaluated histologically to see the degree of damage caused by LD-PS.

Post-contrast MRI of the WAS rat bladder wall showed deeper penetration of contrast enhancement (Fig 1-b) compared to control rats. Post-contrast T1 values of WAS rats were also significantly increased with respect to precontrast values (Fig 1-c). Evaluation with cystometry showed that control rats did not show the changes in inter-contraction intervals (ICI) whereas WAS rats showed a significant decrease of ICI after LD-PS infusion (Fig 2-a) although other cystometric parameters were not different between two groups.  WAS rats also showed a significant increase in PS-induced bladder pain behaviors compared to controls (Fig 2-b). In the bladder mucosa of WAS rats, NGF protein and TRPV1/TRPA1 mRNA levels were increased whereas mRNA levels of junction molecules (Cx43, ZO-1) were decreased compared to controls (Fig 2-c).  Histological evaluation showed the bladder epithelial damage with LD-PS in WAS rats, but not in control rats.

These results indicate that psychological stress induced by WAS increases bladder permeability as detected by MRI and enhances bladder pain sensation evident as shortened ICI and bladder pain behaviors and that increased bladder permeability and bladder hypersensitivity in WAS rats are associated with increased expression of NGF and pain-related receptors such as TRPV1/TRPA1 as well as decreases of gap junction molecules.

The new contrast-enhanced MRI method is useful to quantitatively detect changes in bladder permeability following WAS-induced psychological stress.  Furthermore, in the psychological stress condition, NGF upregulation and junction molecule downregulation in the bladder mucosa under psychological stress may contribute to the urothelial damage induced by bladder insults, leading to enhanced bladder pain sensation and bladder overactivity in BPS/IC.

Tyagi P, et al. Novel contrast mixture achieves contrast resolution of human bladder wall suitable for T1 mapping: applications in interstitial cystitis and beyond. Int Urol Nephrol (2018);50:401-409.
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Lutgendorf SK, et al. Stress and symptomatology in patients with interstitial cystitis: a laboratory stress model. J.Urol (2000);164:1265-1269.