Interstitial cystitis / bladder pain syndrome (IC/BPS) affects 3.5 to 8.6 million women in the United States. The clinical hallmark of this disease is a chronic painful bladder which is typically worse with bladder filling, has pain poorly relieved with bladder emptying, and marked urinary urgency / frequency. There is considerable overlap between IC/BPS, and the overactive urinary urgency/frequency of overactive bladder (OAB). The underlying pathophysiology of BPS is unknown but may include chronic inflammation, autoimmune dysregulation, bacterial cystitis, urothelial dysfunction, deficiency of the glycosaminoglycan barrier, and urine cytotoxicity. Women with BPS often report a herald noxious event which results in the initial onset of painful bladder, such as cystitis, pelvic surgery, or sexual trauma. The overarching aim of our research was to evaluate an acute intravesical exposure in a pilot animal model using cystometry, organ bath myography and histology for 6 common environmental exposures and one novel agent implicated in mast cell stabilization. For this phase of our pilot study, we sought to characterize the cystometric dose response threshold in rats, for six common environmental exposures associated with overactive and painful bladder dysfunction in humans with IC/BPS.

Following institutional animal protocol approval, seven female adult wild type Sprague Dawley rats (Charles River Laboratories) underwent urethane anesthetized bladder cystometry (CMG). CMG under anesthesia was performed using solutions implicated in overactive and painful bladder dysfunction. Common environmental exposures were assessed, including caffeine (0.0001 to 10 mg/mL), ethanol (0.001 to 30 %), capsaicin (0.001 to 10 uM), citric acid (0.0001 to 100 mg/mL), acetic acid (0.01 to 3 %), saccharin (0.0001 to 1 mg/mL) and one novel agent implicated in mast cell stabilization, compound 48/80 (0.01 to 100 ug/mL). CMG was performed at 50 uL/min infusion rate through PE-50 tubing placed into the dome of the bladder. Bladders were filled at a constant rate until stable cystometric contraction patterns were demonstrated with normal saline (0.9% NaCl). Baseline fills with saline were repeated at least 2 to 7 times in order to demonstrate reproducibility of baseline for each animal, and post void residual (PVR) was checked after each fill cycle. Following saline infusion, the infusion was changed to intravesical dose-escalating solutions of: caffeine, ethanol, capsaicin (the active compound in chili peppers), citric acid, acetic acid, saccharin and compound 48/80 (a mast cell secretagogue). Animals were continuously observed through all CMG cycles, non-voided contractions were annotated, and voided volume was manually measured for each void by weighing each void by the change in weight of an absorbent paper before and after each void. PVR was recorded in uL based on the weight of the residual volume removed from the bladder through the PE-50 tubing at the end of each fill cycle. PVR was used to calculate cystometric capacity and voiding efficiency for each fill cycle. All animals were comfortably anesthetized throughout each CMG cycle and there were no animals which required early euthanasia.

The results from CMG for the intravesical agents with known irritant (Figure 1) and suspected (Figure 2) effects were graphed over time, with intravesical exposure concentration noted on the x-axis over time, with a separate data point noted for each complete CMG fill cycle. Concentrations of zero represent saline infusion. Mean voided volumes are graphed in 1x or 10x scale in uL where indicated. PVR volumes are graphed in uL for all animals. Pre-void bladder volume was calculated for each individual void by adding the voided volume to the PVR at the end of each fill cycle in order to determine beginning bladder capacity for each measured void, with the assumption that PVR was stable throughout each CMG fill cycle. Voiding efficiency for each void was calculated by dividing pre-void bladder volume by each unique void volume, and then a mean voiding efficiency calculated for each fill cycle. In order to normalize data for graphing on a single chart, voiding efficiency (e.g. 0.01 for 1%) for each intravesical exposure are graphed in 1,000x (1k) or 10,000x (10k) scale where indicated. There were no rats and no data points excluded from any of the figures.

With respect to the acids and irritants (Figure 1), intravesical acetic acid in various concentrations has been used to induce overactivity in many animal models of overactive bladder. On the other hand, citric acid, a common component of citric and acidic juices, is implicated as a known bladder irritant which exacerbates the painful symptoms of IC/BPS. Following exposure to acetic acid, there were stable reductions in voided volume which were 10 fold lower than that observed following exposure to citric acid. Acetic acid resulted in dose-wise reductions in PVR, with a pronounced acute exposure threshold beginning at 0.5 to 1% acetic acid, associated with increases in mean voided volume and mean voiding efficiency. Following exposure to citric acid, there was a universal increase in voided volume with marked improvement in voiding efficiency beginning at 0.001 mg/mL, continuing to 0.1 mg/mL. After the 1 mg/mL exposure threshold, there was a sudden decrease in voiding efficiency with a dose response effect which continued to worsen voiding efficiency and raise PVR, and progressively worsened with higher concentrations. Looking at the exposure to caffeine, which is implicated as a bladder irritant in humans with overactive bladder, there was a dose response threshold beginning at the 0.001 to 0.01 mg/mL concentration. With increasing concentrations of caffeine, mean voiding efficiency progressively increased and there was a dose independent loss of functional bladder capacity. Following exposure to capsaicin, there were initial improvements in voiding efficiency which began at the lowest concentration of 0.001 uM, and then beginning at the 1 uM exposure level, there was a progressive worsening in voiding efficiency concurrent with time dependent increase in PVR.

With respect to the artificial sweetener saccharin and ethanol (Figure 2), each of these agents, when administered intravesically, appear to exert a physiologic effect on bladder function. Following exposure to saccharin, beginning at the 0.01 mg/mL threshold, there appears to be a dose dependent increase in voiding efficiency as a result of loss of functional capacity and dose sensitive decrease in PVR. Ethanol has been implicated in humans as inducing diuresis as a result of increased intake volume (polydipsia) and subsequent increased urine output (polyuria), which may manifest as overactive bladder during the acute phase, or as urinary retention in the state of inebriation. Following intravesical exposure to ethanol in the rat, we found a slow loss in voiding efficiency which appeared to be dose dependent for intravesical concentrations higher than 0.1 % ethanol, meanwhile PVR was relatively stable throughout ethanol exposure. Following exposure to intravesical compound 48/80, a mast cell secretagogue implicated in mast cell destabilization, we found no obvious physiologic effect on cystometry up to 100 ug/mL in wild type rats.

Cystometric effect was demonstrated by all of the intravesical agents except compound 48/80. Regarding the acids, more consistent irritative effects were noted following exposure to citric acid. More compliant bladder filling and lower end fill pressures were noted after exposure to caffeine, ethanol and capsaicin, which appeared to have effects on both the bladder and the outlet.

Further research is needed to understand the complex dose response mechanism by which intravesical exposures contribute to overactive and painful bladder dysfunction in humans with IC/BPS.