Pelvic malignancy accounts for a third of new cancer cases and up to half receive radiation. Little is known about the time course of chronic radiation induced bladder dysfunction in rats. Our aim was to evaluate changes in ambulatory voiding function and bladder cystometry during the chronic phase of radiation induced bladder dysfunction, with at least 50 days of recovery following acute bladder irradiation.

Thirty-six adult female Sprague Dawley rats were divided into 3 groups. After institutional animal protocol approval, bladders were identified by computed tomography and irradiated on day 0 [0 Gy (n=7); 20 Gy (n=25); 30 Gy (n=4)] using image-guided external beam radiotherapy (PXi X-Rad SmART). Nocturnal ambulatory (overnight, 12-hours per cage cycle) micturition frequency and voided volume were recorded using a 12-channel 100-gram load cell array (sensitivity 50 uL per void) and metabolic cages at baseline (day 0) and weekly following radiation (up to 4 months). Void frequency and volume were recorded in 24 hour intervals to allow animals to habituate to each cage cycle. Bladders were then assessed by urethane anesthetized cystometry and H&E histology. Cystometric voiding efficiency (VE = void volume / capacity) was calculated as the percent void volume as a proportion of bladder capacity. Data were analyzed in SAS (Cary, NC, USA) using t-test, Pearson correlation, Spearman correlation, and mixed effects models to evaluate animals over time.

There were 6,078 ambulatory voids, representing 362 cage cycles and up to 16 time points. On Spearman analysis (0, 20, 30 Gy; n=4/group), void volume correlated with food intake (r=-0.41, p<0.01), rat weight (r=0.27, p<0.01) and stool output (r=-0.58, p<0.01). Meanwhile overnight urine output (r=0.05, p=0.54) and water intake (r=0.03, p=0.74) were independent of void volume. Using a mixed effect model to evaluate within and between group differences over time, there was a significant decrease in mean void volume after 20 Gy (p=0.014). High morbidity was associated with sticky stool. Recovery of void volume was noted after 20 Gy only (3 months).

When comparing our larger cohort of rats who received either 0 or 20 Gy, there were three 20 Gy rats who died of radiation proctitis prior to bladder cystometry. On cystometry there was a heterogeneous response to radiation noted [0 Gy (n=7) versus 20 Gy (n=22)]. Radiated bladders (20 Gy) on a whole tended to be smaller capacity (p=0.095), had higher threshold (p=0.925) and peak pressures (p=0.888), similar contraction amplitudes (p=0.768), and wide variability in post void residual (p=0.063). There were clear differences in void volume when expressed as a percent of bladder capacity, as seen in Figure 1 (p<0.001). Pearson correlation results on cystometry [0 Gy (n=7) versus 20 Gy (n=22)] were notable for low threshold pressure associated with larger void volume (p=0.023) and higher amplitude contraction (p<0.001). Whereas small volume voids were associated with elevated post void residual (p=0.001).

Radiation (20 Gy, Day 50+) induced two distinct phenotypes at a threshold of 10% voiding efficiency, as seen in Figure 2 [VE <10% (n=8) vs. VE >10% (n=9)]. Small capacity end-stage bladders (>10% VE) demonstrated lower threshold pressure (<20 cmH2O, p=0.025), higher amplitude (>20 cmH2O, p=0.009) contractions, consistent with the overactive end-stage phase of bladder dysfunction. Underactive (<10% VE) bladders had high threshold pressures (>20 cmH2O), weak amplitude (<10 cmH2O) and elevated post void residual (>700 uL, p=0.005). On H&E histology, irradiated bladders (20 Gy, Day 50) demonstrates sloughing of the urothelium, smooth muscle atrophy, interstitial expansion and urothelial hemorrhage.

Radiation (20 Gy, Day 50+, n=17) induced two distinct phenotypes on urethane anesthetized cystometry at a threshold of 10% voiding efficiency. A heterogeneous effect of radiation needs to be considered when assessing bladder response to therapy in future animal models of external beam radiation cystitis.

After bladder radiation (20 Gy), acute dysfunction subsided by 1 month and there were two distinct phenotypes of chronic dysfunction noted at 50+ days, a small capacity end-stage overactive bladder and a large capacity underactive bladder. Further investigation is needed to understand the mechanisms of heterogeneity following external beam bladder irradiation.