Modeling of chronic radiation-induced cystitis in mice

Abstract

Purpose: Radiation cystitis (RC), a severe inflammatory bladder condition, develops as a side-effect of pelvic radiation therapy in cancer patients. There are currently no effective therapies to treat RC, in part due to the lack of preclinical model systems. In this study, we developed a mouse model for RC and used a small animal radiation research platform (SARRP) to simulate the targeted delivery of radiation as used with human patients.

Methods and materials: To induce RC, C3H mice received a single radiation dose of 20Gy delivered through two beams. Mice were subjected to weekly micturition measurements to assess changes in urinary frequency. At the end of the study, bladder tissues were processed for histology.

Results: Radiation was well tolerated as no change in weight was observed in the weeks post treatment, and there was no hair loss at the irradiation sites. Starting at 17 weeks post treatment, micturition frequency was significantly higher in irradiated mice versus control animals. Pathological changes include fibrosis, inflammation, urothelial thinning and necrosis. At a site of severe insult, we observed telangiectasia, absence of Uroplakin-3 and E-cadherin relocalization.

Conclusions: We developed a RC model that mimics the human pathology and functional changes. Furthermore, radiation exposure attenuates the urothelial integrity long-term allowing for potential continuous irritability of the bladder wall from exposure to urine. Future studies will focus on the underlying molecular changes associated with this condition and investigate novel treatment strategies.

Figure 1

Small animal radiation research platform setup. (A) Bladder computed tomography imaging in the presence and absence of instilled contrast fluid. (B) Small Animal Radiation Research Platform treatment computed tomography with typical treatment beam arrangement and isodose distribution for 20 Gy. A ventral beam entry was chosen to reduce dose to the long bones of the legs. Two beams of 5 × 5 mm were used to spread dose to skin on entry and exit points. The bladder has been outlined with a black dotted line for ease of visualization.

Figure 2

Delayed increase in micturition frequency after radiation exposure. (A) Average weekly mouse weights throughout the experimental phase were graphed. (B) Timeline of the conducted animal study. (C) Representative images of pH-sensitive paper at the end of a micturition study at 17 weeks after irradiation (IR) exposure. (D) The moving average of weekly measurements of micturition frequency and (E) volume per micturition event over 19 weeks were calculated and plotted. n = 20 per treatment group; *P < .05 (multiple t test); error bars = standard error of the mean.

Figure 3

Irradiation (IR)-induced chronic pathologic damage to the bladder. Bladder tissues were scored by a pathologist for fibrosis, inflammation, urothelial thinning, and ischemic necrosis on a scale from 0 to 4 (0 = absent, 4 = severe; n = 6 per treatment group).

Figure 4

Hematoxylin and eosin staining of normal (A) and irradiated bladder (B-D) tissues are shown with indicated pathological changes. Arrowheads point at sites of hemorrhaging. Black bar, 100 μm.

Figure 5

Accumulation of fibrous tissue in response to radiation treatment. Untreated and irradiated (IR) bladder samples were stained with Masson trichrome stain, thereby giving fibrous tissue a blue color. Bladder tissues were scored for the degree of fibrosis by 2 scientists blinded to the process and classified as normal/mild or moderate/severe fibrosis. n = 8 and n = 7 for non-IR and IR groups, respectively.

Figure 6

Influx of mast cells in areas of severe radiation-induced urothelial damage. Normal and irradiated bladder tissues were stained with Toluidine blue (nuclei = blue; mast cells = purple). Total number of mast cells was counted in two full bladder cross sections per sample. Arrow indicates purple stained mast cell. N=8 and n=7 for non-IR and IR groups respectively; black bar = 200μm.

Figure 7

Irradiation (IR)-induced bladder damage correlates with hypervascularization and loss of bladder integrity. (A) Normal and IR bladder tissue were stained for uroplakin-3. Six images per tissue sample were scored (1-10) blinded by 2 independent scientists for the presence or absence of uroplakin-3. n = 8 and n = 7 for non-IR and IR treatment groups, respectively. (B) Normal tissues and areas of severe radiation-induced damage to the urothelium were stained for blood vessels (CD31) and E-cadherin. Gray bar, 100 μm, black bar, 200 μm; *P < .05 (multiple t test).