Hypothesis / aims of study
Exposure of the lower urinary tract (LUT) to ionizing radiation for the treatment of pelvic tumors can cause the development of radiation cystitis (RC). The acute phase of RC is characterized by irritative symptoms including urinary frequency and mild hematuria. However, up to 10% of patients can develop a severely debilitating form of chronic RC which can include; decreased bladder compliance and voiding function due to extensive fibrosis and severe hematuria . The mechanisms that lead to chronic RC are unclear and the aim of this study was to determine the physiological and metabolic consequences of ionizing radiation on the mouse urinary bladder.
Study design, materials and methods
Primary mouse urothelial cell isolation: Adult female C57BL/6 mice were sacrificed, urinary bladders isolated and opened along the ventral aspect to expose the mucosal surface. The bladders were washed with sterile Hank’s balanced salt solution (HBSS) and incubated overnight at 4ºC in minimal essential medium (MEM) with 200 units of dispase. The bladder surface was washed with fresh MEM and the mucosal surface gently scraped and urothelial cells collected. The cells were washed twice by centrifugation and plated on collagen coated coverslips at a density of 20,000 cells per coverslip and kept in culture for three days before experiments. Coverslips were exposed to 0 or 4 Gray of X-ray irradiation (Precision Instruments X-RAD320 biological irradiator, 1 Gray = 100 Rads) following preincubation with mitochondrial targeted free radical scavenger, XJB-5-131 (1 µM) or vehicle (DMSO) for 60 minutes before irradiation. Cells were examined 15 min, 1 hour, 6 hours and 24 hours after irradiation.
Mitochondrial membrane potential and reactive oxygen species (ROS) fluorescence imaging: Mouse urothelial cells were loaded with either tetramethylrhodamine, Methyl Ester (TMRM) or dihydrorhodamine 123 (DHR123) to record mitochondrial membrane potential and ROS production, respectively. The changes in fluorescence intensity for each dye in response to irradiation was evaluated using an Olympus IX71 fluorescence microscope.
Selective mouse bladder irradiation: Mice were anesthetized with 300 mg/kg of 2,2,2-tribromoethanol (intraperitoneal injection) and a lower midline incision made to externalize the urinary bladder. The mice were mounted on lexan platforms that allowed the bladders to be held outside the abdominal cavity by a suture attached to the urachus in the path of a focused X-ray beam (10 Gray). Following irradiation, the bladders were internalized, the incisions sutured and animals allowed to recover up to three days with prophylactic analgesic and antibiotic treatments.
Immunofluorescence: Mice were sacrificed, bladders isolated and fixed with 4% paraformaldehyde. Tissue were cryopreserved in 30% sucrose solution, frozen in optimal cutting medium and sectioned on a cryostat. Bladder sections were probed with antibodies targeting cytokeratin (CK)-20 to determine integrity of the apical urothelial layer. Tissue were additionally stained with DAPI (nuclear marker) and rhodamine-phalloidin (actin stain).
Interpretation of results
These data demonstrate that urothelial cells are radiosensitive and that exposure to ionizing radiation causes acute disruption of the urothelial layer. This correlated with enhanced mitochondrial ROS generation and depolarization of the mitochondrial membrane potential. These changes may account for the irritative symptoms of early stage RC. Pretreatment of urothelial cells with the mitochondrial-targeted free radical scavenger, XJB-5-131 (developed by our group), prevented irradiation induced damage and could be a potential protective agent against RC. Although the urothelium can recover from the initial insult, there may still be long-term consequences to urothelial mitochondria function that contribute to the development of chronic stage RC. These could include persistent enhancement of ROS production and/or impairment of mitochondrial clearance (i.e., mitophagy).