Twelve-week-old male Sprague Dawley rats were randomly divided into three groups: control group (n=5), ischemia only group (I, n=5) and ischemia/reperfusion group (I/R, n=25). Acute BOO followed by bladder distension (ischemia) and drainage (reperfusion) was induced as described elsewhere (15) with slight modifications. Briefly, the rats were anesthetized by an intraperitoneal injection of 50 mg/kg zoletil (tiletamine/zolazepam combination), and a 3-0 a silk ligature was placed around the prepuce of the rat penis. To promote bladder overdistension, 12 mL/kg Ringer solution was given intraperitoneally with intramuscular administration of furosemide (12 mg/kg) in I and I/R groups. A capacity of 3±0.5 mL was reached 15 min after this application. After the bladder was kept overdistended for 60 min, it was exposed via a lower midline abdominal incision and drained by a syringe with 18 G needle, until the rat woke up from anesthesia. Five rats in the I/R group was allowed to reperfuse for 30 min, 60 min, 120 min, 3 days and 2 weeks, respectively (n=5 in each I/R subgroup). The control group was anesthetized for 60 min by zoletil without administration of Ringer solution and furosemide, and the I group was anesthetized and overdistended for 60 min without reperfusion. After this, the rats were sacrificed and the bladders were extirpated. The bladder dome was maintained overnight in 10% formaldehyde solution and embedded in paraffin for histological studies. The remaining bladder tissue was rapidly frozen in liquid nitrogen and stored at -80℃ until processing. The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the Clinical Research Institute at the Seoul National University Hospital (AAALAC accredited facility) (IACUC number: 08-0259).

For detection of apoptosis, the terminal deoxynucleotidyl transferase-medited dUTP-biotin nick end labeling (TUNEL) method was used. Briefly, 5 µm thick bladder tissue sections were deparaffinized and re-hydrated in graded series of xylene and ethanol. Then, the slides were pretreated with 20 µg/mL proteinase K and quenched in 3% hydrogen peroxidase in PBS at room temperature for 5 min. After the equilibration, all sections were incubated in a humidified chamber at 37℃ for 1 hr with a terminal deoxynucleotidyl transferase enzyme. The reaction stopped and then the slides were incubated with an anti-digoxignenin-peroxidase conjugate at room temperature for 30 min. After stained by 0.05% diaminobenzidine in staining buffer, the color development was monitored under the microscope. When the dark brown apoptotic bodies were detected, the slides were counterstained in 0.5% methyl green for 10 min. The slides were then washed with 100% N-butanol, and mounted. In each slide, three high-power (×400) fields were randomly selected and the apoptotic index was expressed as the percentage of apoptotic cells relative to the number of total cells in a given area (non-apoptotic nuclei plus apoptotic cells).

After bladder tissues were homogenized in an ice-cold lysis buffer, whole cell homogenates were centrifuged at 14,000 g, 4℃ for 20 min and the supernatants were collected. After quantitation of protein concentration, equal amounts of proteins (20-50 µg) were electrophoresed on SDS-polyacrylamide gel, and transferred onto polyvinyl difluoride. The membranes were blocked with 5% nonfat dry milk in TBST for 1 hr at room temperature, and incubated overnight at 4℃ with primary antibodies (poly [ADP-ribose] PAR, Trevigen, Gaithersburg, MD, USA; caspase 3, Cell Signaling Technology, Beverly, MA, USA; mitochondrial manganese superoxide dismutase (MnSOD), Stressgen, Ann Arbor, MI, USA; Bcl-2 and Bax, Santa Cruz Biotechnology, Santa Cruz, CA, USA, diluted 1:1,000, respectively). Membranes were then incubated with appropriate horseradish peroxidase-linked secondary antibodies (Pierce, Rockford, IL, USA) for 2 hr at room temperature, and visualized using the ECL detection system (Pierce, Rockford, IL, USA). The results of western blots were quantified by densitometry using Bio-Rad imaging software Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA).

All results are expressed as means±SD. Because of the modest sample size, the nonparametric Kruskal-Wallis test was performed to analyze the total groups, and the Mann-Whitney U-test was conducted to compare between two groups. A P value <0.05 was considered statistically significant.

At 30 min, 60 min, and 120 min following bladder emptying, tissue sections from the I/R group exhibited a significant increase in the number of TUNEL positive cells than the controls. However, compared to the control group, there was no statistical difference in the number of TUNEL positive cells in the I group, I/R groups at 3 days and 2 weeks after bladder emptying (Fig. 1).

Western blot analysis of the expression of MnSOD, caspase 3, Bcl-2 and Bax and amounts of PAR showed time-dependent changes. Expression of MnSOD significantly decreased in the I/R group at 30 min, 60 min, and 120 min after bladder emptying compare to the control group, and began to recover thereafter. Expression of caspase 3 significantly increased in the I/R group at 30 min, 60 min, and 120 min after bladder emptying compared to the control group, and significantly reduced to the control level at 3 days and 2 weeks after bladder emptying. When compared to the control group, the I/R group showed significant decrease in Bcl-2 expression and significant increase in the Bax expression at 30 min, 60 min, and 120 min after bladder emptying. As a result, the ratio of Bcl-2/Bax was significantly reduced. The expression of Bcl-2 and Bax, and the ratio of Bcl-2/Bax recovered to the control level at 3 days and 2 weeks after bladder emptying. On the other hand, the amounts of PAR (a marker of PARP activity) significantly increased at 30 min, 60 min, and 120 min after bladder emptying compared to the control group. Three days and 2 weeks after bladder emptying in the I/R group, there were no statistical difference in the expression of MnSOD, capase 3, Bcl-2 and Bax and the amounts of PAR between the I group and the control group (Fig. 2).