We used 40 male Sprague-Dawley rats, weighing 450 to 500 g (16 weeks old). They were divided into control group (n=10) and BOO-induced group (n=30). For the control group, a sham operation was performed. In the BOO-induced group, a partial BOO was attempted. Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and experimental protocols were approved by the Institutional Animal Care (CUMC-2008-0012-02) and Use Committee of the Catholic University of Korea.

Experimental animals were anesthetized using an intramuscular injection of 15-mg/kg ketamine (Yuhan Co., Seoul, Korea) and 5-mg/kg xylazine (Rompun, Bayer Korea Co., Seoul, Korea). With the rats in the supine position, a longitudinal incision was made in the lower abdomen. The bladder neck and periurethral area were dissected out. A 25-G needle sheath was inserted into the urethra and the bladder neck was ligated using a 3-0 silk, after which the sheath was removed. In the sham-operated control animals, the bladder neck was very loosely ligated to not induce any obstruction.

Three weeks following the obstruction, cystometrogram was performed in the control and BOO-induced groups. Of the 30 rats with BOO, 10 were given an intravesicular administration of the α3 nAChR antagonist, hexamethonium (HM group); 10 were administered the α7 nAChR antagonist, methyllycaconitine citrate (MLC group), and 10 were received only saline infusion (BOO group).

A suprapubic midline incision was made under urethane anesthesia (12 mg/kg) and the bladder was identified. A 25-G needle connected to polyethylene tubing was placed in the bladder dome. The tubing was connected to a pressure transducer and an infusion pump using a 3-way stopcock. The bladder was emptied and saline was infused into the bladder at 0.04 mL/min using a Harvard syringe pump. Intravesical pressure was recorded using a polygraph apparatus (Grass 7D, Grass Instrument Co., Quincy, MA, USA); the contraction interval, contraction pressure, and presence of nonvoiding contraction (NVC) were examined. NVCs were defined as the rise in intravesical pressure exceeding 4 cmH₂O during the filling phase but not during voiding of urine. The Harvard syringe pump was stopped in the HM and MLC rats and the bladder was emptied for the administration of the nAChR antagonists. Voiding activity was prevented by clamping the penis. Saline infusions (0.2 mL) containing 80mM HM or 1mM MLC were instilled using a 25-G needle placed superior to the bladder. One hour later, saline was infused into the bladder at 0.04 mL/min using a Harvard syringe pump. The BOO group also received the same treatment without the drugs for 1-hour bladder retention and the intravesicular pressure was recorded.

After the bladder function test, the bladder and urethra of the control and BOO-induced groups were dissected from the adjacent tissue. The bladder was cut above the trigone region and was used for immunofluorescent staining and Western blot analysis.

Thawed tissue samples (stored in liquid nitrogen) were fixed in 4% paraformaldehyde and placed in 0.5M sucrose at 4°C. Using the optimal cutting temperature solution, polyethylene glycol, the tissues were sectioned at 3 μm. The tissue sections were then rinsed with phosphate buffered saline (PBS; pH 7.4). To inhibit nonspecific immunofluorescence staining, slides were exposed to a blocking solution (1.5% normal goat serum, 1.5% normal horse serum, 1% BSA, 0.1% Triton X-100 in PBS) at room temperature for 1 hour. Antibodies to α3 nAChR (sc- 1771; 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and α7 nAChR (sc-5544; 1:200; Santa Cruz Biotechnology Inc.) were then applied and incubated for 2 hours at room temperature. The slide was rinsed with PBS three times and then incubated in the secondary antibody, Alexa Fluor 488 labeled goat antirabbit IgG (1:300; Molecular Probes, Eugene, OR, USA) at room temperature for 1 hour. The sample was rinsed with PBS three times. Slides were stained with 4’,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA), and mounted for examination using a light microscope (Axiovert 200, Zeiss, Oberkochen, Germany). The immunoreactivity was expressed as mean±standard error of the mean (SEM) using a scoring system according to the degree of staining (5, very strong; 4, strong; 3, intermediate; 2, weak; 1, very weak). Thus, the distribution and intensity of α3 and α7 nAChRs were measured by 1 urologist and 2 pathologists.

The tissue was ground in liquid nitrogen and processed using a Qproteome Mammalian Protein Prep Kit (Qiagen, Hilden, Germany). The prepared sample was centrifuged at 4°C and 14,000 rpm for 10 minutes. Total protein was measured using a Bradford dye-binding protein assay kit (Bio-Rad Laboratories Inc., Hertfordshire, CA, USA) according to the manufacturer’s instructions.

Protein (30 μg) and 2× sample buffer (Bio-Rad Laboratories Inc.) were mixed at a ratio of 1:1. The reaction was performed at 100°C for 5 minutes and then stopped at 4°C. Electrophoresis was performed at 4°C and 70 V for 2 hours, using a Mini-PROTEAN three-cell unit (Bio-Rad Laboratories Inc.). Isolated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA, USA) using a transblot SD semidry system (Bio-Rad Laboratories Inc.) based on six gels at 4°C at a voltage of 20 V for 90 minutes. The membrane was blocked using 10% skim milk. Primary antibodies to α3 nAChR (57 KDa: ab65181, Abcam PLC, Cambridge, UK), α7 nAChR (56 KDa: ab24644, Abcam PLC), and glyceraldehyde 3-phosphate dehydrogenase (36 KDa: ab37168, Abcam PLC) were diluted 1:500 using 5% skim milk. The reaction was run at room temperature for 1 hour. After the membrane was rinsed, enzyme-conjugated secondary antibodies, diluted in 5% skim milk at a ratio of 1:10,000, to α3 nAChR and glyceraldehyde 3-phosphate dehydrogenase were reacted with rabbit IgG-HRP (ab6721: Abcam PLC) and that of α7 nAChR was reacted with rat IgG-HRP (ab6734: Abcam PLC) at room temperature for 30 minutes. After the membrane was rinsed, the reaction using enhanced chemiluminescence solution (Amersham Biosciences, Buckinghamshire, UK) was performed for 1 minute. The membrane was placed on a photographic film and developed. Using a densitometer (LAS-3000, Fuji Film, Tokyo, Japan), quantitative analyses of α3 and α7 nAChRs levels were performed for the urothelium and detrusor muscle in the control and BOO groups.

All data were analyzed using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). The data were expressed as mean±SEM. For comparisons among the groups, one-way analysis of variance and Newman-Keuls multiple comparison test were performed with P<0.05 as an indication of statistical significance.

In cystometrogram, the contraction interval of the BOO group (4.13±0.15 minutes) decreased significantly compared with the control group (10.25±0.17 minutes) (P<0.05). NVCs were observed in the BOO group. In the HM group, the contraction interval was 4.65±0.24 minutes, and there was no significant difference compared with the BOO group. However, the contraction interval of 7.55±1.12 minutes in the MLC group was significantly longer than that in the BOO group (P<0.05) (Fig. 1A). In the HM and MLC groups, NVCs were almost lost compared with the BOO group.

The contraction pressure of the BOO group was 30.12±1.38 cmH₂O and significantly higher compared with the control group (20.95±0.92 cmH₂O) (P<0.05). The contraction pressures of the HM and MLC groups were 19.08±1.53 and 13.53±1.02 cmH₂O, respectively, and significantly lower compared with the BOO group (P<0.05) (Fig. 1B).

The α3 nAChR expression was observed mainly in the urothelium but had weak expression in the detrusor muscle layer. In the control group, it was observed mainly in the luminal surface of the urothelium. In the BOO group, the expression of α3 nAChR was significantly increased in the urothelium compared with the control group. However, there was no significant difference from the control group in the detrusor muscle layer (Fig. 2). The α7 nAChR expression was observed in both the urothelium and detrusor muscle, unlike α3 nAChR. In the control group, α7 nAChR expression was observed in all layers of the urothelium. In the BOO group, α7 nAChR expression further increased in the urothelium and detrusor muscle compared with the control group (Fig. 3).

Expressions of α3 nAChR and α7 nAChR were confirmed in the mucosa and detrusor muscle. Alpha3 nAChR expression was significantly increased in the mucosa of the BOO group compared with the control group (P<0.05). However, the expression levels in the detrusor muscle were similar for the BOO and control group. Expression of α7 nAChR in the BOO group significantly increased both in the mucosa and detrusor muscle compared with the control group (P<0.05) (Fig. 4). These results were in accordance with the immunofluorescence result.