Female Sprague–Dawley rats (12 weeks old, 230–240 g, N?=?45) were divided into three groups: control (Con, N?=?15), bilateral ovariectomy (Ovx, N?=?15), and bilateral ovariectomy plus subcutaneous injection with 17β-estradiol (Sigma-Aldrich, St. Louis, MO, USA) (Ovx?+?Est, N?=?15). The Con group underwent a sham operation. The Ovx group underwent a bilateral ovariectomy with intramuscular injection of zolazepam/tiletamine cocktail (4.4 mg/kg) and was treated with a subcutaneous injection of an oil vehicle. The Ovx?+?Est group underwent a bilateral ovariectomy, followed by treatment with subcutaneous estradiol daily (50 ug/kg/day) from 7 days after ovariectomy. All experimental animals were fed a standard diet up until the day before the experiment. Four weeks after ovariectomy and 3 weeks after hormonal replacement, animals with an estrous cycle confirmed via a vaginal smear were anesthetized with an intramuscular injection of a zolazepam/tiletamine cocktail (4.4 mg/kg) for tissue dissection after the cystometry. The study was approved by the Ethics Committee of Chonnam National University Medical School.
Four weeks after the ovariectomy, 5 rats in each group were anesthetized with 1.2 g/kg urethane injected subcutaneously. A suprapubic midline incision was performed to expose the bladder, a transvesical catheter with a fire-flared tip (polyethylene catheter-50) was inserted into the dome of the bladder and secured with a ligature, and the abdomen was closed. The catheter was connected to a pressure transducer and syringe pump via a 3-way stopcock to record intravesical pressure and to infuse saline into the bladder. After the bladder was emptied, cystometry was performed with saline infused at a rate of 0.04 ml/min. The contraction pressure and contraction interval were recorded.
The tissue sections (N?=?5 in each group, eight sections in each tissue) were rinsed in phosphate-buffered saline (PBS), and then treated with 3% hydrogen peroxide in 60% methanol for 30 min to quench endogenous peroxidase activity. After washing in PBS, tissue sections were treated with normal chicken serum for 30 min to block nonspecific binding. After being washed in PBS, the sections were incubated with a 1:50 dilution in PBS of antibody for caveolin 2 or caveolin 3 (Chemicon, Temecula, CA, USA) for 12–14 h at 4°C. Immunoreactivity for caveolin 2 and caveolin 3 were detected with the use of Alexa-Fluor 594 chicken anti-mouse IgG (1;100, H?+?L; Invitrogen, Carlsbad, CA, USA). Tissues were mounted with the use of a mounting solution containing 4-6-diamidino-2-phenylindole. For a negative control, tissues were prepared in a similar manner, except that caveolin 2 and caveolin 3 were omitted from the incubation solution. Tissues were examined with a model LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) with an excitation wavelength appropriate for Alexa-Fluor (405 or 594 nm). Final images were constructed with the use of LSM Image Examiner software.
All minced tissues were homogenized in ice-cold isolation solution with a Tissumizer homogenizer (Tekmar, Cincinnati, OH, USA). Tissues were homogenized with five bursts of five strokes of a micro-sawtooth generator. Tissue homogenates (N?=?5 in each group, 30 μg of protein) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, England, UK). The blots were then washed with Tris-Buffered Saline Tween (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20). Each membrane was blocked with 5% skimmed milk for 1 h and incubated with the appropriate primary antibody. Monoclonal rabbit antibodies for caveolin 2 and caveolin 3 (1;1500, Chemicon) and polyclonal rabbit antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1;4000, Cell Signaling Technology, Danvers, MA, USA) were used. The membrane was then washed, and caveolin-2, caveolin-3, and GAPDH were detected with goat anti-mouse-IgG and goat anti-rabbit-IgG conjugated to horseradish peroxidase, respectively. Antibody incubations were performed in a 4°C incubator. The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK). GAPDH was used as an internal control. Densitometry analysis was performed with a Studio Star Scanner using NIH image V1-57 software.
The results are expressed as mean?±?standard deviation, except for the data for the cystometric parameters, which are expressed as mean?±?standard error of the mean. Analysis of variance was used to test the null hypothesis that there would be no differences in the mean expression levels between the three groups. Differences were considered significant at P?<?0.05.