All protocols in this study adhered to the National Institute of Health Guide for the Care and Use of Laboratory Animals. The study received approval from the Institutional Animal Care and Use Committee of the clinical facility. The BOO model used in this study was developed by replicating the study protocol previously conducted by the authors [10,11].

Twenty female Sprague-Dawley rats, aged 6 weeks, were divided into 4 groups, with each group containing 5 rats. Group 1 (n=5) served as the control, while group 2 (n=5) underwent sham surgery. Groups 3 (n=5) and 4 (n=5) underwent BOO surgery and were monitored for 1 week and 8 weeks, respectively. BOO surgery was performed on the relevant rats under isoflurane (BK Pharm, Goyang, Korea) anesthesia. A skin incision was made in the lower abdomen, followed by urethral dissection. Subsequently, 4-0 silk sutures were used to encircle the urethra, with a 1-mm external diameter metal rod placed outside the lumen. Once the sutures were placed, the rod was removed and the abdominal wound was closed. For infection prophylaxis, cephalosporin (Flomoxef, BK Pharm, Goyang, Korea, 10 mg/kg per day) was administered interperitoneally.

Eight weeks after BOO surgery, the voiding response was evaluated in all rats by inserting a PE-50 catheter into the bladder. After the administration of an isoflurane anesthetic, a midline abdominal incision was made to expose the bladder. A collar was formed by warming the bladder end of the catheter, which was then positioned in the bladder via a tiny incision in the bladder dome and held in place by a suture. The opposing catheter end was threaded through the subcutaneous tissue to exit the skin. The wound was closed by suturing muscular and dermal layers. Anesthesia was then terminated.

The rats were housed in a restraining cage for 6 hours, which included a 2-hour recovery period following the surgical anesthesia. The dimensions of the cage were designed to allow the rats to crouch comfortably, but not to turn around. Once the rats had fully recovered from the isoflurane anesthesia, a pump was connected to the bladder catheter via a T-stopcock. This setup facilitated the continuous infusion of physiological saline, maintained at room temperature, into the bladder at a rate of 0.04 mL/min. The pump was also linked to a pressure transducer. The saline infusion triggered multiple voiding responses in the rats. The urination reflexes induced by volume loading were studied in the restrained rats. The parameters that were recorded included the intravesical pressure necessary for micturition, the pressure threshold (PT), the maximal voiding pressure (MVP), and the intercontraction interval (ICI). The ICI was defined as the time elapsed between voiding episodes or reflex bladder contractions.

After cystometry, the rats were euthanized via an intracardiac infusion of 100 mL of cold saline and 100 mL of 4% paraformaldehyde in phosphate-buffered saline, while under anesthesia. The bladder was subsequently extracted from each rodent and preserved in 4% paraformaldehyde for 48 hours. The viscera were then cryoprotected using 30% sucrose for 72 hours. Subsequently, a cryostat (Leica CM 1900, Leica, Wetzlar, Germany) was employed to cut sections of 10-μm thickness. These sections were then subjected to fluorescent staining and immunofluorescence processing. The samples were examined using an Olympus laser confocal fluorescence microscope.

The samples were subjected to immunostaining to detect the presence of von Willebrand factor (vWF) (1:200, Abcam, Cambridge, UK). Antigens were extracted following a 10-minute warming period with 9mM sodium citrate at a pH of 5. To inhibit intrinsic peroxidase activity, a peroxidase-blocking reagent (Dako, Carpinteria, CA, USA) was utilized. Free-floating tissue sections were incubated overnight at 4°C using a mixed primary antibody solution for vWF. Subsequently, Alexa Fluor 488-conjugated anti-mouse IgG (1:200, Molecular Probe, Eugene, OR, USA) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:200, Molecular Probe) were added, followed by an additional 60-minute incubation at room temperature. A negative control specimen was prepared in the same manner, but without the inclusion of primary antibodies. The stained samples were examined using an Olympus laser confocal fluorescence microscope. Quantitative analysis of the histological examinations was conducted using an image analyzer system (National Institutes of Health Image J 1.34, http://rsbweb.nih.gov/ij/).

As stated above, the rodents were sacrificed after cystometry was completed. The bodies of the bladders were removed and dissected away from any attached soft tissues prior to weighing. They were placed directly into a freezer in liquid nitrogen until additional biochemical and molecular biological assessments were required.

The Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total DNAs from cells and samples. Real-time polymerase chain reactions (PCRs) were carried out. The threshold cycle value was the basis for assaying relative gene expression. Normalization was performed against β-actin as a housekeeping gene. Data averaged over 3 experiments are presented. The PCR procedure comprised the following stages: 95°C for 10 seconds, and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Lastly, melting curve analysis over 60°C–95°C was carried out.

To isolate and extract total RNA, an RNA extraction kit (RNeasy Mini Kit, QIAGEN, Hilden, Germany) was used following the manufacturer’s instructions. The extracted RNA solution was treated with RNase-free DNase (QIAGEN, Hilden, Germany) to ensure there was no DNA contamination. Using an RT2 First Strand Kit (QIAGEN) and 2 μg of total RNA, the initial cDNA strand was synthesized in accordance with the manufacturer’s recommendations. This was then combined with RT2 quantitative PCR master mixes. Small quantities of this mixture were placed in a 96-Well RT2 Profiler PCR Array for rat hypoxia (QIAGEN). This array included SYBR green-optimized primer assays for a carefully selected panel of genes relevant to the pathway, consisting of 84 genes associated with the hypoxic response and 12 genes serving as a reference for the quality of loading and cDNA. The real-time PCR assay was conducted by heating the plate for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Quality assurance was provided by genomic DNA, reverse transcription, and positive PCR controls. β-actin served as the housekeeping gene. Any changes in gene expression were represented as a factor of increase or decrease, with a cutoff value of changes over a factor of 2 defining upregulated or downregulated expression. The primers used for real-time PCR for hypoxia are listed in Table 1.

Data are presented as mean±standard error. The nonparametric Mann-Whitney U-test was carried out to detect any differences between data from various groups. A P-value <0.05 was deemed to represent statistical significance.

The mean body mass of the rats in the sham surgical group and groups receiving BOO were equivalent (266±3.3 g and 235±7.1 g, respectively). The rats that underwent sham surgery had a significantly lower bladder weight (86.9±8.5 mg vs. 242.2±7.8 mg) than those that underwent a BOO procedure (242.2±7.9 mg) (P<0.05) (Fig. 1).

In rats that underwent BOO, the ICI was considerably higher (1,384±214 seconds) than in the sham surgical cohort (390±58 seconds, P<0.05). The PT values for the 2 groups were comparable (7.8±0.7 cm H₂O in the sham surgical group and 8.0±0.8 cm H₂O in the BOO cohort). The BOO group showed a lower MVP than the sham surgery group (23.7±4.1 cm H₂O vs. 34.7±7.4 cm H₂O) with elevated RU (6.4±1.5 mL vs. 0.1±0.08 mL) (Fig. 2).

Relative to the control group, HIF-1α mRNA expression levels were similar in rats that underwent BOO at week 1, but increased at 8 weeks following the BOO procedure (P<0.05). VEGFA and VEGFR2 mRNA expression levels at 1 week and 8 weeks following BOO were similar to those in the control group.

Compared with rats in the sham surgery group, the group at 1 week after BOO demonstrated higher levels of angiopoietin-1 (Ang-1), Ang-2, Ang-4, and Tie2 mRNA expression (Fig. 3). However, these levels were diminished at 8 weeks after BOO (P<0.05).

Fewer vWF-positive blood vessel configurations were identified on immunofluorescence staining in rats that underwent BOO (P<0.05) (Fig. 4).

Hif3a (-39.6) and Per1 (-5.1) were among the HIF and cotranscription factors. Additional HIF interactors included cyclin-dependent kinase inhibitor 2A (Cdkn2a, 11.0). Early growth response 1 (Erg1, 4.2), matrix metalloproteinase 9 (Mmp9, 74.7), serpin peptidase inhibitor, clade E, member 1 (Serpine1, 11.0), lysyl oxidase (LOX, 6.0), and heme oxygenase 1 (Hmox1, 6.1) were related to angiogenesis (Fig. 5).