Briefly, rats were anesthetized intraperitoneally with ketamine (75 mg/kg; Ketamine 50, Yuhan Corp., Seoul, Korea) and xylazine (15 mg/kg; Rompun, Bayer Korea Ltd., Seoul, Korea). Through a lower midline abdominal incision, the para-urethral space was exposed, and a silk stay suture (usually 4-0) was placed about 2–3 mm lateral to the urethra to pull up the vaginal epithelium, allowing an easy incision to be made. Through a small incision in the vaginal epithelium, a needle with nylon ligature (3-0) was inserted into the other side of the urethra via the vaginal space. The ligature was tied tightly around the urethra with a 1-mm steel rod. After the knot was made, the rod was removed, and the knot was rolled down and positioned in the vagina, allowing the knot to be easily removed through the vaginal approach, without another abdominal incision, 2 weeks after the obstruction procedure [9].

Catheters to monitor the IVP and IAP were inserted 3 days before cystometry. To record the IVP, a polyethylene catheter with a cuff (PE-50, Becton Dickinson, Franklin Lakes, NJ, USA) was inserted into the dome of the bladder and held in place by fixation with a purse-string suture, through a low midline abdominal incision. To record the IAP, a 0.05-mL balloon (Latex, Dawoo Medical, Incheon, Korea) was placed at the tip cuff of a PE-50 polyethylene catheter for each experiment, as described previously [10]. The balloon was placed directly above the bladder, just after the IVP catheter was inserted into the bladder.

Both catheters were subcutaneously tunneled and fixed to the back skin with a silk ligature. The free end of the catheter was finally sealed. Each rat was individually housed after surgery, and water and food were given ad libitum. After surgery, the animals were left to rest for at least 24 hours under controlled conditions.

Cystometric investigations were performed 2 weeks after the sham operation and BOO surgery in the sham and BOO groups and 1 week after obstruction surgery in the deobstructed group. Awake rats were put in metabolic cages (Nalgene metabolic cage, Nalge, Rochester, NY, USA). The catheter from the bladder was connected to a 2-way valve that was linked to an infusion pump (PHD 22/2000 programmable syringe pump, Harvard Apparatus, Holliston, MA, USA) and a pressure transducer (Research Grade Blood Pressure Transducer, Harvard Apparatus, Holliston, MA, USA). The balloon catheter for IAP measurement was connected to another pressure transducer. Micturition volume (MV) was simultaneously recorded using a fluid collector connected to a force displacement transducer (Research Isometric Transducer, Harvard Apparatus). Bladder infusion was performed with normal saline at a rate of 20 mL/hr in all groups. Data were stored digitally and analyzed using Acq Knowledge 3.8.1 software (Biopac Systems, Goleta, CA, USA) at a sampling rate of 100 Hz and an MP150 data acquisition system (Biopac Systems). For each animal, the mean values from 3 reproducible micturition cycles were analyzed. IAP was defined as the balloon pressure corrected by subtracting the lowest balloon pressure in each entire voiding cycle for zeroing. The detrusor pressure (DP) was defined as the IVP minus the IAP. The DOs were defined as increments of the IVP during the filling phase that exceeded 2 cm H₂O from baseline without simultaneous changes in the IAP or fluid expulsion from the bladder (Fig. 1). Values are expressed as mean±standard error (SE). All pressure parameters are expressed as the DP, not the IVP. After performing the urodynamic tests, the rats were sacrificed by cervical dislocation. The bladder and urethra were removed together and their weight was measured. The bladder and urethra were separated and a microarray analysis of the bladder was performed.

The following cystometric parameters were quantified: basal pressure (BP), threshold pressure (TP), maximal pressure (MP), MV, residual volume (RV), bladder capacity (BC; MV+RV), and micturition interval (MI). The cystometric parameters used to evaluate DO during the filling phase were the time of the filling phase (interval between the initiation of saline infusion and micturition), the total number of instances of DO during the filling phase, the frequency of DO instances (per minute), and the average pressure difference between the peak and the base of the DO (DO pressure). Compliance was calculated by the formula (P2−P1)/(V2−V1), where P2 is the pressure of the stable curve just before the bladder contracts, and V2 is the infused volume at that time. P1 is the baseline pressure, and V1 refers to the corresponding infused volume.

Bladder tissue was removed from the rats in all groups, RNAlater (RNA stabilization solution, Ambion Inc., Foster City, CA, USA) was added, and the samples were then stored at 4°C for 1 day. Then, the RNAlater was removed, the tissue was disrupted using liquid nitrogen and a mortar and pestle, and total RNA was isolated using TRI REAGENT (Molecular Research Center Inc., Cincinnati, OH, USA).

Synthesized Cy3 or Cy5 fluorescent cDNA was mixed at the same concentration as that of the single-stand DNA, followed by the addition of 0.05 mg/mL of yeast tRNA and denaturation at 95°C. The hybridization slide glass was scanned using a GenePix 4000B scanner (Axon Instrument, Union City, CA, USA) and the scan image was analyzed using GenePix Pro 4.0 software (Axon Instrument). Analysis related to expression profiling, clustering, and ontology was also performed using Gene-Spring (Silicon Genetics, Redwood City, CA, USA) software. Using GeneSpring GX 7.3.1, global normalization, intensity normalization, and block scale normalization were performed to assess the accuracy and reliability of the experiments.

Scatter plots, intensity values, and ratio plots of RI and M versus A plots were created using the intensity values of the normalized genes in the control and experimental groups. Variation in overall gene expression was analyzed. Ontology classification, hierarchical clustering, and linkage analysis for each experiment were performed using GeneSpring GX 7.3.1. In our study, differences of gene expression that were greater than 2 fold in the BOO, DDO, and non-DDO groups compared to the sham group were analyzed, and the number of genes showing such between-group differences was identified and compared for each group.

The results are given as mean±SE. The normality of the distributions was checked using the Shapiro-Wilks W test. The statistical significance of the differences was calculated with the unpaired Student t-test or 1-way analysis of variance followed by the Tukey post hoc test for multiple comparisons. All analyses were performed with GraphPad Prism ver. 7.00 (Graph Pad Software, San Diego, CA, USA) and SigmaStat for Windows version 3.5 (Systat Software Inc., San Jose, CA, USA), at a significance level of 0.05 and a confidence interval of 95%.

Among the genes that showed greater than 2-fold changes in gene expression in the experimental groups, 220 were associated with apoptosis pathways. In particular, the expression levels of inhibin alpha (INHA) and interleukin 1-alpha (IL1A) increased much more in the non-DDO group than in the BOO group. Furthermore, the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A), zinc finger protein 162 (ZFP162), and vascular endothelial growth factor A (VEGFA) decreased in the BOO group, but decreased to an even greater extent in the non-DDO group. In contrast, the expression levels of these genes were not significantly different in the DDO group, or they showed a slight increase (Table 2).

The results of this functional analysis showed that the expression of genes related to cell adhesion pathways mostly decreased in the BOO group and non-DDO group. In the DDO group, there was no change in the expression of genes such as catenin-beta1 (CTNNB1), adhesion regulating molecule 1 (ADRM1), and procollagen type I alpha 1 (COL1ALPHA1). In the DDO-group, the expression levels were significantly reduced to a level more than 10 times lower than baseline. In addition, the expression of genes such as cadherin 3 (CAH3) and integrin-alpha 6 (ITGA6) was decreased by 2-5 times in the BOO and non-DDO groups. However, the degree of expression of thrombospondin 4 (THBS4) increased in the BOO group, but differentially decreased in both the DDO and non-DDO groups (Table 2).

A total of 30 genes related to this functional pathway were overexpressed (approximately 70% of these genes), mainly in the BOO and non-DDO groups. In particular, the degree of expression of genes such as chemokine ligand 2 (CXCL2), chemokine ligand 4 (CCL4), CCL3, CCL5, and CXCL10 increased to 4.3–26.4 times their baseline levels in the non-DDO group, but in the DDO group had normalized to 1.1–1.7 times the baseline values. This tendency was similar to the above results for other chemokine ligands, except for the CXCL10 gene (Table 2).

The degree of expression of genes confirmed to be part of the pathway involving the extracellular matrix was generally decreased, but the expression of matrix metallopeptidase (MMP) increased in all 3 experimental groups (Table 2).

Eighty-nine genes involved in the inflammatory response pathway showed expression changes. The genes belonging to this group are almost identical to those found in the chemokine and receptor and immune response pathways (Table 2).

The degree of gene expression associated with the mechanism of ischemia-reperfusion injury may be highly relevant for the patterns of obstruction and deobstruction in the present experimental model. This is because when BOO occurs, the bladder expands and the blood vessels of the bladder wall are compressed and become ischemic, after which reperfusion takes place in some components of the vascular system when the bladder is deobstructed. As shown in Table 2, in these functional categories, almost all gene expression levels were normalized in the DDO group, similarly to the results of other categories, and there was little change in comparison with the sham group. It was also found that the expression of these genes was normalized in the DDO group, rather than the non-DDO group.

Through our investigation, the results of 3-dimensional principal component analysis (PCA) of the whole and functional categories of the genotypes analyzed were confirmed. When all genes were examined, the differences in the gene expression patterns of the BOO, DDO, and non-DDO groups were clearly visualized in the 3-dimensional PCA comparison space (Fig. 5).

In the present study, gene expression patterns were clearly distinguished in all 3 experimental groups when all genes and functional categories were examined. Those of DDO (0.066) than of non-DDO (0.694) in the 2 deobstructed groups were distinguished clearly observed in the bladder of the BOO group, and the difference between the DDO and non-DDO groups was also 0.027, and these results were easily confirmed by the correlation plot.