Experimental Animals and Study Design
Approval for all experiments was granted by the Institutional Animal Care and Use Committee at the University of California, San Francisco. Fourteen female Sprague-Dawley rats, aged 24 and 48 weeks, were procured from Charles River Laboratories (Wilmington, MA, USA). These rats were randomly assigned to two cohorts: (A) Young (24 weeks) and (B) middle-aged (48 weeks). Each cohort was subdivided into 3 groups: sham control (SHAM group), vaginal balloon dilation and ovariectomy (VBDO group), VBDO + BAPN (BAPN group). Each group consists of 7 rats: 6 for functional study and 1 for single cell RNA sequencing. The aging sham control group underwent a sham procedure without vaginal balloon dilation and ovariectomy. The VBDO procedure was performed following established protocols18. In brief, under appropriate anesthesia (ketamine/xylazine, 90 mg/kg and 10 mg/kg respectively, intraperitoneally), an 18Fr latex Foley catheter's balloon was inserted into the rat's vagina and filled with 4 ml of water. A 130-g weight was placed at the suspended end of the catheter, creating a consistent force directed toward the pelvic floor. The balloon was retained for 4 hours. After a week, the rats were anesthetized, and bilateral ovaries were surgically removed. Starting from the second week following the initial VBDO, rats in the BAPN groups received intraperitoneal injections of 300 mg/kg BAPN twice a week for 4 weeks. Subsequent to a 1-week washout period, all rats underwent measurement of leak point pressure (LPP). Upon completion of LPP measurement, the rats were euthanized, and their urethras were collected for single-cell RNA sequencing and histological analysis.
Leak Point Pressure Measurement
Leak point pressure (LPP) was measured using established methods 33. In brief, rats were anesthetized with urethane (i.p), and a polyethylene-90 tube was introduced into the bladder dome and secured using a purse string suture. The bladder was gradually filled with heated phosphate-buffered saline (PBS) while recording the volume. The bladder's capacity was recorded at the point of urine leakage. This process was repeated thrice, and the mean bladder capacity was calculated. The bladder was then emptied through aspiration and manual pressure. Changes in intravesical pressure were captured using LabView 6.0 software (National Instruments, Austin, TX, USA) at a sampling rate of 10 samples/sec. The bladder was filled to 40% of its capacity, and incremental manual extravesical pressure was applied until leakage occurred. This process was repeated six times, and the LPP values were documented. Subsequently, the rats were humanely euthanized, and their entire urethras were collected for further analysis.
Immunofluorescence Staining
Tissue samples were initially fixed using a cold solution containing 2% formaldehyde and 0.002% saturated picric acid in a 0.1 M phosphate buffer at a pH of 8.0. This fixation process lasted for 4 hours, followed by immersion in a buffer solution containing 30% sucrose overnight. Subsequently, the specimens were embedded in OCT Compound (Sakura Finetek USA, Torrance, CA) and stored at a temperature of -70°C. To prepare the fixed frozen tissue specimens for analysis, sections of 10 microns were cut and carefully placed onto SuperFrost-Plus charged slides (Fisher Scientific, Pittsburgh, PA), where they were allowed to air dry for a duration of 5 minutes. These prepared slides were then treated with a mixture of 0.3% H2O2 in methanol for 10 minutes, followed by two consecutive 5-minute washes with PBS. Next, the slides were subjected to a 30-minute incubation at room temperature with a solution containing 3% horse serum in PBS and 0.3% Triton X-100. After the excess solution was removed from the tissue section, the slides were left to incubate overnight at a temperature of 4°C with primary antibodies, including anti-myosin skeletal heavy chain (MHC; 1:500; Mouse, [MY-32] (ab51263), Abcam), anti-smooth muscle actin (SMA; 1:1000; Mouse, Abcam), and anti-Uroplakin-III (UP-III; 1:500; mouse, Santa Cruz Biotechnology, Inc.).For the subsequent steps, secondary antibodies tagged with Alexa-488 and Alexa-594 (1:500; Invitrogen) were used. Nuclei were stained using DAPI, followed by phalloidin (1:500; Invitrogen). The resulting stained tissues underwent examination using fluorescence microscopy.
Masson's Trichrome Staining, Neuromuscular Junctions, and Phalloidin Staining
To perform Masson's trichrome staining, sections of urethral tissue were initially placed in warm Bouin solution (58°C) for a duration of 20 minutes. Following this, the sections were rinsed and subjected to Weigert Hematoxylin staining for 10 minutes, ensuring that only the nuclei remained stained after thorough rinsing. The subsequent steps included staining with Biebrich Scarlet-Acid Fuchsin for 3 minutes, followed by immersion in phosphomolybdic acid for 45 minutes. Aniline Blue staining was then performed for 3 minutes, succeeded by a 2-minute distilled water rinse and a 2-minute immersion in 1% acetic acid. This was followed by two rounds of a 2-minute distilled water rinse. The sections were then dehydrated through a series of increasing ethanol concentrations, air-dried, and eventually mounted. Neuromuscular junctions were stained using α-bungarotoxin (1:500, Invitrogen), followed by phalloidin. For image analysis, five random fields per tissue section were photographed and documented using a Retiga digital camera along with ACT-1 software (Nikon Instruments Inc., Melville, NY, USA).
Assay for Senescence-Associated β-Galactosidase Activity
To perform SA-β-Gal staining on cryosections of the urethral tissue, the samples were embedded in OCT, then sectioned at a thickness of 10 µm and allowed to air-dry. After rehydration in PBS, the staining procedure was carried out utilizing the Cell Signaling kit (#9860). This involved an initial fixation period of 12 minutes, followed by incubation in the staining solution at 37°C for a duration of 12 hours.
Single-cell RNA-seq library preparation and sequencing
Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (PN-1000094, 10× Genomics) according to the manufacturer’s instructions. Final libraries were sequenced on an Illumina NovaSeq 6000. The raw sequencing reads were processed by Cell Ranger (v.3.1.0) with the default parameters. The reference genome version was Rattus norvegicus mRatBN7.2.106.
Single-cell RNA-seq library preparation and sequencing
Quality control and sample integration
The gene–cell matrix of each sample was used to create a Seurat object with the Seurat package in R. Cells were further filtered according to the following threshold parameters: the total number of expressed genes range from 500 to 9,000; total UMI count range from 0 to 25,000; and proportion of mitochondrial genes expressed, < 20%. Batch correction was performed using the IntegrateData function in the Seurat package according to the package manual (https://satijalab.org/seurat/v3.1/pbmc3k_tutorial.html).
Cell identification and clustering analysis
The merged Seurat objects were scaled and analysed by principal component analysis (PCA). The first 20 PCs were also used to get clustering and perform t-distributed stochastic neighbour embedding (tSNE) dimensionality reduction. The FindClusters function in Seurat package with the resolution parameter set as 0.5 was used to cluster the cells. For further analysis of each cluster, we isolated them and performed the above two steps again to get subcluster information.
Differentially expressed gene calculation and gene enrichment analysis
The Seurat function FindAllMarkers (test.use = wilcox; min.pct = 0.1; logfc.threshold = 0.25) was used to identify differentially expressed genes (DEGs) based on the normalised UMI count. Unless otherwise noted, the DEGs in each selected subcluster were calculated based on comparison between that subcluster and the rest of the dataset. GO analysis was performed using the WebGestalt website (http://www.webgestalt.org), the Over-Representation Analysis (ORA) or Gene Set Enrichment Analysis (GSEA) was chosen as Method of Interest, and only Biological Process was chosen in Functional Database. The pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software based on the log2 (FC) and P-values of the DEGs.
Ligand–receptor interaction and transcription factor network construction
The CellChat package was used for ligand–receptor interaction analysis. The cell–gene matrix was divided according to the six major clusters or five major combined with the subclusters of the other major cluster. The “secreted signalling”, “ECM–receptor” and “cell–cell contact” paired datasets were chosen to analyse the cell communication.
For the transcription factor (TF) regulation network analysis, a total of 1,479 Rat TFs in AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) and a cell–gene matrix was taken as input for the GENIE3 package. In the output regulator–target table, only pairs with weights greater than 0.1 were retained and the first 8 TFs were displayed further.
Statistics and Reproducibility
Statistical significance was calculated by GraphPad Prism 8 software with Tukey's multiple comparisons test of ANOVA. The confidence interval was 95%. Results were considered significant at P < 0.05. Statistical analysis between one group and the rest of the scRNA-seq data were made by Wilcoxon (Mann-Whitney) rank sum test; two-tailed, with the Seurat package in R, and the confidence interval is 95%. Statistical parameters are reported in the respective figures and figure legends.