Fig 1.
Strategy of the STC and subsequent analysis.
(A) Schematic outlining how the STC surgery was performed and sutured. (B) Schematic indicating how the linear incision surgery was performed and sutured.
Fig 2.
Recovery of voiding volume after STC.
(A) Representative images of VSOP performed on sham, incisional sham and STC bladders, 1d PS, 1wk (7d PS), 2wk (14d PS), 4wk (28d PS) and 8wk (56d PS) after surgery. (B) Mean maximum voiding volume determined from VSOP analysis after determining a standard curve using mouse urine. Graph demonstrating results for maximum voided volume per void in linear incision (blue line) STC (green line) and sham (red line) bladders. The y-axis indicates mean volume in μL and the x-axis indicates the time after surgery that mice were subjected to VSOP in days. Error bars are standard error of the mean (SEM). (C) Graph of ratio of maximum voided values in (B) for linear incision to sham (blue line) and STC to sham surgery (green line). By 56d PS the volume of STC bladders had reached 63% of age-matched sham control bladders. (D) Graph indicating the mean number of voids as indicated by spots on the blotting paper after 2h. Error bars are SEM.
Fig 3.
Bladder wall thickness and collagen deposition are altered after STC.
(A-D) Representative images of sham operated (A, C) and STC-operated (B, D) bladders 1wk (7d PS), 2wk (14d PS), 4wk (28d PS) and 8wk (56d PS) after surgery stained with hematoxylin and eosin (H&E). (E) Bar graph indicating mean wall thickness in μm. Black bars represent sham operated bladders and gray bars represent STC operated bars. Error bars represent SEM. Statistical significance (P < 0.05) is indicated by an asterisk.
Fig 4.
Increased collagen to smooth muscle ratio in STC bladders.
(A-D) Representative images of sham operated (A, C) and STC-operated (B, D) bladders 1wk (7d PS), 2wk (14d PS), 4wk (28d PS) and 8wk (56d PS) after surgery stained with Masson’s trichrome stain. (E) Bar graph indicating the collagen to smooth muscle ratio in sham and STC bladders at the same time points. Black bars represent sham operated bladders and gray bars represent STC operated bars. Error bars represent SEM. Statistical significance (P < 0.05) is indicated by asterisks.
Fig 5.
Regrowth of the detrusor muscle 8wk after STC surgery.
Tiled confocal images of sham operated and STC operated bladders 1wk (7d PS), 2wk (14d PS), 4wk (28d PS) and 8wk (56d PS) after surgery. (A-E) The top row of panels shows staining for smooth muscle myosin (SMM) in red with nuclei counterstained in blue (DAPI). (F-J) Row of panels shows staining for cytokeratin-5 (CK5) marking the urothelium (green). (K-O) Row of panels shows staining for CD34 marking BMSCs (green). (P-T) Row of panels shows staining for Sca-1 marking BMSCs (green). Magnification bars are 500μm and are shown in the lower portion of panels (E, J, O, T). Arrow in (E) points to gap in smooth muscle regrowth at 8wk.
Fig 6.
Regenerating cells originate primarily from the suture site.
(A) Tiled confocal images of bladder sections stained with anti-calponin (green), EdU (yellow), and DiI (red). Time after STC surgery is indicated above each set of panels. The white lines forming a cross indicate the regions that were analyzed for fluorescent pixels. DiI was applied to the suture immediately after the bladder was sealed. (B) The same images in (A) showing only EdU (yellow) and DAPI (blue) pixels. This view allows us to see the EdU pixels clearly. (C) EdU incorporation in a sham operated bladder at 1wk showing little incorporation of EdU. (D) Quantification of pixels in images such as those shown in (A). The bar graph indicates the ratio of EdU pixels to DAPI pixels in the indicated regions at the indicated times after surgery. Significance is indicated above bars (P < 0.05). Error bars are standard error of the mean.
Fig 7.
Quantification of EdU incorporation in smooth muscle, urothelial and BMSCs.
(A-C) Representative confocal images of EdU treated STC bladders stained with anti-calponin (A), anti-CK5 (B) and anti-Sca-1 (C) all in green. All nuclei are stained with DAPI (blue) and EdU incorporation is shown in pink. Quantification of co-localization of EdU+ nuclei with antibody stain. This is indicated on the y-axis as %EdU incorporated. The number shown is the mean number of %EdU+/antibody+ cells divided by the total number of antibody+ cells x100 per section counted. Significance (*) is indicated above bars (P < 0.05). Error bars are standard error of the mean.
Fig 8.
Transcripts associated with regeneration and scarring are expressed in the STC bladder.
(A-D) Quantitative polymerase chain reaction (QPCR) analysis of total RNA isolated from sham operated or STC operated bladders 1wk (7d PS, light blue bars), 2wk (14d PS, green bars), 4 wk, (28d PS, aqua bars) and 8wk (56d PS, dark blue bars). All bars were standardized to the level of GAPDH transcripts in each sample. Bars represent the fold-change relative to sham for each gene. (A) Quantification of representative stem cell and tissue remodeling transcripts: Abbreviations are as follows: CD34-sialomucin; Sca-1-stem cell antigen 1; FN1-fibronectin; MMP2-matrix metalloproteinase 2; LN5-laminin 5; and TNC-tenascin C. (B) Quantification of representative detrusor muscle transcripts: SMM-smooth muscle myosin; SRF-serum response factor; ACTA2-smooth muscle alpha actinin; and P2RX1-purinergic receptor P2X1. (C) Quantification of representative fibrosis transcripts: Col1α1-collagen1 alpha1; Col3α1- collagen3 alpha1; Col7α1-collagen7 alpha1, FSP-1-fibroblast specific protein-1; Vim-vimentin; and ZEB2-zinc finger E-box binding homeobox-2. (D) Quantification of representative neural and urothelial transcripts: NeuN- Hexaribonucleotide Binding Protein-3, NF200-neurofilament 200; UpkII-uroplakin II; Ck5- cytokeratin-5; and SHH-sonic hedgehog. Fold change on graph indicates log2 fold change as determined by the ΔΔCt method. Data for the graphs is found in S2–S5 Tables. Statistical significance was determined by comparing the distribution of ΔCt values for shams versus STC surgery using unpaired Student’s T-Tests. Error bars on ΔΔCt values were calculated using error propagation. Asterisks indicate statistical significance (P < 0.05) as indicated in S2–S5 Tables.
Fig 9.
Inflammatory cell invasion into the regenerating portion of the bladder is characterized by a pro-inflammatory cytokine response.
Tiled confocal images of bladder sections stained with antibodies to CD68 (A-E) and Ly-6B.2 (F-J). Anti-CD68 marks infiltrating macrophages and anti-Ly-6B.2 marks infiltrating neutrophils. Leftmost panels (A, F) are sham bladders. Other panels are from STC surgery and are labeled as above. (K) Representative images of cytokine arrays interrogated with protein isolated from sham surgery bladders (top) and STC surgery bladders (bottom) one week after surgery. Cytokines of interest are indicated next to each set of duplicate spots. (L) Densitometric quantification of images similar to and including those shown in (K). Only bars that demonstrated statistically significant changes (P < 0.05) are shown. Bars are grouped by color: blue bars represent chemokines, purple bars represent Th1 cytokines, yellow bars represent pleiotropic cytokines and red bars represent TIMP and ICAM-1.