Proliferation of Interstitial Cells in the Cyclophosphamide-Induced Cystitis and the Preventive Effect of Imatinib

Abstract

Cyclophosphamide- (CYP-) induced cystitis in the rat is a well-known model of bladder inflammation that leads to an overactive bladder, a process that appears to involve enhanced nitric oxide (NO) production. We investigated the changes in the number and distribution of interstitial cells (ICs) and in the expression of endothelial NO synthase (eNOS) in the bladder and urethra of rats subjected to either intermediate or chronic CYP treatment. Pronounced hyperplasia and hypertrophy of ICs were evident within the lamina propria and in the muscle layer. IC immunolabeling with CD34, PDGFRα, and vimentin was enhanced, as reflected by higher colocalization indexes of the distinct pairs of markers. Moreover, de novo expression of eNOS was evident in vimentin and CD34 positive ICs. Pretreatment with the receptor tyrosine kinase inhibitor Imatinib prevented eNOS expression and ICs proliferation, as well as the increased voiding frequency and urinary tract weight provoked by CYP. As similar results were obtained in the urethra, urethritis may contribute to the uropathology of CYP-induced cystitis.

Figure 1

Increased vimentin/CD34 colabeling induced by CYP in the bladder and urethra and the preventive effect of Imatinib. Bladder sections of control (a), CYP-chronic (b), Imatinib-control (c), and Imatinib-chronic (d) treated animals. Labeling for CD34 (green) and vimentin (Vim, red) increased in intensity and extension in the CYP treatments and Imatinib prevented these effects. (e-f) Labeling for CD34 (e) and the merged image Vim-CD34 (f) of a urethral section from a CYP-chronic treated rat. The nuclei were counterstained with DAPI (blue) in (e). Long arrows indicate double-labeled bipolar cells with long prolongations situated parallel to smooth muscle fibers. Short arrows show large double-labeled multipolar big cells in between muscle fibers and in the serosal coat. Asterisks indicate large cells that were only vimentin-immunoreactive. U, urothelium; Su, suburothelium; lp, lamina propria; m, muscle layer. Scale bars = 40 μm.

Figure 2

Increased vimentin/PDGFRα colabeling induced by CYP in the bladder and urethra and the preventive effect of Imatinib. (a–c) Bladder sections of control (a), CYP-intermediate (b), and CYP-chronic (c) treated animals. (d-e) Urethral sections of control (d) and CYP-intermediate (e) treated animals. Vimentin (Vim, red) and PDGFRα labeling (green) increased in intensity and extension in the CYP treatments, effects that were prevented by Imatinib. (f–h) vimentin-ir (f) and PDGFRα-ir (g) and the merged images (h) in sections of the bladder from a CYP-chronic treated animal. The nuclei were counterstained with DAPI (blue) in (f) and (g). Long arrows indicate double-labeled bipolar cells with long prolongations. Short arrows show double-labeled multipolar cells present in between muscle fibers and in the lamina propria. Asterisks indicate large cells that were only vimentin-ir. U, urothelium; Su, suburothelium; lp, lamina propria; m, muscle layer. Scale bars = 40 μm except in (b), (c), and (d) (80 μm).

Figure 3

Increased CD34/PDGFRα colabeling induced by CYP in the bladder and urethra and the preventive effect of Imatinib. (a–c) Bladder sections of control (a), CYP-intermediate (b), and Imatinib-intermediate (d) treated animals. Labeling for CD34 (green) and PDGFRα (red) increased in intensity and extension in the CYP treatments, effects that were prevented by Imatinib. Labeling for PDGFRα (d) and CD34 (e) and the merged image (f) in the bladder lamina propria after CYP-intermediate treatment. Nuclei were counterstained with DAPI (blue) in (d) and (e). Long arrows indicate double-labeled bipolar TCs with long prolongations. Short arrows show large double-labeled multipolar cells present in between muscle fibers and in the lamina propria. U, urothelium; Su, suburothelium; lp, lamina propria; m, muscle layer. Scale bars = 40 μm.

Figure 4

Quantification of vimentin (a), CD34 (b), and PDGFRα (c) immunoreactivity in the bladder and urethra of rats given CYP and Imatinib. The staining area above the intensity threshold was measured in hand drawn fields of bladder and urethral sections. Measurements were made independently in the lamina propria and the muscle layer of both organs. Values represent the mean ± SEM (n = 6-7 different fields from at least 4 different animals): ^∗∗P < 0.01 and ^∗∗∗P < 0.001 compared to controls; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 compared to the corresponding treatment without Imatinib (one-way ANOVA followed by Dunnett's multiple comparison test).

Figure 5

Changes in voiding frequency and in urinary tract wet weight in CYP-treated rats and the reversion following Imatinib administration. The total number and the number of small-volume voids per hour were measured (a), as well as the complete lower urinary tract wet weight after sacrificing the animals (b). Data are expressed as the mean ± SEM (n = 6-7 per group): ^∗∗P < 0.01 and ^∗∗∗P < 0.001 compared to controls; ^#P < 0.05 and ^###P < 0.001 compared with the corresponding treatment without Imatinib (one-way ANOVA followed by Dunnett's multiple comparison test).

Figure 6

The eNOS expression induced by CYP in ICs within the rat bladder and urethra and the preventive effect of Imatinib. (a–f) Representative images showing the labeling in the urethra for eNOS (green) and vimentin (vim, red) in the muscle layer after CYP-intermediate (a) and in the lamina propria after CYP-chronic treatment (b). Similar labeling in the bladder of CYP-chronic (c), control (d), Imatinib-intermediate (e), and Imatinib-chronic (f) rats. The colocalization of eNOS with vimentin was only evident in CYP-treated animals (a–c), while it was restricted to the endothelium in controls (d) and in animals pretreated with Imatinib (e, f). (g-h) Representative images showing eNOS (red) and CD34 (green) colabeling of ICs in the lamina propria (g) and the muscle layer (h) of CYP-intermediate treated bladders. (i–k) Labeling for vimentin (red, (i)) and eNOS (j) and the merged image (k) of ICs in the lamina propria of a CYP-intermediate treated bladder. The nuclei were counterstained with DAPI (blue) in (i) and (j). Long arrows indicate double-labeled bipolar cells with long prolongations. Short arrows show double-labeled multipolar cells located between muscle fibers and in the lamina propria. Asterisks indicate the labeling of intramural vessels. U, urothelium; lp, lamina propria; m, muscle layer. Scale bars = 40 μm except in (c) and (d) (20 μm) and (f) (80 μm).