Delayed toxicity of cyclophosphamide on the bladder of DBA/2 and C57BL/6 female mouse

Abstract

The present study describes the delayed development of a severe bladder pathology in a susceptible strain of mice (DBA/2) but not in a resistant strain (C57BL/6) when both were treated with a single 300 mg/kg dose of cyclophosphamide (CY). Inbred DBA/2 and C57BL/6 female mice were injected with CY, and the effect of the drug on the bladder was assessed during 100 days by light microscopy using different staining procedures, and after 30 days by conventional electron microscopy. Early CY toxicity caused a typical haemorrhagic cystitis in both strains that was completely repaired in about 7-10 days. After 30 days of CY injection ulcerous and non-ulcerous forms of chronic cystitis appeared in 86% of DBA/2 mice but only in 4% of C57BL/6 mice. Delayed cystitis was characterized by infiltration and transepithelial passage into the lumen of inflammatory cells and by frequent exfoliation of the urothelium. Mast cells appeared in the connective and muscular layers of the bladder at a much higher number in DBA/2 mice than in C57BL/6 mice or untreated controls. Electron microscopy disclosed the absence of the typical discoidal vesicles normally present in the cytoplasm of surface cells. Instead, numerous abnormal vesicles containing one or several dark granules were observed in the cytoplasm of cells from all the epithelial layers. Delayed cystitis still persisted in DBA/2 mice 100 days after treatment. These results indicate that delayed toxicity of CY in female DBA/2 mice causes a bladder pathology that is not observed in C57BL/6 mice. This pathology resembles interstitial cystitis in humans and could perhaps be used as an animal model for studies on the disease.

Figure 1

a) Photomicrograph of untreated DBA/2 mouse bladder stained with trichrome modified stain, showing normal transitional epithelium, mucosa (m) and muscle bundles (arrowhead). b) Bladder of untreated C57BL/6 mouse, stained with haematoxylin-eosin showing normal transitional epithelium, mucosa (m) and muscle bundles (arrowhead). c) Bladder of DBA/2 mouse 58 days after treatment with CY. Stained with trichrome modified stain, showing the urothelium partially detached (star) and intense infiltration of leucocytes in the oedematous connective tissue of the mucosa (m). d) Bladder of C57BL/6 mouse 70 days after treatment with CY. Stained with haematoxylin-eosin, showing normal transitional epithelium, mucosa (m) and muscle bundles (arrowhead). e) Bladder of DBA/2 mouse 87 days after CY, stained with acidified toluidine blue. Numerous mast cells are observed in the connective tissue of the mucosa, and also between muscle fibres and in the adventitial tissue. f) Photomicrograph of bladder of C57BL/6 mouse 70 days after CY, stained with acidified toluidine blue. Only one mast cell is seen in the whole section. Original magnification × 200.

Figure 2

a) Electron photomicrograph of bladder of untreated DBA/2 mouse. The transitional epithelium shows a characteristic scalloped appearance in the luminal surface and discoidal vesicles are observed (DV). The intermediate layer (IL) presents whorl-like structures surrounded by layers of membranes (W) as well as mitochondria and a few lysosomes. In the basal layer (BL) the cells are small and contain numerous mitochondria. L: lumen. b) Bladder of DBA/2 mouse 58 days after CY. Multiple granules of different sizes filled the dark (DC) and light surface cells which are completely depleted of discoidal vesicles. In the intermediate layer (IL), the cells present many round vesicles containing one or various round granules (V) and in some cases also lipid droplets (LD); the complete disappearance of whorl-like structures is very characteristic. L: lumen. Original magnification × 5000. Bars: 2 µm.

Figure 3

Electron photomicrograph of DBA/2 mouse bladder 57 days after CY. A group of mast cells (MC) with homogeneous electron-dense granules are seen in the connective tissue of the lamina propria. E: eosinophil leucocyte. Original magnification × 5000. Bars: 2 µm.

Figure 4

Electron photomicrograph of C57BL/6 mouse bladder 70 days after CY. The ultrastructure of the transitional epithelium is similar to that of the untreated DBA/2 mice except the cells are larger than those observed in Fig. 2a, and a higher number of discoidal vesicles (DV) appears in the surface cells. W: whorl-like structure. L: lumen. Original magnification × 5000. Bars: 2 µm.