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. 2020 Jan;190(1):108–124. doi: 10.1016/j.ajpath.2019.09.015

Figure 1.

Figure 1

Representative images of mouse bladder urothelium after cyclophosphamide injection. A–E: Hematoxylin and eosin (H&E) images from 0 to 28 days after cyclophosphamide administration. A: Uninjured adult mouse bladder with three to four cell layers, including large polyploid superficial (arrowhead) and smaller deeper (arrow) cells. B: Mouse bladder 1 day after cyclophosphamide administration shows significant urothelial cell sloughing (black arrowheads), leaving regions of denuding (arrow) and hemorrhage (green arrowheads). C: Mouse bladder 3 days after cyclophosphamide administration shows urothelial regeneration with cellular hyperplasia (arrow). D: Mouse bladder 10 days after cyclophosphamide administration shows ongoing urothelial cell regeneration with increased cellular hyperplasia (arrow). E: Mouse bladder 28 days after cyclophosphamide administration shows apparent restoration of normal urothelial cell layers, including superficial cells (arrowhead) and smaller deeper cells (arrow). F–J: Images at 2 hours after cyclophosphamide administration. F: H&E-stained bladder shows some sloughing of urothelial cells (arrow). G: Immunofluorescence (IF) for superficial cell marker, KRT20 (red), in a section adjacent to F shows that the sloughing cells (arrow) are KRT20+ superficial cells and that regions of remnant urothelium have lost KRT20+ superficial cells (arrowhead). H: IF for the superficial cell and intermediate cell subset marker, uroplakin 3a (UPK3; white) in a section adjacent to G shows coexpression with KRT20 in sloughing superficial cells (arrow) but preserved expression on luminal urothelial surface, even in regions without KRT20 (arrowhead), consistent with preserved UPK3+ intermediate cells. I: IF for the intermediate cell and basal cell marker, KRT5 (green), in the same section as H shows intact KRT5+ intermediate and basal cells. J: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (green) in a section adjacent to H and I is negative throughout the urothelium. K–O: Images at 12 hours after cyclophosphamide administration. K: H&E-stained bladder showing detachment of sheets of urothelial cells (arrow). L: KRT20 IF (red) in an adjacent section to K is completely negative, even on detaching urothelial cells (arrowhead), consistent with previous loss of superficial cells. M and N: UPK3 IF (white; M) and KRT5 IF (green; N) in a section adjacent to L show injury and detachment of sheets of UPK3+ and KRT5+ intermediate cells (arrows). O: TUNEL staining (green) in a section adjacent to M and N shows widespread apoptosis throughout the detaching and remnant urothelium (arrowheads). A–E, J, and O: Dotted lines indicate demarcation between urothelial layer and underlying lamina propria. G–J and L–O: Blue indicates DAPI. Scale bars = 50 μm (A–O). L, lumen.