Table 1.
Age-related differences in the levels of antioxidants, lactate dehydrogenase and malondialdehyde in the urothelium and serum of young and aging C57BL/6JOlaHsd female mice.
Figure 1.
Immunolabeling of iNOS and COX in aging and young mice urothelium.
(a) The intense brown colour shows a strong positive immunoreaction against iNOS in the cytoplasm of superficial, intermediate and basal urothelial cells of aging mice. Nuclei are stained with haematoxylin (blue). (b) Only occasional iNOS-positive superficial cells with pale brown-coloured cytoplasm are detected in the urothelium of young mice, while intermediate and basal cells are almost completely iNOS-negative. Nuclei are stained with haematoxylin (blue). (c) Weak immunoreaction (green fluorescence) against COX in all urothelial cells of aging mice. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. (d) Intense and extensive dotty reaction (green fluorescence) against COX in the cytoplasm of superficial urothelial cells of young mice. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. SC-superficial cells, IC-intermediate cells, BC-basal cells, L-lumen of the urinary bladder.
Figure 2.
Immunolabeling of Ki-67, active caspase 3, uroplakins and cytokeratin 20 in aging mice urothelium.
(a) Individual Ki-67 positive cells with red fluorescent signal (asterisk) are present in the urothelium of aging mice. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. (b) No apoptotic cells (red fluorescence) were detected in the urothelium. Apoptotic fibroblast in connective tissue is marked with asterisk. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. (c) Immunoreaction against uroplakins shows typical labeling (red fluorescence) of the apical plasma membrane and the membranes of specific vesicles - fusiform vesicles in the cytoplasm of superficial and intermediate cells. Nuclei are stained with DAPI (blue). (d) Immunoreaction against cytokeratin 20 (green fluorescence) reveals typical network of this cytokeratin in the apical cytoplasm of superficial cells. Nuclei are stained with DAPI (blue). Arrows show lipofuscin granules. SC-superficial cells, IC-intermediate cells, BC-basal cells, L-lumen of the urinary bladder.
Figure 3.
Structure of aging and young mice urothelium.
(a) Semi-thin section of urothelium in aging mice showing normal structure of three-layered epithelium with numerous lipofuscin granules in the cytoplasm of superficial cells (arrows). (b) Semi-thin section of urothelium in young mice showing normal structure of three-layered epithelium with no lipofuscin granules in the superficial cells. (c) Ultrathin section of aging urothelium. Ultrastructure of superficial cell fulfiled with specific fusiform vesicles and numerous osmiophilic lipofuscin granules. (d) Ultrathin section of young urothelium. Typical ultrastructural appearance of superficial cell with large amounts of fusiform vesicles in the cytoplasm but no lipofuscin granules. SC-superficial cells, IC-intermediate cells, BC-basal cells, N-nucleus, L-lumen of the urinary bladder.
Figure 4.
Electron micrographs of lipofuscin in superficial cells of aging mice urothelium.
(a) Ultrastructural appearance of lipofuscin granules (arrowheads), heterogeneous in their size and structure. (b) Lipofuscin surrounded by double membrane (arrowheads), indicating that the structure is an early autophagic vacuole (autophagosome). (c) Lipofuscin in late autophagic vacuole (autophagolysosome), limited by single membrane (arrow). (d) The lipofuscin granule (arrow) is filled with electron-dense precipitate as the reaction product of enzyme histochemistry for detection of acid phosphatase activity as the main lysosomal enzyme. Precipitate at the sites of acid phosphatase activity proves that lipofuscin-loaded autophagolysosome contains active lysosomal enzymes trying to degrade the undegradable lipofuscin. FV-fusiform vesicles, typical structures of superficial urothelial cells, L-lumen of the urinary bladder.
Figure 5.
Autofluorescence of lipofuscin granules in superficial cells of aging mice urothelium.
Images were taken using ultraviolet (a), blue (b), green (c), ultraviolet and blue (d) excitation light. Image (d) shows that lipofuscin granules appear exclusively in superficial cells and mostly in the vicinity of their nuclei. Arrows shows clusters of lipofuscin granules. In a, d: Nuclei are stained with DAPI (blue). L-lumen of the urinary bladder.
Figure 6.
Ultrastructural appearance of mitochondria in superficial cells of aging mice urothelium.
(a) Enlarged and dilated mitochondria with no visible cristae inside. (b) Mitochondria of normal size and shape but with electron-dense inclusions (arrows). (c) Mitochondria of normal size and shape but with partial or complete destruction of cristae (star frames). L-lumen of the urinary bladder.