Age-related changes in murine bladder structure and sensory innervation: a multiphoton microscopy quantitative analysis

Abstract

Our study aimed to examine and quantify age-related structural alterations in the healthy mouse bladder using ex vivo two-photon laser scanning microscopy (TPLSM). Freshly dissected bladders from 25-, 52-, and 85-week-old C57bl/6J mice were examined, and morphological analyses and quantification of cell layers and nerves were performed. The numbers of stretched, curled, branched, and total number of nerves in volume units of the stained muscle layer were quantified. We observed differences in the bladder wall architecture and innervation with age. Especially in 85-week-old mice, age-related changes were found, including detachment of urothelial cells and an increase in connective tissue, intermingled with the smooth muscle fibers in the muscle layer (collagen-smooth muscle ratio of 1.15 ± 0.29). In 25- and 52-week-old mice, the collagen-smooth muscle ratios were 0.20 ± 0.04 and 0.31 ± 0.11, respectively, and a clear separation of collagen and muscle was observed. The overall number of nerves and the number of curled nerves were significantly higher in the 85-week-old mice (74.0 ± 13.0 and 25.9 ± 4.8, respectively), when comparing to 25-week-old mice (26.0 ± 2.7 and 6.7 ± 1.2, respectively) and 52-week-old mice (43.8 ± 4.3 and 22.1 ± 3.3, respectively). Significant age-related alterations in bladder morphology and innervation were found, when comparing freshly dissected bladder tissue from 25-, 52-, and 85-week-old mice. The higher number of curled nerves might be an indication of an increased neurotransmitter release, resulting in a higher nerve activity, with a part of the nerves being possibly mechanically impaired. This study shows that two-photon laser scanning microscopy of healthy aging male mice is a useful method to investigate and quantify the age-related changes in the bladder wall.

Keywords: Aging; Mice; Multiphoton microscopy; Sensory innervation; Urinary bladder.

Fig. 1

Autofluorescent, label-free images of the bladder mucosa (imaged from the urothelial side) of a, b 25-week-old, c 52-week-old, and d 85-week-old mice. a Transepithelial lining of the urothelium (s superficial layer, i intermediate layer, and b basal layer). Cytoplasm of urothelial cells (white arrow) was visible in the green channel, with non-fluorescent nuclei (white asterisk). b Typical hexagonally-shaped umbrella cells of the superficial urothelial layer (white arrow), showing cell components (red arrowhead). c Detachment of superficial urothelial cells from the epithelial lining (white arrow heads) and degeneration of urothelial cells (red asterisk). d Increased degeneration of urothelial cells (red asterisk). Cells appeared more deformed and showed a loss of cytoplasm. The multi-layered epithelial lining of the urothelium was distorted; the underlying collagen in the lamina propria/sub-urothelium was visible (Co). Collagen was visualized in the blue channel due to second harmonic generation. Scale bars, 20 μm

Fig. 2

Autofluorescent, label-free images of the lamina propria (LP) (imaged from the urothelial side) in the bladder wall of a 25-week-old, b 52-week-old, and c 85-week-old mice. a Collagen, below the urothelium, could be seen in the second, spectral channel (Co). Vessel structures with a diameter ranging from 10 to 40 μm were found (white asterisk). b Collagen could be detected with a strong second harmonic generation signal (Co, white asterisk). Also, blood vessel could be seen in the LP (white arrow). c An increase of amount and density of collagen was noticeable, reaching from the sub-urothelium, throughout the LP to the smooth muscle layer (Co). The connective tissue appeared multi-layered, with a layer existing of rather straight-shaped collagen (Co, white asterisk) bundles (20–40 μm), followed by a thick layer of curled collagen (Co, red asterisk) bundles (40–80 μm). Vessel structures in the LP were more dilated and were showing a diameter of up to approx. 70 μm (white arrow). Macrophages were also found (red arrow) and confirmed with MOMA-1 immuno-histochemical staining. Scale bars, 20 μm

Fig. 3

Label-free images (imaged from the adventitial side) of the muscle layer and collagen-smooth muscle ratio in the bladder wall of a 25-week-old and b, c 85-week-old mice. Collagen was seen in the blue spectral channel (indicated by Co and white broken lines) and could be separated from the muscle layer, seen in the green spectral channel (indicated by M). a–c The amount of blue connective tissue (Co) increased significantly (p = 0.001) in 85-week-old mice in comparison to that in 25-week-old mice. It appeared more intermingled with the green bundles of the muscle layer (M). a is taken from a z-stack of the muscle layer. LP and urothelium can be found below the muscle layer in the z-stack, but not shown in the image. c 3D visualization of b: tissue volume showing that the collagen-smooth muscle ratio of the aged mice (collagen indicated by broken lines and Co, muscle indicated by M), which also increased significant (p < 0.001) in contrast to the 25-week-old mice. Macrophages were found and indicated by a red arrow. Scale bars, 20 μm

Fig. 4

Quantification of layer thickness: a Collagen layer, b muscle layer, c collagen-smooth muscle ratio. Squares indicate means, whiskers indicate ±1SE. SE standard error of the mean. In producing panel c, firstly, collagen-muscle ratios of all stacks were computed. Secondly, from thus array of ratios, mean and the ±SE were computed

Fig. 5

Images of label-free nerve fibers (imaged from the adventitial side) in the stained muscle layer of the bladder wall. Nerves (white arrowhead) and cell nuclei (SYTO 13 staining) of macrophages (red arrow) and smooth muscle cells (white arrow) could be detected in the green channel. Collagen (Co) was seen in the blue channel, and stained muscle fibers (M, Sulforhodamine B staining) in the red channel. a Straight-shaped muscle bundles (M) with smooth muscle cells (white arrow) and nerves (white arrowhead). b, c Magnification of stretched nerves (white arrowhead), showing a branching point (asterisk). d, e Magnification of curled nerves (white arrowhead) with branching point (asterisk). Scale bars, 20 μm

Fig. 6

Quantification of nerves in the muscle layer of the dome region: a stretched nerves, b branched nerves, c curled nerves, and d total number of nerves. Squares indicate means, whiskers indicate ±1SE. SE standard error of the mean