The prevalence of lower urinary tract symptoms (LUTS), characterized by problems regarding storage and/or voiding of urine, is known to significantly increase with age [1]. Multiple bladder components may become dysfunctional with age including mucosal, muscular, stromal and neural physiology [2]. Optimal execution of visceroceptive processes is essential for the maintenance of a proper homeostatic balance and is achieved by interactions between the central and peripheral nervous systems at multiple levels. Interestingly, the process of aging decreases visceroceptive awareness. Currently, it is unclear how this interaction is organized, and how the aging process negatively impacts these functions. 
The correct execution of communication between the lower urinary tract and the central nervous system is crucial for optimal bladder functioning. Moreover, this system is highly dependent on effective interaction between the bladder urothelium and afferent nerve fibers located in the lamina propria in close proximity to the urothelium. In the current study we quantified aging-related differences in the expression of calcitonin gene-related peptide (CGRP, an established marker for sensory nerve fibers) in the trigonal mucosal layers of young (3 – 4 months) and aged (25 – 30 months) rats. We hypothesized that trigonal mucosa obtained from aged rats shows significantly less CGRP expression compared to young rats.

We collected bladders from 3 animals per age group. The bladder was cut open longitudinally and pinned flat with the lumen side upwards. Tissue was post-fixed for 60 minutes in 4% PFA, immersed in 30% sucrose for > 24 hours. The trigonal region between the ureteral orifices and the urethra was isolated, embedded in optimum cutting temperature compound medium (Tissue-Tek OCT, Sakura Finetek, Torrance, CA, USA) and stored at -80°C until cutting. Tissue was then serially sectioned at 10 μm and mounted on glass slides (Fisher Scientific, Pittsburgh, PA) with 3 sections per slide. Sections were air dried >1hour, washed with PBS, incubated with permeabilizing block solution (5% normal donkey serum, 1% bovine serum [Sigma-Aldrich, USA] and 0.2% Triton X-100) for 1 hour, followed by incubation with primary antibody (anti-CGRP, 1:2000, Sigma) overnight at 4°C. The following day, slides were washed with PBS and incubated with secondary Cy3 goat anti-rabbit (Alexa Fluor 555, 1:500) in blocking solution for 2 hrs at room temperature. DAPI (1:2500) 15 min at room temperature was then used to counterstain the nuclei. Slides were post fixated with 4% PFA for 10 minutes and washed with PBS. Slides were then cover slipped using mounting medium (Immuni-Mount) and dried >24hours at room temperature in the dark. Control experiments included omission of primary antibody from blocking solution.
Images were taken so that the full length of each section was fully imaged. The images (N = 160) were binarized in ImageJ using the triangle algorithm for optimal thresholding and median filtering was applied to reduce single pixel noise. A region of interest detection threshold was applied to detect a minimum of 10 connected CGRP positive pixels. For each image we computed the total CGRP positive area (μm2) and the median value for each animal was used for further analysis.

Representative images of CGRP expression in young and aged trigonal mucosa are shown in figure 1.A and B. Upon statistical analysis the trigonal mucosa of aged animals shows a significantly lower CGRP positive area compared to young animals (p = 0.0049) (Fig. 1.C). These results indicate that aging has a negative effect on the area of CGRP positive signal in the trigone. The structural and functional integrity of the web of sensory nerves in the trigonum of aged animals is significantly affected, and communication between the bladder urothelium and the central nervous system is highly likely compromised as a result.

Based on the findings reported here, we argue that damage to the sensory system in the trigonal mucosa may be a likely contributor to the development or exacerbation of LUTS. To our knowledge, the detrimental effects of aging on sensory nerve fibers in the bladder has not been quantified before. These new insights advance our understanding of LUTS in older adults and improve treatment selection strategies and therapeutic developments.

The structural and functional integrity of the web of sensory nerves in the trigonal mucosa of aged animals is negatively impacted by the aging process (in part due to increased oxidative stress over time [3]), and communication between the periphery and the CNS is highly likely compromised as a result. These detrimental changes to the sensory system are highly likely associated with the reported decrease in visceroceptive awareness in older adults and may be a contributor to the development or exacerbation of LUTS. Future research should assess potential regional differences within the bladder in aging-related changes in sensory innervation with the potential to identify novel therapeutic targets which might improve the quality of life and self-reliance of many older adults suffering from LUTS.

Gibson W, Wagg A. Incontinence in the elderly,'normal'ageing, or unaddressed pathology? Nature Reviews Urology. 2017;14(7):440-448.
Suskind AM. The aging overactive bladder: A review of aging-related changes from the brain to the bladder. Curr Bladder Dysfunc. 2017;12(1):42-47.
Birder, L. A., Kullmann, A. F., & Chapple, C. R. (2018). The aging bladder insights from animal models. Asian journal of urology, 5(3), 135-140.

Continence 2S2 (2022) 100463
DOI: 10.1016/j.cont.2022.100463