Background: Aged nerves present a series of features that trigger abnormal nerve functionality in the form of slower conduction velocity, abnormal neurotransmitter release, and reduced regeneration capacity. While these age-related changes are known to induce skeletal muscle weakness and degeneration, how cardiac nerves are affected by age, and whether nerve ageing impacts cardiac muscle functionality, remains unknown. One of the reasons for this knowledge gap is the limited availability of experimental tools to study the intracardiac nervous system (ICNS). In the present work, we introduce a novel imaging approach for studying thick tissue sections that improves the quality of structural information that can be obtained on the ICNS. We apply this approach to describe so-far hidden features of peripheral nerve aging.
Methods:
For immunofluorescence (IF), sciatic nerves (n=6) and hearts (n=4) from 2-3 and 18-24-month-old mice were harvested. Sciatic nerves were then fixed and sectioned into 3 pieces for whole-mount IF. Hearts were embedded in OCT, snap frozen in liquid nitrogen, and 20-μm sections were fixed for IF. The total number of nuclei (stained with DAPI), Sox10+ nuclei (Schwann cells, SC), PH3+ nuclei (cell cycling cells), and Sox10+/PH3+ (cell cycling SC) nuclei were quantified using a semi-automatic routine for ImageJ. CD68 was used to detect phagocytes, and the number of CD68+ cells with cytoplasmic Sox10 positive signal was counted to study the SC-phagocytosis index. Finally, a thick tissue (0.5 mm) 3D imaging protocol, that does not require detergent-based tissue clearing, was developed. For this, DAPI was incorporated into the washing and fixing solutions of Col1a1-eGFP hearts. After coronary perfusion-based chemical fixation, the heart was sectioned with a vibratome to obtain 0.5 mm slices. The slices were then incubated with DAPI in phosphate-buffered saline at 4oC for 5 days in a nutator. Sections were embedded in a glycerol-based mounting medium and cured in a humidity-controlled chamber to optimize the refractive index to reduce scattering. Finally, samples were mounted in-between 2 cover slides and imaged using a multiphoton microscope.
Results:
Sciatic nerves from old mice show reduced numbers of total nuclei, proliferative cells, SC, and proliferative SC compared to young mice. At the same time, a higher number of phagocytic cells (CD68+) with cytoplasmic Sox10 signal was found in aged nerves. In the hearts from old mice, a reduction in the number of SC was observed, compared to young mice. Finally, using the above protocol for thick tissue imaging, fluorescent signals from GFP-positive cells and DAPI stained nuclei were obtained through the whole atrium and 0.5 mm of ventricular sections from the Col1a1-eGFP hearts.
Conclusions and future directions:
The reduction in cell numbers found in aged nerves may be explained by reduced cell cycling activity of the remaining cells, and/or increased SC phagocytosis. In ongoing work, we are exploring whether these changes also take place in the ICNS. So far, a reduction in the number of SC has been found. Our findings, together with a recent report by Elia et al. (J Geriatr Cardiol, 2021), wherein a depletion of nerve fibres was described in human old hearts, makes it worth exploring if these neural changes alter on cardiac electrophysiology and the propensity for arrhythmias.