Methods for Quantitative Analyses of Nerve Fiber Deformation in the Myenteric Plexus Under Loading of Mouse Distal Colon and Rectum.

Visceral pain in the large bowel is a hallmark of irritable bowel syndrome (IBS) and the primary reason patients seek gastroenterological care. Notably, mechanical distension of the distal colon and rectum (colorectum) reliably evokes abdominal pain and thus understanding mechanotransduction of sensory nerve endings (nerve fibers) in the colorectum is crucial for understanding and treating IBS-related bowel pain. To facilitate such understanding we aimed to establish novel methods to mechanically test, image, and analyze large-strain deformations of networks of nerve fibers in the myenteric plexus of the colorectum, and thus enable quantitative analyses. We successfully delivered circumferential deformation (force or displacement driven) to intact segments of colorectum while maintaining the myenteric plexus in focus during fluorescent imaging to capture the deforming nerve fibers. We also established a semi-automated method to recapitulate the network morphology and a code to calculate the stretch ratios of individual nerve fibers deforming within the myenteric plexus of mouse colorectum. Our code allows plotting of stretch ratios for each fiber, stretch ratios vs. fiber angle, and stretch ratios vs. fiber length. Our methods not only facilitate analyses of deformations of networks of colorectal nerve fibers in the context of visceral nociception but are also applicable to analyzing the in-plane deformation of other two-dimensional fiber networks. We provide free, public access to our analysis code for MATLAB, including input files for a simple test case, at github.uconn.edu/imLab/Fiber-Network_Analyses .