Differential biomechanical properties of mouse distal colon and rectum innervated by the splanchnic and pelvic afferents

Abstract

Visceral pain is one of the principal complaints of patients with irritable bowel syndrome, and this pain is reliably evoked by mechanical distension and stretch of distal colon and rectum (colorectum). This study focuses on the biomechanics of the colorectum that could play critical roles in mechanical neural encoding. We harvested the distal 30 mm of the colorectum from mice, divided evenly into three 10-mm-long segments (colonic, intermediate and rectal), and conducted biaxial mechanical stretch tests and opening-angle measurements for each tissue segment. In addition, we determined the collagen fiber orientations and contents across the thickness of the colorectal wall by nonlinear imaging via second harmonic generation (SHG). Our results reveal a progressive increase in tissue compliance and prestress from colonic to rectal segments, which supports prior electrophysiological findings of distinct mechanical neural encodings by afferents in the lumbar splanchnic nerves (LSN) and pelvic nerves (PN) that dominate colonic and rectal innervations, respectively. The colorectum is significantly more viscoelastic in the circumferential direction than in the axial direction. In addition, our SHG results reveal a rich collagen network in the submucosa and orients approximately ±30° to the axial direction, consistent with the biaxial test results presenting almost twice the stiffness in axial direction versus the circumferential direction. Results from current biomechanical study strongly indicate the prominent roles of local tissue biomechanics in determining the differential mechanical neural encoding functions in different regions of the colorectum. NEW & NOTEWORTHY Mechanical distension and stretch-not heat, cutting, or pinching-reliably evoke pain from distal colon and rectum. We report different local mechanics along the longitudinal length of the colorectum, which is consistent with the existing literature on distinct mechanotransduction of afferents innervating proximal and distal regions of the colorectum. This study draws attention to local mechanics as a potential determinant factor for mechanical neural encoding of the colorectum, which is crucial in visceral nociception.

Keywords: biaxial test; irritable bowel syndromes; mechanotransduction; second harmonic generation; visceral pain.

Fig. 1.

The schematic of the sensory innervations of mouse distal colon and rectum (colorectum) by lumbar splanchnic and pelvic nerves. Considering the anus as zero coordinate, the distal 30 mm of the colorectum is divided into rectal (0–10 mm), intermediate (10–20 mm), and colonic (20–30 mm) segments.

Fig. 2.

Biaxial tissue testing of specimen from mouse colorectum and local strain measurement. A: specimen (7×7 mm² squares) were harvested from three segments of the colorectum (rectal, intermediate, and colonic) and mounted onto a custom-built adaptor for biaxial stretch tests. B: adaptor fabricated by 3-D printing was further trimmed and configured as in A to allow lateral displacement of the specimen during biaxial stretch with minimal resistance force (0.5 mN/mm). C: photo images of a specimen sprayed with carbon dots for local strain measurement by optical tracking. D: magnified view of the carbon dots in the white square region in C, showing speckle patterns of micrometers in size.

Fig. 3.

Cauchy stress-stretch ratio results from quasi-static biaxial stretch test at multiple stretch rates. Linear ramped loading (0–80 mN) and unloading (80–0 mN) forces were applied to the specimen at multiple rates: 0.4, 1.2, and 2 mN/s. Stretch rates below 1.2 mN/s provide repeatable and robust results, and ramped forces of 1.2 mN/s were used as the testing protocol throughout.

Fig. 4.

Colorectal stress-strain behavior determined by biaxial tissue stretch tests. A: representative Cauchy stress-stretch ratio curves (solid colored lines) calculated as the average of the last three loading/unloading cycles of the total 30 cycles. Displayed in gray lines were the first 27 loading/unloading cycles for tissue preconditioning. The Cauchy stress-stretch ratio curves in the loading cycle at colonic, intermediate, and rectal segments from male (B) and female (C) mice, respectively. Repeatable stretch forces were delivered to all specimens, and the average stretch ratios were plotted along with the standard error of the mean. For male colorectum, data from 8 colonic, 10 intermediate, and 9 rectal specimens were analyzed. For female colorectum, data from 8 colonic, 10 intermediate, and 9 rectal specimens were analyzed.

Fig. 5.

Colorectal tissue viscoelasticity quantified as a fraction of energy loss. A: force-displacement curves were used to calculate the fraction of energy loss using the gray area enclosed by the loading and unloading curves normalized by the total area under the loading curve (gray plus hatched area). B: fraction of energy loss from 16 colonic, 20 intermediate, and 18 rectal segments. The horizontal bars indicate the average fraction of energy loss.

Fig. 6.

Residual stresses in the colorectum quantified by opening angle measurements. A: photographs of tissue rings (2 mm thick) harvested from rectal, intermediate, and colonic segments before and after cutting open. The opening angles are labeled as θ. B: average opening angles measured from seven colons are 42.4, 58.4, and 119.4°, gradually increasing from proximal to distal locations in the colorectum.

Fig. 7.

Planer axial (A), circumferential (B), and shear strain (C) distribution during biaxial tissue testing on the 7 × 7 mm² colorectal specimen verifies homogeneous strain distribution. Average shear strain is one order of magnitude lower than strain in axial and circumferential directions.

Fig. 8.

Histological studies by hematoxylin-and-eosin (H&E) staining (A) and second harmonic generation (SHG) imaging (B), respectively. The H&E staining confirms the tissue integrity after the biaxial mechanical stretch protocol. Representative SHG images show different through-thickness fiber orientations and thicknesses for the corresponding layers. C. muscular, circular muscular; L. muscular, longitudinal muscular. Scale bars indicate 200 µm in A and 50 µm in B.