Load-bearing function of the colorectal submucosa and its relevance to visceral nociception elicited by mechanical stretch

Abstract

Mechanical distension beyond a particular threshold evokes visceral pain from distal colon and rectum (colorectum), and thus biomechanics plays a central role in visceral nociception. In this study we focused on the layered structure of the colorectum through the wall thickness and determined the biomechanical properties of layer-separated colorectal tissue. We harvested the distal 30 mm of mouse colorectum and dissected this tissue into inner and outer composite layers. The inner composite consists of the mucosa and submucosa, whereas the outer composite includes the muscular layers and serosa. We divided each composite axially into three 10-mm-long segments and conducted biaxial mechanical extension tests and opening-angle measurements for each tissue segment. In addition, we quantified the thickness of the rich collagen network in the submucosa by nonlinear imaging via second-harmonic generation (SHG). Our results reveal that the inner composite is slightly stiffer in the axial direction, whereas the outer composite is stiffer circumferentially. The stiffness of the inner composite in the axial direction is about twice that in the circumferential direction, consistent with the orientations of collagen fibers in the submucosa approximately ±30° to the axial direction. Submucosal thickness measured by SHG showed no difference from proximal to distal colorectum under the load-free condition, which likely contributes to the comparable tension stiffness of the inner composite along the colorectum. This, in turn, strongly indicates the submucosa as the load-bearing structure of the colorectum. This further implies nociceptive roles for the colorectal afferent endings in the submucosa, which likely encode tissue-injurious mechanical distension.

NEW & NOTEWORTHY Visceral pain from distal colon and rectum (colorectum) is usually elicited from mechanical distension/stretch, rather than from heating, cutting, or pinching, which usually evoke pain from the skin. We conducted layer-separated biomechanical tests on mouse colorectum and identified an unexpected role of submucosa as the load-bearing structure of the colorectum. Outcomes of this study will focus attention on sensory nerve endings in the submucosa that likely encode tissue-injurious distension/stretch to cause visceral pain.

INTRODUCTION

Visceral pain is the cardinal symptom of irritable bowel syndrome (IBS), which affects 15–20% of the US population, and is one of the most frequent reasons for patients to seek medical attention (8). IBS-related visceral pain arises from the distal colon and rectum (colorectum) with several unique clinical characteristics that can be linked to the biomechanics. First, nonmechanical stimuli to the colorectum (e.g., burning) often fail to evoke pain or other sensory perceptions (37). In contrast to the various pain stimuli experienced at the skin (e.g., burning, cold, pressure, cutting, etc.), visceral pain from the colorectum lacks well-defined modalities and often presents as a dull or cramped sensation (2). Second, certain injurious mechanical stimuli, such as cutting or pinching the intestine, cause no pain (8). IBS pain is usually unrelated to injury; the patients’ colorectums lack apparent structural damage or inflammation and appear “normal” compared with colorectums in healthy subjects (16). Third, it is mechanical distension of the colorectum that reliably evokes pain in both healthy controls and patients with IBS (37). Indeed, heightened perception of pain during rectal or colonic distension (visceral hypersensitivity) is considered a biomarker for IBS (7, 9).

Sensory afferent neurons with endings embedded in the colorectal wall encode mechanical stimuli within the colorectum. Macroscopic mechanical stimuli in the colorectum trigger microscopic mechanical stress/strain at the afferent endings and surrounding tissues. This causes the opening of putative mechanosensitive ion channels in the lipid bilayer membrane of the afferent endings to depolarize the membrane potential, which leads to the generation of a train of action potentials informing the central nervous system (20). Thus, the biomechanics of the colorectum both at the macroscopic and microscopic levels significantly influences this mechanotransduction process.

In contrast to the plethora of knowledge about the neurophysiology of colorectal afferents to encode different modalities of stimuli (e.g., 11, 12, 14, 15, 17, 18, 20, 28), our biomechanical understanding of the colorectum is limited. A recent study by us revealed differential biomechanical properties from proximal to distal regions of the colorectum, an area predominantly innervated by the lumbar splanchnic nerves (LSNs) in the proximal colonic region and the pelvic nerves (PNs) in the distal rectal region (42). We reported that the distinct mechanical properties between the colonic and rectal regions in the colorectum are consistent with the differential neural-encoding functions of LSN and PN afferents that dominate the colonic and rectal innervations, respectively. This is compelling evidence that strongly indicates the determinant roles of tissue biomechanics in afferent neural encoding of mechanical stimuli.

The colorectum also has a layered structure through the thickness of the wall, consisting of two major composites loosely connected at the interstitial space between the submucosa and circular muscular layers. The inner composite includes the mucosal and submucosal layers, whereas the outer composite includes the circular muscular, intermuscular, longitudinal muscular, and serosal layers. Sparse neural-tracing studies intricately revealed the locations and morphology of PN afferent endings in different layers of the colorectum, showing a concentration of afferent endings in the submucosal and muscular layers (6, 45). Using nonlinear imaging, i.e., second-harmonic generation (SHG), we revealed thick and dense collagen fibers in the submucosa that form a sheetlike network with orientations approximately ±30° from the axial direction (42). Furthermore, we recently conducted systematic quantification of the SHG signals and determined the morphology, density, and orientation of collagen fibers through the thickness of flattened mouse colorectum (B. Feng, F. Maier, S. Siri, and D. M. Pierce, unpublished observations).

On the basis of our evidence of structural heterogeneity through the thickness of the colorectal wall, we focus here on determining the biomechanical properties of separated colorectal layers. We intend to reveal the load-bearing structures of the colorectum, which will likely be strategic locations for the afferent endings to detect noxious colorectal distension/stretch. We separated mouse colorectum into the inner (mucosal-submucosal) and outer (muscular-serosal) composites and conducted biaxial tension tests on harvested mouse tissues of 7 × 7 mm² from both inner and outer composites along three distinct longitudinal locations. We also quantified residual stresses in the inner mucosal-submucosal and outer muscular-serosal composites of the colorectum by measuring opening angles (21, 32).

METHODS

All experiments were reviewed and approved by the University of Connecticut Institutional Animal Care and Use Committee.

Colorectal tissue harvesting.

We used mice of both sexes in this study (C57BL/6; Taconic, Germantown, NY), aged 8–12 wk and weighing 20–30 g. Our previous study indicates no significant differences in biomechanical properties of the colorectum between male and female mice (42). Thus, data from both sexes were pooled together. All mice were raised in individually ventilated cages (up to 5 mice per cage), bedded with aspen Sani-Chip bedding, fed an irradiated Teklad global diet, and provided light from 7 AM to 9 PM. For experiments, mice were anesthetized by isoflurane inhalation, euthanized by exsanguination via perforation of the right atrium, and transcardially perfused with oxygenated Krebs solution (in mM: 117.9 NaCl, 4.7 KCl, 25 NaHCO₃, 1.3 NaH₂PO₄, 1.2 MgSO₄·7H₂O, 2.5 CaCl₂, 11.1 d-glucose, 2 butyrate, and 20 acetate) bubbled with carbogen (95% O₂-5% CO₂). A midline laparotomy was performed, and the pubic symphysis was transected to expose the pelvic floor organs. The distal 30 mm of the large bowel, including the distal colon and rectum, were dissected free of connective tissues and transferred to modified Krebs solution, to which was added nifedipine (4 µM; L-type calcium channel antagonist to block muscle activities), penicillin-streptomycin (100 U/ml), and protease inhibitors (P-2714; Sigma-Aldrich, St. Louis, MO). To be consistent with prior electrophysiological and behavioral studies that implemented colorectal distension (e.g., 17), the tissue was cannulated as shown in Fig. 1A and distended by phosphate-buffered saline (PBS) at ascending levels of graded intraluminal pressure: 15, 30, 45, and 60 mmHg, 10 s for each distension. Graded distension was performed at least four times on each colorectum in modified Krebs solution at room temperature.

Fig. 1.

Schematics of layer-separated biaxial tissue testing on mouse colorectum. A: distension of mouse colorectum leads to both circumferential and axial deformations, justifying the use of biaxial tensile tests. B: image of a mouse colorectum chromatically stained with hematoxylin and eosin, indicating the interstitial space underneath the submucosa that allows separation of colorectum into inner (mucosal-submucosal) and outer (muscular-serosal) composites. C: biaxial tensile tests were conducted on 7 × 7-mm² tissue specimens harvested from three axial regions of the colorectum, i.e., rectal (0–10 mm), intermediate (10–20 mm), and colonic (20–30 mm). Specimens were separated into inner and outer composites and tested individually.

Biaxial stretch test for separated layers.

The presence of interstitial space below the submucosa as shown in Fig. 1B allows separating the colorectal wall into inner and outer composites by fine blunt dissection. The 30-mm colorectum was divided into three even segments: 0–10 mm (rectal), 10–20 mm (intermediate), and 20–30 mm (colonic), as shown in Fig. 1C. We then harvested tissue patches of ~7 × 7 mm² from each of the three segments and separated them into inner and outer composites from the interstitial space while maintaining the tissue integrity. The tissue patches were then mounted onto a custom-built stretch device and subjected to a quasistatic biaxial tensile test. We implemented the same test procedure on intact mouse colorectal tissue, with details reported previously (42). Briefly, we used a computer-controlled, force-displacement actuator with sub-milli-Newton force resolution (model 300-D; Aurora Scientific, Aurora, ON, Canada) to perform the biaxial tensile stretch test. A custom 3-D-printed adaptor consisting of long and narrow cantilevers permitted free lateral deformations during the biaxial stretch tests. From each colorectum, six square-shaped specimens (~7 × 7 mm²) from inner and outer composites at three different longitudinal segments were tested as illustrated in Fig. 1B. Throughout the experiments, the tissue remained submerged in the ~120 ml of aforementioned modified Krebs solution at room temperature containing nifedipine, penicillin-streptomycin, and protease inhibitor. The test protocol for each specimen consisted of 30 cycles of quasistatic ramped loading (0–40 mN) and ramped unloading (40–0 mN) at 1.2 mN/s. Thus, the first 27 cycles are for tissue preconditioning, and the data from the last 3 cycles were averaged as the steady-state force-displacement response of the specimen (42).

To calculate the Cauchy stress, we measured the specimen thickness in the load-free condition for both the inner and outer composites. We conducted histological staining on four colorectums at three axial segments and used the average thickness for calculating the Cauchy stress. Colorectum was cut open, flattened in load-free condition, and fixed with 4% paraformaldehyde for 60 min. The tissue was submerged in 20% sucrose overnight for cryoprotection, embedded in optimum cutting temperature compound (Sakura Finetek, Tokyo, Japan), frozen, and sectioned transversely at 10 µm. After staining with hematoxylin and eosin was completed, the tissue was examined on an Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) provided with appropriate filters and a Hamamatsu ORCA-ER, C4742-80 digital camera. The thickness of the inner and outer composites was measured using Hamamatsu Photonics Wasabi 150 software (Hamamatsu Photonics, Hamamatsu, Japan).

Measuring residual stress.

After performing the graded distension, we measured the residual stresses in both inner and outer composite layers of the colorectum under load-free conditions. To measure circumferential residual stress, we cut open a 2-mm ring of tissue from the tubular colorectum and separated the interstitial space below the submucosa into the inner and outer composites. To quantify the amount of residual stress in the tubular colorectum, we used the opening angle (21, 32), which is defined as the angle subtended by two radii drawn from the midpoint of the inner wall to the inner tips of two ends of the sector (Fig. 2A; 42). We allowed a 30-min period for complete release of the residual stress (the zero-stress state) and took photographs of the stripes of inner and outer composites for subsequent measurement of their opening angles. The axial residual stress was measured by taking a 2-mm-wide tissue strip along the whole length of the colorectum, resting for 30 min for complete release of the residual stress, and measuring any changes in length and curvature compared with those of the tubular colorectum.

Fig. 2.

Colorectal residual stresses measured by opening angles. A: a ring of the colorectal tissue (2 mm thick) was cut open, separated into inner (mucosal-submucosal) and outer (muscular-serosal) composites to achieve a stress-free state, and measured for opening angles as labeled with θ. B: photographs of inner and outer composite tissues in the stress-free state ready for measuring the opening angles. C: average opening angles measured from 9 colorectums. D: absence of axial residual stresses in the colorectum. The intact colorectum and dissected 2-mm-thick longitudinal strips show no apparent difference in axial length or curvature. Scale bars, 5 mm.

Quantifying the submucosal thickness by nonlinear imaging of SHG.

We quantified the thickness of the dense network of collagen fibers in the submucosa along different axial locations of the colorectum. Colorectums were cannulated and distended by graded hydrostatic pressure (15, 30, 45, and 60 mmHg, 10 s each) via PBS columns four times. Then, colorectums were distended with either 0 (load-free condition)- or 60-mmHg intraluminal pressure (distended condition), fixed with 4% paraformaldehyde for 60 min, cut open, flattened, and mounted onto glass slides (Permount; Fisher Scientific, Hampton, NH), with the serosal side facing the coverslip (no. 1.5), for nonlinear two-photon imaging of SHG. For imaging, we used an LSM 780 (Carl Zeiss, Oberkochen, Germany) with a ×40 objective (C-Apochromat ×40/1.2 W Corr) with a working distance of 280 µm. We used a tunable two-photon light source (Chameleon; Coherent, Santa Clara, CA) to excite the SHG at 900 nm and collected signal at 450 nm. This setup allowed label-free imaging, highly specific to collagen fibers, at every micrometer through the entire thickness of our specimens (cf. 41). The macroscopic layer thickness of the collagen fiber network was quantified post hoc from the image stacks using ImageJ (National Institutes of Health, Bethesda, MD). SHG signals in each image were identified and quantified following a procedure described previously (24). Briefly, the 8-bit grayscale SHG images were set with a threshold that excluded at least 99.99% of background intensity based on its Gaussian distribution using ImageJ. The threshold value was calculated from the standard normal distribution (Z) equation: threshold = 3.72·SD + m, where 3.72 was the Z-score at P = 0.9999 and SD and m were the standard deviation and mean of background intensities in 8-bit grayscale (0–255), respectively. The thickness was determined from the start to the end of the images in the stack with SHG signals occupying >20% of total image area.

Data analyses.

Cylindrical coordinates (r, θ, z) are assumed for the colorectum in radial, circumferential, and axial directions, respectively. We denote T as the mean thickness in the unloaded reference configuration measured from histological staining of the colorectum. L_θ and L_z are the measured lengths of the unloaded specimens in the circumferential and axial directions, respectively. The biaxial stretch forces are denoted as f_θ and f_z. The stretch ratios (λ_θ, λ_z) were calculated using the specimen geometry after the 27 cycles of the preconditioning as the reference coordinates (X_θ, X_z) normalized to the deformed coordinates (x_θ, x_z): λ_θ = x_θ/X_θ and λ_z = x_z/X_z. Considering the inner and outer composites as thin layers, we computed tension (t) assuming incompressibility and negligible shear using t_θθ = f_θ/(λ_zL_z), t_zz = f_z/(λ_θL_θ). We computed Cauchy stresses (σ) also assuming incompressibility and negligible shear using σ_θθ = λ_θf_θ/TL_z, σ_zz = λ_zf_z/TL_θ. Data are presented throughout as means ± SE unless specifically noted. One-way and two-way analyses of variance (ANOVA) or repeated-measures ANOVA were performed as appropriate using SigmaPlot v13.0 (Systat Software, San Jose, CA). Bonferroni post hoc multiple comparisons were performed when F values for main effects were significant. Differences were considered significant when P < 0.05. Before data collection, the sample sizes for the experiments were determined by estimated power analysis using standard deviations from prior tissue biomechanical studies by us and others. The statistical power of the study was further validated after the data collection to be >0.8.

RESULTS

Presence of circumferential residual stress in both inner (mucosal-submucosal) and outer (muscular-serosal) composites.

The opening angles were measured after cutting open the 2-mm-thick tissue rings and separating them into inner mucosal-submucosal and outer muscular-serosal composites as shown in Fig. 2B. Opening angles from nine colorectums were averaged and are displayed in Fig. 2C, showing significantly lower opening angle in the rectal segments than in the intermediate and colonic segments for the inner composite (1-way ANOVA, F_2,24 = 14.55, P < 0.001, post hoc comparison, P < 0.05 for rectal vs. colonic and rectal vs. intermediate). Opening angles are comparable in all three segments of the outer composites (1-way ANOVA, F_2,24 = 1.18, P = 0.32). Collectively, opening angles are higher in the outer composite than in the inner composite (2-tailed t-test, t₃₃ = 11.36, P < 0.001), suggesting more residual compression in the outer muscular-serosal layers than the inner mucosal-submucosal layers. Longitudinal strips of tissue (2 mm wide) were taken from the colorectum as shown in Fig. 2D, which showed no apparent changes of length and curvature compared with the tubular colorectum. This indicates the absence of axial residual stress in the colorectum.

Tension-stretch behaviors of the inner (mucosal-submucosal) and outer (muscular-serosal) composites.

Biaxial tests (0–40 mN at 1.2 mN/s) were conducted on ~7 × 7-mm² specimens harvested from 20 colorectums. Specimens showing apparent tissue tearing and abrupt change in the force-displacement curve were excluded. The force-displacement relations from 11 specimens in each category (inner or outer composite at colonic, intermediate, or rectal segment) were averaged and are plotted in Fig. 3A. The precise tissue geometry (i.e., lateral length of the specimen) in the load-free condition was measured by analyzing the picture of the specimens before the tensile test to allow calculation of the tension in the inner and outer composites. The tension-stretch relations of specimens from colonic, intermediate, and rectal segments were averaged and are plotted in Fig. 3B. For statistical comparison, only data from the loading cycles were included and compared in Fig. 3B. Specimens from all longitudinal segments showed significant anisotropy between the circumferential and axial directions in the inner mucosal-submucosal composite (2-way ANOVA, F_1,10 = 50.9, P < 0.001 for colonic; F_1,10 = 209, P < 0.001 for intermediate; F_1,10 = 233, P < 0.001 for rectal), as well as in the outer composite (F_1,10 = 39, P < 0.001 for colonic; F_1,10 = 368, P < 0.001 for intermediate; F_1,10 = 148, P < 0.001 for rectal). In addition, the axial tension-stretch relations showed significant difference between the inner and outer composites in Fig. 3B (2-way ANOVA, F_1,600 = 10.3, P = 0.004 for colonic; F_1,600 = 27.4, P < 0.001 for intermediate; F_1,600 = 56.1, P < 0.001 for rectal). The circumferential tension-stretch relations differ between the inner and outer composites only at the rectal segment (2-way ANOVA, F_1,600 = 4.36, P = 0.051 for colonic; F_1,600 = 3.93, P = 0.061 for intermediate; F_1,600 = 9.09, P = 0.007 for rectal). The higher axial stiffness and comparable circumferential stiffness collectively indicate more pronounced tissue anisotropy of the inner mucosal-submucosal composite than the outer muscular-serosal composite.

Fig. 3.

Force-displacement relations from biaxial tensile tests conducted on both inner (mucosal-submucosal) and outer (muscular-serosal) composites. A: average force-displacement data from 11 colorectums. B: considering both inner and outer composites as thin-walled material, we calculated the wall tension-stretch relations, and results are displayed as averages and standard error bars. The results from colonic, intermediate, and rectal segments are displayed separately. Here, circumf, circumferential. *P < 0.05.

Consistent axial tension-stretch behaviors from colonic to rectal regions.

As shown in Fig. 4A, the axial tension-stretch relations across different longitudinal segments are consistent with no significant variation at colonic, intermediate, and rectal regions (2-way ANOVA, F_2,900 = 2.36, P = 0.11 for the inner composite; F_2,900 = 1, P = 0.37 for the outer composite). In contrast, as shown in Fig. 4B, the circumferential tension-stretch relations showed significant difference between the colonic, intermediate, and rectal segments in both the inner composite (2-way ANOVA, F_2,900 = 4.83, P = 0.015, post hoc comparison, P = 0.02 for colonic vs. rectal) and the outer composite (F_2,900 = 20.8, P < 0.001, post hoc comparison, P = 0.01 for rectal vs. intermediate, P = 0.009 for rectal vs. intermediate, and P < 0.001 for rectal vs. colonic).

Fig. 4.

Comparison of tension-stretch relations across different axial segments of the colorectum. A: axial tension-stretch relations showed no statistical differences between colonic, intermediate, and rectal segments for either the inner or outer composite. B: circumferential tension-stretch relations were significantly different for both composites. *P < 0.05.

Differential stress-strain behaviors from colonic to rectal regions.

From the force-displacement data in Fig. 3A, we followed the same procedure reported previously (42, 43) to calculate the Cauchy stress-stretch relations (strain) from each specimen and plotted the average in Fig. 5, which only includes the loading stress-strain data for clearer comparison. The tissue anisotropy is apparent in both inner mucosal-submucosal and outer muscular-serosal composites as evidenced by statistical differences between the axial and circumferential stress-strain relations in all axial segments (2-way ANOVA, F_1,260 = 32, P < 0.001 for inner colonic; F_1,200 = 328.6, P < 0.001 for inner intermediate; F_1,150 = 342.9, P < 0.001 for inner rectal; F_1,850 = 272.5, P < 0.001 for outer colonic; F_1,490 = 162.2, P < 0.001 for outer intermediate; F_1,100 = 92.9, P < 0.001 for outer rectal). Axial stress-strain properties of the outer muscular-serosal composite layer showed significant difference between all three segments (2-way ANOVA, F_2,300 = 307.9, P < 0.001, post hoc comparison, P < 0.05 for rectal vs. intermediate, rectal vs. colonic, and intermediate vs. colonic), whereas no significant difference was observed in the inner mucosal-submucosal composite (2-way ANOVA, F_2,300 = 3.025, P = 0.064). Significant difference was observed in the circumferential stress-strain properties for both inner (2-way ANOVA, F_2,300 = 35.48, P < 0.001) and outer composites (F_2,300 = 718.8, P < 0.001) between the three longitudinal segments. Because of the significant increase in wall thickness from colonic to rectal regions, the stress-strain relations indicate progressive increase of tissue compliance from proximal to distal colorectum, more pronounced than the tension-stretch curves in Fig. 3B.

Fig. 5.

Cauchy stress-stretch relations. Progressive increase of colorectal wall thickness from colonic to rectal segments leads to significant reduction of effective tissue stiffness. Here, circumf, circumferential. *P < 0.05.

Differential viscoelastic behaviors between the inner (mucosal-submucosal) and outer (muscular-serosal) composites.

Following our previously reported method (42; S. Siri, F. Maier, S. Santos, D. M. Pierce, and B. Feng, unpublished observations), the viscoelastic behaviors of the specimen were quantified from the force-displacement relations as the area between the loading and unloading curves normalized by the total area under the loading curve, indicating the fraction of energy dissipation during the symmetric loading-unloading cycle. Displayed in Fig. 6 is the energy loss of tested specimens from six categories (colonic, intermediate, and rectal segments in inner and outer composites) in axial and circumferential directions, respectively. There was a significant difference in the fractions of energy dissipation between axial and circumferential directions in both the inner composite (2-tailed t-test, t = −8.5, P < 0.001) and the outer composite (2-tailed t-test, t = −4.4, P < 0.001), consistent with our previous findings from intact colorectal wall (42). In addition, the inner mucosal-submucosal composite showed significantly lower energy loss than the outer muscular-serosal composite in both axial and circumferential directions (2-tailed t-test, t = −4.6, P < 0.001 for axial; t = −2.04, P = 0.046 for circumferential). Overall, colorectal tissue is more elastic in the axial direction and more viscous in the circumferential direction, and the inner mucosal-submucosal composite is more elastic than the outer muscular-serosal composite.

Fig. 6.

Colorectal tissue viscoelasticity as fraction of energy loss in colonic, intermediate, and rectal segments. The horizontal bars indicate the average fraction of energy loss. (Here, n = 11 specimens for all categories.)

Submucosal thickness measured by SHG.

We measured the thickness of the submucosa by SHG detection of the collagen fiber network in the image stacks (Fig. 7A). The thicknesses of submucosa were measured at colonic, intermediate, and rectal regions from eight colorectums in the load-free condition and five colorectums in the distended (60-mmHg) condition, and the average thicknesses are plotted in Fig. 7B. Distension of 60 mmHg did not significantly change the thickness of the submucosa compared with the load-free condition (2-way ANOVA, F_1,29 = 0.08, P = 0.77). In the load-free condition, there was no statistical difference in submucosal thickness across different longitudinal regions of the colorectum (1-way ANOVA, F_2,18 = 1.13, P = 0.35). In the distended condition, submucosal thickness is significantly thinner at the colonic segment than at the intermediate and rectal segments (1-way ANOVA, F_2,11 = 10.8, P = 0.003, post hoc comparison, P < 0.05 for colonic vs. rectal and colonic vs. intermediate).

Fig. 7.

A: representative second-harmonic generation (SHG) image showing collagen fibers aligned at ~30° off the axial direction (vertical in the SHG image). B: average thickness of submucosa as measured by SHG signals in the image stacks for colonic, intermediate, and rectal segments. *P < 0.05.

DISCUSSION

The wall of the colorectum is a multilayered structure including mucosal, submucosal, circular muscular, intermuscular, longitudinal muscular, and serosal layers from the inside to the outside of the lumen. In mice, the gap between the basal submucosa and the circular muscular layer is apparent (Fig. 1B), which allowed in this study the surgical separation of mouse colorectum into the inner (mucosal and submucosal layers) and outer composites (muscular and serosal layers) while keeping the tissue integrity of both composites. The enteric nervous system intrinsic to the colorectum is known to have two major neural plexuses in the inner and outer composites, i.e., the submucosal and myenteric plexuses, respectively (22). Regarding the extrinsic sensory innervation, afferent endings from the PN innervation are also concentrated in the submucosal (32% of total afferent endings) and muscular layers (22% in myenteric ganglion and 25% in circular muscular) accordingly to a recent sparse neural-tracing study (45). It is generally assumed that neuronal tissue in the inner mucosal-submucosal composite participates in normal gastrointestinal functions such as absorption, secretion, and detection of noxious materials at the mucosa (3). Accordingly, neuronal tissue in the outer muscular-serosal composite is considered to play critical roles relevant to mechanical movement (38, 46), including the gut peristalsis from coordinated activities from intrinsic myenteric neurons and the neural encoding of colorectal distension by extrinsic afferents that reliably evokes visceral pain (34). The macroscopic anatomy of the colorectum seems to support this notion as the wavy villi-crypt structure of the mucosa in the inner mucosal-submucosal composite seems unlikely to undertake significant mechanical load compared with the muscular layers in the outer composite with highly aligned muscle bundles.

This is the first study that unequivocally reveals the surprising load-bearing role of the inner mucosal-submucosal composite via direct biomechanical measurement. With careful blunt dissection and our newly established biaxial test protocol that measures in the milli-Newton range (42, 43; S. Siri, F. Maier, S. Santos, D. M. Pierce, and B. Feng, unpublished observations), the present study reveals that the tension stiffness of the inner mucosal-submucosal composite is at least comparable to, if not higher than, that of the outer muscular-serosal composite. Considering distended colorectum as a thin-walled structure, the wall tension (i.e., force per unit length) reflects the local loading situation through the wall following the intraluminal pressure. Our tension-stretch relations reveal comparable stiffness between the inner mucosal-submucosal and outer muscular-serosal composites; the inner mucosal-submucosal composite has slightly higher axial stiffness than the outer composite, whereas the outer muscular-serosal composite has higher circumferential stiffness. Hence, the wall tension resulting from colorectal distension will be undertaken by both composites with inner composite taking slightly more axial tension and outer composite more circumferential tension. As a consequence, nerve endings in the mucosal and submucosal layers (i.e., the inner composite) are likely to experience significant mechanical stress during noxious colorectal distension just as endings in the muscular layers do. In the present study we removed smooth muscle activities with nifedipine (L-type calcium channel blocker), which is consistent with prior neurophysiological studies of visceral afferents that remove the confounding factor of smooth muscle tone (e.g., 4, 5, 12). Determining the effect of smooth muscle tone on visceral nociception awaits further physiological and biomechanical studies.

Despite the vast difference in wall thickness from proximal to distal colorectum, colorectal distension usually causes comparable increase in circumference and length of the tubular structure across the longitudinal locations (e.g., in Fig. 1A), which agrees with the small variations of the tension-stretch relations measured from the colonic, intermediate, and rectal regions in the present study (Fig. 4). This suggests that the load-bearing structures are comparable in strength and geometry along the axial locations despite the vast increase in wall thickness from proximal to distal colorectum. In support, we found comparable thickness of the submucosa with a rich network of collagen fibers from the proximal to distal colorectum under load-free conditions, which can be reliably detected by the nonlinear imaging method of SHG (31, 40). Collagen fibers serve as the major load-bearing structures for many biological tissues [e.g., the skin (39), tendon cartilage (27), and blood vessels (25)]. Studies by us and others have indicated the presence of thick collagen fibers in the intestinal submucosa that form a network by crisscrossing two families of helical fibers ±30° from the axial direction (36, 42), which is in good agreement with the present biaxial stretch data indicating that the axial stiffness of the inner mucosal-submucosal composite is approximately twice the circumferential stiffness. Together, this evidence strongly indicates the submucosa as the load-bearing structure of the colorectum, especially for axial loading. Despite the clear tissue heterogeneity, we derived bulk stress-strain relations for the inner mucosal-submucosal and outer muscular-serosal composites assuming homogenous material composition that are within the same range as the stress-strain relations measured with intact colorectum in our previous report (42). On the basis of the stress-strain relations, we found that mechanical stiffness is significantly lower in the distal rectal region than in the proximal regions, which is likely caused by the progressive increase in the nominal thickness of the inner and outer composites from proximal to distal colorectum. In contrast, the tension-stretch relations show much less variation from proximal to distal colorectum, which reflects the relatively homogeneous deformation of mouse colorectum in both axial and circumferential directions during distension (e.g., Fig. 1A). Since wall tension is not affected by the nominal wall thickness, it is a more suitable metric than Cauchy stress for the colorectum.

To effectively detect injurious colorectal distension, it is reasonable to assume that the nociceptive nerve endings are strategically located at the concentrations of mechanical stress in the colorectum. Muscular layers are the logical location, and rectal intraganglionic laminal endings (rIGLEs) that encode rectal tension were found in the intermuscular layers of guinea pig rectum (34, 47), making them potential candidates of colorectal nociceptors. In support, these rIGLEs reliably encode rectal tension rather than displacement (34, 47), suggesting their roles in detecting stress concentration in the rectum. However, rIGLEs tend to have a low threshold for rectal stretch and are absent in the proximal colonic region, and thus they are unlikely to represent all of the colorectal nociceptors. Results of this study indicate that the submucosa is also a load-bearing structure and likely has nociceptive endings to detect mechanical stresses, a hypothesis with no direct experimental evidence supporting it in the present literature to the best of our knowledge. Indirect evidence comes from aortic baroreceptors (which detect a wide range of mechanical distension of the aorta) with endings in the adventitia of the artery (29), which is essentially the same type of tissue as the colorectal submucosa according to a recent study, i.e., a fluid-filled interstitial space supported by a network of thick collagen bundles (1). This fiber-reinforced fluidlike structure of submucosa and adventitia actually agrees with our present finding that the submucosal thickness is not changed by 60-mmHg colorectal distension (Fig. 7). Similarly, the adventitia is the load-bearing structure of the blood vessel, functioning as a “stiff tube” to prevent overstretch and rupture of arteries at noxious blood pressures (23). Furthermore, colorectal afferent endings from the lumbar splanchnic innervation were reported to concentrate in the submucosa close to the mesenteric arteries and arterioles (26). Thus, nociceptive endings that detect the proximal colonic distension may be preferentially located in the submucosa, although this requires extensive further testing. All the above evidence collectively suggests the presence of colorectal nociceptors in the submucosa that detect concentrations of mechanical stress. One potential candidate is the muscular-mucosal afferent class that encodes both colorectal stretch and fine mucosal stroking with endings likely in the mucosal and submucosal layers (5, 12). Further studies are required to identify the morphology, function, and molecular profiles of these submucosal nociceptors.

It is worth emphasizing that pressurizing hollow tubular structures such as the colorectum causes not only circumferential stretch but also significant elongation in the axial direction (e.g., Fig. 1A). Neurophysiological studies with colorectum cut open and pinned flat (to access the afferent receptive fields from the mucosal side) generally implemented circumferential stretch to characterize colorectal afferent functions, revealing that most afferents responding to circumferential stretch are in the pelvic pathway, which innervates the distal portion of the colorectum (12, 13); very few afferents responded to circumferential stretch in the lumbar splanchnic pathway, which innervates the proximal colorectum (5, 12). However, when the colorectum was kept in tubular form, lumbar splanchnic afferents are reliably activated by colorectal distension (10), which implies their roles in encoding axial stretch of the colorectum. An elegant nerve transection study revealed that the LSNs may not play important roles in visceral nociception because the pseudoaffective behavioral (visceral motor) responses in mice evoked by noxious colorectal distension were not affected by transecting the LSNs (30). However, the visceral nociceptive roles of lumbar splanchnic afferents are supported by clinical studies in humans indicating strong visceral pain perception when activating the lumbar splanchnic pathway (33, 35). Thus, further studies are required to focus on the lumbar splanchnic afferents that encode noxious axial colorectal stretch, an understudied area that could reveal new mechanisms of peripheral visceral nociception.

In summary, biaxial stretch testing reveals that the tension stiffness of the inner mucosal-submucosal composite is comparable to, if not higher than, that of the outer composite, which is against the general notion that the wavy structures of colorectal mucosal-submucosal layers are low in mechanical strength. The opening-angle measurements reveal the presence of modest circumferential prestress, but no axial prestress in the colorectum. Nonlinear imaging by SHG reveals that the thickness of submucosa is consistent from proximal to distal colorectum in contrast to the vast difference in colorectal wall thickness, suggesting the load-bearing roles of submucosa in the inner mucosal-submucosal composite. Outcomes of this study call for focused research on the role of colorectal afferent endings in the submucosa that encode noxious colorectal distension, an understudied area that could potentially provide new mechanisms of visceral hypersensitivity.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant K01-DK-100460 (to B. Feng) and National Science Foundation Grant CMMI-1727185 (B. Feng and D. M. Pierce).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.M.P. and B.F. conceived and designed research; S. Siri, F.M., and S. Santos performed experiments; S. Siri, F.M., D.M.P., and B.F. analyzed data; S. Siri, F.M., S. Santos, D.M.P., and B.F. interpreted results of experiments; S. Siri, F.M., S. Santos, and B.F. prepared figures; S. Siri and B.F. drafted manuscript; S. Siri, F.M., D.M.P., and B.F. edited and revised manuscript; S. Siri, F.M., S. Santos, D.M.P., and B.F. approved final version of manuscript.