Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease.

Inflammatory bowel disease (IBD) continues to increase in prevalence in industrialized countries. Major complications of IBD include formation of fibrotic strictures, fistulas, reduced absorptive function, cancer risk, and the need for surgery. In other chronic gastrointestinal disease models, stiffness has been shown to precede fibrosis; therefore, stiffness may be a reasonable indicator of progression toward stricture formation in IBD patients. Herein, we seek to quantify tissue stiffness and characterize fibrosis in patients with IBD and to compare mechanical properties of unaffected human tissue to common animal species used for IBD studies. Inflamed and unaffected tissue from IBD patients and unaffected tissue from mice, pigs, and cows were indented using a custom device to determine the effective stiffness. Histology was performed on matched tissues, and total RNA was isolated from IBD tissue samples and used for gene expression analysis of pro-fibrotic genes. We observed an increase in the effective stiffness (steady-state modulus, SSM) (p < 0.0001) and increased expression of the collagen type I gene (COL1A1, p = 0.01) in inflamed tissue compared to unaffected areas in our IBD patient cohort. We also found that increased staining of collagen fibers in submucosa positively correlated with SSM (p = 0.093). We determined that unaffected animal bowel stiffness is significantly greater than similar human tissues, suggesting additional limitations on animal models for translational investigations regarding stiffness-related hypotheses. Taken together, our data support development of tools for evaluation of bowel stiffness in IBD patients for prognostic applications that may enable more accurate prediction of those who will develop fibrosis and more precise prescription of aggressive therapies

Quantitative assessment of intestinal stiffness and associations with fibrosis in human inflammatory bowel disease - Fig 8

<p><b>Representative H&E and Masson’s Trichrome of the cow small bowel (A,B), pig small bowel (C,D), pig colon (E,F), and mouse colon (G,H).</b> The mouse colon has a very distinct muscle layer highlighted by the black arrow (G), while the cow small bowel has a very large area of collagen deposition (B, black star) that is absent in the pig small bowel and colon (black stars, D and F). Scale bar is 100 μm.</p

Fibrotic markers are less prevalent in representative staining of the unaffected regions of bowel than inflamed regions.

<p>Adjacent regions of Ileum (A-D) and colon (E-H) have been labeled with H&E (A,C,E,G) and Trichrome (B,D,F,H) as indicated. Black arrows highlight collagen clustering while black stars illustrate disrupted mucosal layer in inflamed tissue compared to unaffected tissue segments. Scale bar is 100 μm.</p

Multi-scale indentation system characterizes mechanical properties of soft tissues.

<p>(A) Custom indentation device with sensor positioned with the cantilever in position to test inflamed ileum. (B) Bovine small bowel positioned under the sensor probe with the mucosa exposed. Hydration was maintained by pooling small volumes of saline solution on and around samples (not seen here to improve visualization of tissue).</p

Tissues with time-dependent mechanical properties reach a quasi-steady-state after stress relaxation during indentation.

<p>Normal force (top, green line) as a function of relaxation time is used in computing the transient modulus (bottom, blue line) based on a modified Hertz contact model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e001" target="_blank">Equation 1</a>). The Steady-State Modulus (SSM) seen at the end of relaxation is reported throughout this work since SSM reduces strain-rate dependencies in determining the properties of a viscoelastic material.</p

The steady-state modulus of human colon and ileum vary with inflammation state.

<p>(A) The difference in stiffness between colon and ileum was not significant (p = 0.30, n = 44 colon measurements on 4 samples from 3 patients and n = 33 ileum measurements on 9 samples from 9 patients). (B) Inflamed regions are stiffer than unaffected regions (p < 0.0001, n = 43 inflamed indentations from 7 patients and n = 34 unaffected indentations from 6 patients). Following this trend, inflamed colonic tissue (n = 31 indentations from 3 patients) is stiffer than unaffected colon regions (n = 13 from 1 patient) and the inflamed ileum (n = 12 from 4 patients) is stiffer than unaffected ileal tissue (n = 21 from 5 patients). Mean stiffness values are represented as a solid black bar.</p

mRNA expression of Col1A1, MMP-1, and CDH-1 is changed in unaffected and inflamed areas of the intestine.

<p>mRNA expression profiles of Col1A1 (A), MMP-1 (B), and CDH1 (C) in inflamed and unaffected areas of IBD patients. N = 9 for each group and is represented as the average of 3 separate experiments. Collagen type I is significantly upregulated in the inflamed areas (*p = 0.0110) along with MMP-1 expression upregulation in inflamed areas (***p = 0.0032). The expression of CDH1, E-cadherin, is downregulated in inflamed areas (*p = 0.0288). Gene expression that resulted in no significant change is detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.s001" target="_blank">S1 Fig</a>.</p

Inflammation affects the behavior of the small and large intestine.

<p>(a) Schematic of GI tract in human and representative locations of tissue resected in ileum (left) and colon (right). Normal tissue should be similar in the two regions, though the ileum has long villi extending into the lumen and the colon can have thicker muscular regions for compacting and moving stool. (b) Endoscope images show unaffected ileum (i) and inflamed ileum (ii) regions and unaffected colon (iii) and inflamed colon (iv) are clearly visible by optical inspection.</p

Comparison of the steady-state modulus (SSM) and effective total modulus of human tissue and control animal tissue.

<p>Inflamed human tissue is displayed in orange while unaffected human and normal animal tissue is displayed in blue. (a) The SSM is representative of the steady-state behavior of a viscoelastic material after stress relaxation (Eqs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e002" target="_blank">2</a>). (b) The effective total modulus is determined from using the Winkler contact model which assumes that the tissue is a foundation of purely elastic springs (Eq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200377#pone.0200377.e003" target="_blank">3</a>). Group means are displayed as a solid black line within each group. Each dot represents one indentation. SI = small intestine, LI = large intestine. * p < 0.0001, ** p = 0.0004, ‡ p<0.0001, ‡‡ p = 0.0043. Note: Scales of y-axes are different for each graph as effective total modulus yields higher values than SSM.</p