Figure 1.
Endoscopic images demonstrating the effect of different doses of TNBS in a fixed concentration of 60 mg/ml.
Severe colitis with a yellowish membrane and coprostasis was noted at day 3, and mucosal shedding, stenosis and necrosis was evident at day 7 in rats given TNBS 17.5 mg and 22.5 mg (0.29 ml and 0.38 ml, respectively). The bottom panel depicts examples of normal rat colon.
Figure 2.
Endoscopic images demonstrating the effect of TNBS at different concentrations instilled rectally in a total volume of 0.7 ml.
TNBS 10.5 mg (15 mg/ml) was associated with minimal mucosal inflammation and edema. TNBS 21.0 mg (30 mg/ml) resulted in an erythematous and edematous mucosa at day 3 and 7. At day 12 mucosal granulation and ulcerations were seen. TNBS 24.5 mg (35 mg/ml) and 31.5 mg (45 mg/ml) resulted in a more severe acute inflammation, and at day 7, larger areas of ulceration with fibrin cover were visible. At day 12, stenotic strictures developed in the rat receiving TNBS 24.5 mg. The rat receiving 31.5 mg was euthanized after the second endoscopy and consequently no endoscopic picture from day 12 can be shown. In the right column, histological pictures corresponding to mild, moderate and severe TNBS-colitis are included. TNBS 10.5 mg (15 mg/ml) resulted in only minimal inflammation with some architectural changes. TNBS 21.0 mg (30 mg/ml) resulted in ulceration that bordered the mucosa with inflammatory cell infiltration. In TNBS 24.5 mg (35 mg/ml), crypt distortion and abscesses (arrows) with mucosal and submucosal inflammatory infiltration were visible. Severe TNBS colitis is seen in the lower hitstologic picture with transmural inflammatory cell infiltration and total denudation of the mucosa. Objective x10 in all histological images.
Figure 3.
Representative endoscopic images of TNBS (30 mg/ml, 0.6 ml) associated changes at different time points and MEICS-score at T0, T3, T7 and T12.
(A) Granulated and edematous mucosa at T3 and T7, and ulcerations at T12 were evident in Rat 1. At T7, small ulcerations/erosions were identified in Rat 4. In Rat 5, an ulceration is visible at T7 and at T12, a stricturing ulcer has developed. (B) The MEICS-score at T3 was significantly different from T12 (p = 0.02).
Figure 4.
Histologic appearance of an endoscopic biopsy and whole colon specimen.
(A) Endoscopic biopsy collected at T7 showing evidence of a crypt abscess (arrow), and mucosal gland distortion. Objective x40. (B) Histologic image of a whole colon specimen collected at termination of the study (T12) identifying distorted mucosal glands, and an ulceration with completed reepithelialization and underlying submucosal inflammation (arrow). Objective x4. The slides were stained with hematoxylin and eosin.
Figure 5.
Differential expression analysis of the TNBS transcriptome.
(A) Scatter plot of the Principal Component Analysis of 8316 genes in response to TNBS. (B) Expression profiles of genes assigned to the 13 clusters of similar expression (see Table S1). Only differentially expressed genes are included. (C) Heat map visualizing functional enrichment of the 13 clusters for over-represented Gene Ontology Biological Process terms. Darker shades represent a greater degree of enrichment.
Figure 6.
Concordance analysis between TNBS-colitis and IBD transcriptomes at the level of single gene loci.
T3 vs. T0, T7 vs. T0 and T12 vs. T0 compared to CD vs. normal (top) and UC vs. normal (bottom). (A–F) Scatterplots of FCs in rat data (x-axis) compared to FCs in the human data (y-axis). Rho values correspond to Spearman correlation coefficients with p-values representing the level of significance of the correlation. Concordant up- and down-regulated genes are represented as red and blue circles respectively. Non-concordant genes are shown as grey circles. Numbers in the legend correspond to the total number of genes assigned to each category. B, C, E and F are only shown in the supplemental section (Figure S1).
Figure 7.
Concordance analysis between TNBS-colitis and IBD transcriptomes at the level of biological pathways.
T3 vs. T0 compared to CD vs. normal (top) and UC vs. normal (bottom). Left, scatter plots of pathway activity scores of KEGG pathways in TNBS and IBD samples. Right, scatter plots of pathway activity scores of Reactome pathways in TNBS and IBD samples. Rho values correspond to Spearman correlation coefficients with p-values representing the level of significance of the correlation. Numbers in the legend correspond to the total number of pathways assigned to each category. A similar figure (Figure S2) for concordant analysis of T7 vs. T0 and T12 vs. T0, compared to CD vs. normal and UC vs. normal is shown in the supplementary section (KEGG and Reactome score change at the top and bottom, for T7 vs. T0 and T12 vs. T0, respectively).
Figure 8.
In situ hybridization (ISH) of significantly regulated genes in TNBS colitis mucosal biopsies.
(A and C) The expression of TLR2 and TLR4 is scattered in the epithelium and submucosal immune cells at T0. (B and D) Increased TLR2 and TLR4 expression was noted in the epithelium at T7. (E) PRNP expression in submucosal immune cells at T0. (F) Intense PRNP expressing cells were evident in the submucosa at T12 and also to some degree in the epithelium. (G) Intense clusters of PPARγ expression in epithelial cells at T0. (H) At T12, the expression is almost abolished. Objective x40 in all pictures.
Table 1.
P-values and FCs for the target genes at every time point.
Figure 9.
Graph displaying confirmatory PCR.
Graph displaying FC for T3 vs. T0, T7 vs. T0 and T12 vs. T0 for SPINK4, ADA and FABP1 for both microarray (MA) and confirmatory PCR. FC is log2 transformed for illustrative purposes. All p-values are highly significant.