A guide to histomorphological evaluation of intestinal inflammation in mouse models

Abstract

Histomorphology remains a powerful routine evaluating intestinal inflammation in animal models. Emphasizing the focus of a given animal study, histopathology can overstate differences between established models. We aimed to systematize histopathological evaluation of intestinal inflammation in mouse models facilitating inter-study comparisons. Samples of all parts of the intestinal tract from well-established mouse models of intestinal inflammation were evaluated from hematoxylin/eosin-stained sections and specific observations confirmed by subsequent immunohistochemistry. Three main categories sufficiently reflected the severity of histopathology independent of the localization and the overall extent of an inflammation: (i) quality and dimension of inflammatory cell infiltrates, (ii) epithelial changes and (iii) overall mucosal architecture. Scoring schemata were defined along specified criteria for each of the three categories. The direction of the initial hit proved crucial for the comparability of histological changes. Chemical noxes, infection with intestinal parasites or other models where the barrier was disturbed from outside, the luminal side, showed high levels of similarity and distinct differences to changes in the intestinal balance resulting from inside events like altered cytokine responses or disruption of the immune cell homeostasis. With a high degree of generalisation and maximum scores from 4-8 suitable scoring schemata accounted specific histopathological hallmarks. Truly integrating demands and experiences of gastroenterologists, mouse researchers, microbiologists and pathologists we provide an easy-to-use guideline evaluating histomorphology in mouse models of intestinal inflammation. Standard criteria and definitions facilitate classification and rating of new relevant models, allow comparison in animal studies and transfer of functional findings to comparable histopathologies in human disease.

Keywords: Histopathology; inflammation; inflammatory bowel disease; mouse models; scoring.

Figure 1

Representative images of H&E-stained colon sections illustrate specifics of inflammatory cell infiltrates. (A) Infiltrating mixed leukocytes in a cross section (×400, scale bar 20 μm). (B) Scattered neutrophils (arrows; ×400, scale bar 20 μm). (C) Multifocal mucosal infiltration of mixed inflammatory cells (arrows; ×100, scale bar 100 μm). (D) Mucosal and submucosal (bracket) infiltrate of inflammatory cells (×100, scale bar 100 μm) and (E) transmural inflammatory cells (×200; scale bar 50 μm).

Figure 2

Representative images of H&E-stained colon sections illustrate the grades of epithelial hyperplasia and of goblet cell loss. Hyperplasia appears as elongated crypts due to increase in epithelial cells (upper row; ×100) and loss of goblet cells that display clear mucus droplets (lower row; ×100).

Figure 3

Representative H&E- and immunohistochemically stained colon sections illustrate epithelial changes and altered mucosa architecture. (A) Cryptitis appears as neutrophils (arrows) between crypt epithelial cells in a cross section (×400, scale bar 20 μm). (B) Crypt abscesses with neutrophils in the lumen and nearly intact epithelium (white arrowhead) or damaged epithelium (black arrowhead) and complete crypt loss (arrow) in cross section (×400, scale bar 20 μm). (C) Loss of surface epithelium marking erosion (arrowheads; ×100, insert ×400). (D) Ulceration (×100, scale bar 100 μm). (E) Ulceration covered by an exudate rich in iNOS⁺ neutrophils (brown; ×100, scale bar 100 μm) and (F) granulocyte-fibrin eschar (×100, insert ×400, scale bar 20 μm). Consecutive sections of (G) granulation tissue (×100, insert ×400, scale bar 20 μm) comprising (H) CD31⁺ endothelial cells (red; ×100, insert ×400, scale bar 20 μm) and (I) apical iNOS⁺ cells (brown; ×400, scale bar 20 μm).

Figure 4

Representative H&E-stained colon sections illustrate altered crypts. (A) Variable diameters in adjacent crypts in a cross section with dilated crypts containing mucus (arrows; ×100, scale bar 100 μm). (B) Slight crypt distortion with crypt abscess (arrow; ×100, scale bar 100 μm). Bifurcated crypts (arrow) in (C) longitudinal section (×100, scale bar 100 μm) and (D) cross section (×400, scale bar 20 μm). (E) Branched crypts (×100, scale bar 100 μm). Mucosa containing only remains of crypts (arrowheads) in (F) longitudinal section (×100, scale bar 100 μm) and (G) cross section (×400, scale bar 20 μm). (H) Herniated crypts (×100, scale bar 100 μm).

Figure 5

Representative H&E-stained sections of the small intestine illustrate villous blunting with normal and altered villi from the duodenum and ileum (×100, scale bar 100 μm).

Figure 6

Histomorphology of colon tissue in DSS-induced colitis in C57BL/6 wild-type mice at day 2 after DSS removal representing scores according to scheme 1 referring to Table 3. A. Sum score 1: mild mucosal inflammatory cell infiltrates (score 1: 1) with intact epithelium (score 2: 0); B. Sum score 2: inflammatory cell infiltrates into mucosa and submucosa (score 1: 2) with undamaged epithelium (score 2: 0); C. Sum score 3: mucosal infiltrates (score 1: 1) with focal ulceration (score 2: 2); D. Sum score 4: inflammatory cell infiltrates in mucosa and submucosa (score 1: 2) and focal ulceration (score 2: 2); E. Sum score 5: moderate inflammatory cell infiltration into mucosa and submucosa (score 1: 2) with extensive ulcerations (score 2: 3); F. Sum score 6: transmural inflammation (score 1: 3) and extensive ulceration (score 2: 3). Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid) and submucosa (dotted); white arrowhead-ulceration; bracket–transmural inflammation.

Figure 7

Histomorphology of colon tissue 6 weeks after intrarectal application of ovalbumin and simultaneous intravenous transfer of ovalbumin-specific T cells into wild-type mice representing scores according to scheme 2 referring to Table 4. A. Score 1: intact epithelium with minimal focal inflammatory cell infiltrates in the mucosa; B. Score 2: scattered inflammatory cell infiltrates in mucosa and submucosa; C. Score 3: diffuse mucosal and submucosal inflammatory cell infiltrates; D. Score 4: moderate inflammatory cell infiltrates in the submucosa which is edematous, focal ulceration. Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid) and submucosa (dotted); white arrowhead-ulceration.

Figure 8

Histomorphology of colon tissue after transfer of CD4⁺CD45RB^hi T cells into Rag1-ko mice representing scores according to scheme 3 referring to Table 5. A. Score 0: normal colon mucosa with intact epithelium; B. Score 1: scattered inflammatory cell infiltrates in the mucosa; C. Score 2: diffuse mucosal infiltrates without submucosal spreading and intact epithelial layer; D. Score 3: moderate infiltration of inflammatory cells into mucosa and submucosa with epithelial hyperplasia and goblet cell loss; E. Score 4: marked inflammatory cell infiltrates in mucosa and submucosa accompanied by crypt abscesses and loss of goblet cells and crypts; F. Score 5: marked inflammatory cell infiltrates within the mucosa spreading to the submucosa going along with crypt loss and hemorrhage. Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid) and submucosa (dotted); black arrowhead-crypt abscess, blue arrowhead-goblet cell loss; yellow arrowhead-crypt loss; red arrowhead-hemorrhage.

Figure 9

Histomorphology of colon tissue in IL-10 ko mice at 4 months of age representing scores according to scheme 4 referring to Table 6. A. Score 1: minimal inflammatory cell infiltrates in the mucosa with intact epithelium; B. Score 2: mild inflammatory cell infiltrates in the mucosa with mild hyperplasia and mild goblet cell loss; C. Score 3: moderate inflammatory cell infiltrates in mucosa and submucosa with moderate goblet cell loss; D. Score 4: marked inflammatory cell infiltration into mucosa and submucosa with marked hyperplasia and marked goblet cell loss, multiple crypt abscesses and crypt loss. Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid) and submucosa (dotted); black arrowhead-crypt abscess; yellow arrowhead-crypt loss; blue arrowhead-goblet cell loss.

Figure 10

Histomorphology of tissue from the terminal ileum in TNF^ΔARE mice starting at 2 weeks of age representing scores according to scheme 5 referring to Table 7. A. Score 0: normal ileum with intact epithelium and short, finger-like villi; B. Score 1: mild mucosal inflammatory cell infiltrate; C. Score 2: mild diffuse inflammatory cell infiltrate in mucosa and submucosa; D. Score 3: moderate inflammatory cell infiltrates in mucosa and submucosa with villous blunting; E. Score 4: marked mucosal, submucosal and transmural inflammatory cell infiltration with lymphoid aggregates predominantely in the submucosa accompanied by villous broadening; F. Score 5: marked transmural inflammatory cell infiltration and villous atrophy (insert: submucosal granuloma; ×400). Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid), submucosa (dotted), and transmural (white); black arrowhead-crypt abscess; circle-lymphoid aggregates.

Figure 11

Histomorphology of duodenal tissue in mice infected with Heligmosomoides polygyrus representing scores according to scheme 6 referring to Table 8. A. Sum score 0: normal duodenum; B. Sum score 2: mild mucosal and submucosal inflammation (score 1: 1) with normal crypts and villi (score 2: 0); C. Sum score 4: moderate infiltration of mucosa (score 1: 2) with mild villous blunting and mild hyperplasia (score 2: 2); D. Sum score 5: transmural inflammation (score 1: 3) with moderate villous blunting and mild hyperplasia (score 2: 2); E. Sum score 6: mucosal inflammatory cell infiltration (score 1: 2) with moderate villous blunting and marked hyperplasia (score 2: 4); F. Sum score 7: marked transmural inflammation (score 1: 4) with moderate blunting and moderate hyperplasia (score 1: 3). Original magnification ×100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid), submucosa (dotted), and transmural (white).

Figure 12

Histomorphology of ileal tissue in mice infected with Toxoplasma gondii at day 7 representing scores referring to Table 9. A. Score 1: minimal and focal inflammatory cell infiltrates in the mucosa with an intact epithelial layer; B. Score 2: mild, diffuse inflammatory cell infiltrates in mucosa and submucosa with flattened epithelium and villous blunting; C. Score 3: moderate, diffuse inflammatory cell infiltrates in mucosa and submucosa with erosions and distorted villlous structure; D. Score 4: necrotic and fibrotic mucosa with distorted villi. Original magnification x100; scale bars 100 μm; arrows-inflammatory cell infiltrates within mucosa (solid) and submucosa (dotted).