Mice Anti-CD40 Colitis Methods

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Histology & Histopathology Methods for Anti-CD40 Colitis in Mice

Processing of Tissues

Proximal and distal colon are submitted individually for histopathology. For each region, three equidistant pieces are cut and embedded in paraffin. Sections are cut and stained with hematoxylin & eosin.

Evaluation of Tissues

Each piece of colon is evaluated individually, and values are averaged separately for the proximal, distal, and total colon.

Edema

Submucosal edema is quantitated by measuring the distance from the muscularis mucosa to the internal border of the outer muscle layer in a non tangential area thought to best represent the severity of this change.

Inflammation Score

The extent of macrophage, lymphocyte and PMN infiltrate is assigned severity scores according to the following criteria:

0 = Normal
0.5 = Very Minimal, one or two small foci, mononuclear inflammatory cells (MNIC) likely background mucosal lymphoid aggregates, if aggregates are definitely Peyer’s patches then they are not scored as abnormal
1 = Minimal, larger focal area with MNIC and neutrophils or minimal diffuse, no separation of glands, may be mostly in areas of submucosal edema or mesentery
2 = Mild, diffuse mild, or multifocal affecting 11 to 25% of mucosa with minor focal or multifocal gland separation, no separation in most areas
3 = Moderate, 26 to 50% of mucosa affected with minimal to mild focal or multifocal separation of glands by inflammatory cell infiltrate, milder in remaining areas of mucosa with some areas having no gland separation by inflammation
4 = Marked, 51 to 75% of mucosa affected with mild to moderate separation of glands by inflammatory cell infiltrate, minimal to mild in remaining areas of mucosa but all glands have some separation by infiltrate
5 = Severe, 76 to 100% of mucosa affected with moderate to marked areas of gland separation by inflammatory cell infiltrate, mild to moderate in remaining areas of mucosa

Gland Loss Score

Crypt epithelial and remaining gland epithelial loss is scored based on the approximate percent of the mucosa that was affected as follows:

0 = None
0.5 = Very Minimal, one or two small focal areas of gland loss or mucosal erosion
1 = Minimal, 1 to 10% of the mucosa affected
2 = Mild, 11 to 25% of the mucosa affected
3 = Moderate, 26 to 50% of the mucosa affected
4 = Marked, 51 to 75% of the mucosa affected
5 = Severe, 76 to 100% of the mucosa affected

Erosion Score

The loss of surface epithelium is scored based on the approximate percent of the mucosa that was affected as follows. This is generally associated with mucosal hemorrhage (reflective of the bleeding seen clinically and at necropsy):

0 = None
0.5 = Very Minimal, one or two small focal areas of gland loss or mucosal erosion
1 = Minimal, 1 to 10% of the mucosa affected
2 = Mild, 11 to 25% of the mucosa affected
3 = Moderate, 26 to 50% of the mucosa affected
4 = Marked, 51 to 75% of the mucosa affected
5 = Severe, 76 to 100% of the mucosa affected

Mucosal Thickness and Hyperplasia Score

Mucosal thickness is measured in a non-tangential area of the section that best represents the overall mucosal thickness. This parameter is indicative of gland elongation and mucosal hyperplasia. A hyperplasia score is derived from the measurement as follows:

0 = Normal: ≤200 µm
0.5 = Very Minimal: 201 to 250 µm
1 = Minimal: 251 to 350 µm
2 = Mild: 351 to 450 µm
3 = Moderate: 451 to 550 µm
4 = Marked: 551 to 650 µm
5 = Severe: >650 µm

Histopathology Sum

A sum of inflammation, gland loss, erosion, and hyperplasia scores is calculated.

PMN Percent and Neutrophil Score

Inflammatory cell infiltrates in the colonic mucosa are evaluated for approximate percent of neutrophils in the total infiltrate, rounded to 0, 10, 25, 50, or 75 percent. This value is then multiplied by the inflammation score to determine the neutrophil score.

Lymphoid Aggregate Count and Diameter

The number of definite mucosal lymphoid aggregates (GALT, Peyer’s patches) is recorded. The largest of these is measured by optical micrometer.