Indomethacin Induced Crohn’s in Rats Histopathology Methods

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Histology & Histopathology Methods for Indomethacin Induced Crohn’s in Rats

Processing of Tissues

Body weights will be taken as well as intestinal weights/10cm of area at risk. Intestines will be scored for gross lesions and multiple sections of gut will be taken for histology (3-5 sections/rat spaced equally along area at risk). Decision to proceed with histopathology will be determined after analysis of live phase/necropsy data.

Gross Scoring Criteria

0 = Normal
0.5 = Very minimal thickening, multifocal in area at risk
1 = Minimal thickening, family diffuse in area at risk
2 = Mild to moderate small intestinal/mesenteric thickening throughout area at risk
3 = Moderate thickening with 1 or more definite adhesions that would be easy to separate
4 = Marked thickening with numerous hard to separate adhesions
5 = Severe intestinal lesion resulting in death

Histologic Scoring Criteria

Preserved (10% Neutral Buffered Formalin) 10cm sections of small intestine are trimmed into 3-5 sections collected at approximately equal intervals along the width of the intestine, without bias toward lesion presence or absence. These trimmed sections are processed through graded alcohols and a clearing agent, infiltrated and embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) by HistoTox, Inc.

Small intestine sections from all animals are examined microscopically by a board certified veterinary pathologist (Dr. Alison Bendele) and observations are entered into a computer-assisted data retrieval system. Three to five cross sections of small intestine are scored for inflammation and necrosis using the following criteria:

Necrosis

0.5 = 1-499 µm width, generally <5% of mucosa affected
1 = 500-1000 µm width, generally 5-10% of mucosa affected
2 = 1001-1500 µm width, generally 11-15% of mucosa affected
3 = 1501-2000 µm width, generally 16-20% of mucosa affected
4 = 2001-3000 µm width, generally 21-25% of mucosa affected
5 = >3000 µm width, >25% of mucosa affected

The area at risk for development of this lesion is generally approximately 30% of the total area of a cross section of jejunum where the mesentery attaches. The total circumference is generally about 10,000 µm and of this total, only about 4000 µm is generally at risk for lesion development, hence the low range for the % mucosa affected.

Inflammation

0.5 = Very Minimal, localized to mesentery, very minimal in intestine (generally associated with necrosis score of 0.5 if necrosis is present)

1 = Minimal, diffuse in mesentery and focal in intestinal lesion if present (generally associated with necrosis score of 1, or higher necrosis scores if there is an anti-inflammatory effect in the absence of a beneficial effect on necrosis)

2 = Mild, diffuse in mesentery and intestinal lesion (generally associated with necrosis score of 2 or higher)

3 = Moderate, diffuse in mesentery and intestinal lesion (generally associated with necrosis score of 3 or higher)

4 = Marked, diffuse in mesentery and intestinal lesion (generally associated with necrosis score of 4 or higher)

5 = Severe, diffuse in mesentery and intestinal lesion (generally associated with necrosis score of 5 or higher)

Inflammation is localized to the mesentery and intestinal area at risk for necrosis with slight extension on either side of it. Therefore the inflammation severity generally tracks with the necrosis and hence has similar scores except where necrosis occurs and inflammation is down modulated by treatments. In this case, inflammation is scored based on overall severity ranging from 0.5 (very minimal) to 5 (severe) without regard for the extent of necrosis.

Histopathology Sum

The mean score for necrosis and inflammation was calculated for each animal and these mean scores were then summed to arrive at a total score. Each section also has a measurement of total width of mucosal necrosis. For each treatment group, the mean ± necrosis, inflammation, and total score, as well as the necrosis width is determined and treatments are compared to the vehicle treated controls.