Microcomp

The fixed intestinal samples were dehydrated, embedded in paraffin wax, cut into serial 5 μm sections, and stained by hematoxylin and eosin. Histological sections were examined by a microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i, Nikon Co., Tokyo, Japan). Three orientated sections cutting vertically from the villus enterocytes to the muscularis mucosa were selected from each sample and the measurements were carried out as follows (19 (link)). The vertical distance from the villus tip to villus–crypt junction level was taken as the intestinal villus height (VH), and the vertical distance from the villus-crypt junction to the lower limit of the crypt as the crypt depth (CD). Ten loci per section were selected for the measurement of the VH, CD, and intestinal wall thickness (IWT). The ratio of VH/CD was calculated as V/C.

Morphometric analysis of the ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction) was evaluated according to Giannenas et al [16 ]. One-centimetre-long segments were taken from the centre of each part and fixed in 10% buffered formalin for morphometrical assays under light microscopy. Formalin-fixed intestinal tissues were processed, embedded in paraffin wax, sectioned at 3 μm and stained the haematoxylin-eosin method. Histological sections were examined with a Nikon phase contrast microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i; Nikon, Tokyo, Japan). Images were viewed using a 4× Eplan objective (40×) to measure morphometric parameters of intestinal architecture. For this purpose, three favourably orientated sections cut perpendicularly from villus enterocytes to the muscularis mucosae were selected from each animal and measurements were carried as follows: villus height was estimated by measuring the vertical distance from the villus tip to villus-crypt junction level for 10 villi per section; crypt depth (the vertical distance from the villus-crypt junction to the lower limit of the crypt) was estimated for 10 corresponding crypts per section.

The lesion scores of the cecum were determined and recorded according to the method of Johnson et al. [15 (link)]. The lesion scores range from 0 (no gross lesion) to 4 (most severe gross lesion). One-cm section of ileum was fixed in 4% paraformaldehyde solution, dehydrated and embedded in paraffin. The section was cut 4 μm long and stained with hematoxylin-eosin. The villus length (VH), crypt depth (CD), and VH to CD ratio (V/C) were measured by a Nikon phase contrast microscope coupled with a Micro Comp integrated digital imaging analysis system (Nikon Eclipse 80i, Nikon Corp., Tokyo, Japan).