Histomorphometric Analysis of Intestinal Samples

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.