Optimetric

For intestinal morphology, duodenum, jejunum, and ileum were collected and drained and washed, and the intestinal segment with a position of about 2 cm in the middle was fixed in 10% formaldehyde–phosphate buffer, then embedded in paraffin, and stained with hematoxylin and eosin for histomorphological analysis. Villus height (VH), crypt depth (CD), and the ratio of villus height to crypt depth (VH/CD) were measured under a 100 × magnification optical microscope (Olympus CX31, Japan) and computer-aided morphometry system (BioScan Optimetric; BioScan Inc., Edmond, WA, USA), as previously described [18 (link)]. VH was measured from the junction of the villus tip to the crypt, and CD was measured from the junction of the villus crypt to the tip of the mucous muscularis [19 (link)].

After a 24 h fixation, the intestinal segments were dehydrated, embedded, and stained with hematoxylin and eosin. Villus height and crypt depth were measured at 200× magnification with a microscope (Olympus CX31, Tokyo, Japan) according to our previous study [19 (link)]. Ten well-oriented and intact villi were selected and determined using a light microscope with a computer-assisted morphometric system (BioScan Optimetric; BioScan Inc., Edmond, WA, USA). Villus height was measured from the tip of the villus to the villus-crypt junction; crypt depth was defined as the depth of the invagination between adjacent villi.

The duodenum, jejunum, ileum, and colon samples were fixed with neutral formalin solution and then embedded in paraffin. After being cut into approximately 5 μm thicknesses, the paraffin sections were stained with hematoxylin and eosin. The intestinal villus height and crypt depth were measured using a light microscope equipped with a calibrated eyepiece graticule (BioScan Optimetric, BioScan Inc., Edmonds, WA, USA) [17 (link)]. Quantitative analysis of the digitally acquired images was performed by ImageJ software. The ratio of villi and crypt was calculated.