Genistein diet does not modify crypt morphology in the ob/ob mouse jejunum: a comparison of cryostat and clearing techniques.

INTRODUCTION

Diabetes is commonly associated with gastrointestinal dysfunction. We have previously shown that transepithelial short circuit current, I (chloride secretion), is significantly reduced in the jejunum from mice vs lean controls, and consumption of 600 mg genistein/kg of diet (600 G) for 4 weeks significantly rescues I. We aimed to evaluate whether morphological changes in the jejunal crypts contribute to the rescue of I.

METHODS

Male mice ( and lean controls) were fed either a genistein-free diet or genistein-containing diet (600 G). Comparisons of crypt morphology were made for crypt depth, length, and numbers of proliferative cells. Assessments of crypt measures using DAPI and 5-ethynyl-2'-deoxyuridine (EdU) were performed using traditional cryostat sectioning and an innovative 3D optical clearing method.

RESULTS

We found that crypt length in the genistein-fed group was significantly greater when measured with cleared tissue (85.19±4.73 µm, <0.05, n=8) compared to lengths measured with cryostat (65.42±3.48 µm, n=8). In addition, proliferative EdU+ counts were approximately fivefold greater with clearing, compared to counts obtained via single plane images from cryostat sections for all groups measured. The average length to EdU+ ratio was unchanged between groups.

CONCLUSION

Thus, we conclude that genistein diet does not affect overall cellular proliferation or crypt morphology, other than for the modest increased crypt length measured via clearing in the genistein group. The increase in crypt length is likely indicative of the greater accuracy of the 3D measures compared to single plane. Genistein diet-induced increases in the intestinal I are therefore likely not attributed to changes in intestinal crypt morphology.