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

Abstract

Introduction

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

Methods

Male mice (ob/ob 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 ob/ob genistein-fed group was significantly greater when measured with cleared tissue (85.19±4.73 µm, P<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 ob/ob 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_sc are therefore likely not attributed to changes in intestinal crypt morphology.

Keywords:

• genistein
• intestine epithelia
• jejunum
• optical clearing
• diabetes

Acknowledgments

This work was supported by a DAREF grant (LA-N), Mid-western University intramural funding (JAK and LA-N), and CHS Biomedical Science funds (NS-S). This article (in preliminary form) was presented at the Experimental Biology 2017 as a poster presentation with interim findings. The poster’s abstract was published in “Poster Abstracts” in The FASEB Journal, abstract 894.2: https://www.fasebj.org/doi/abs/10.1096/fasebj.31.1_supplement.894.2.

Author contributions

LA-N and JAK conceived and designed the experiment. MJC and JAK developed and optimized the protocol for optical clearing and imaging. LA-N and NS-S performed the EdU injections and animal euthanasia. NS-S and MJC processed the samples for optical clearing and performed confocal imaging. NS-S performed all the measures and analyses. LA-N and NS-S wrote the manuscript. All of the authors reviewed the final version of the manuscript and provided their consent before submission. All authors contributed toward data analysis, drafting and critically revising the paper, gave approval for the final version to be published and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.