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

Authors Sandoval-Skeet N, Kaufman JA, Castro MJ, Al-Nakkash L

Received 3 August 2018

Accepted for publication 19 October 2018

Published 29 November 2018 Volume 2018:11 Pages 863—873

DOI https://doi.org/10.2147/DMSO.S182501

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Andrew Yee

Peer reviewer comments 3

Editor who approved publication: Dr Steven F. Abcouwer


Supplementary video 3D reconstruction of optically cleared jejunum stained with DAPI

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Noemy Sandoval-Skeet,1 Jason A Kaufman,2 Monica J Castro,2 Layla Al-Nakkash3

1Department of Biomedical Sciences, 2Department of Anatomy, 3Department of Physiology, Midwestern University, Glendale, AZ 85308, USA

Introduction:
Diabetes is commonly associated with gastrointestinal dysfunction. We have previously shown that transepithelial short circuit current, Isc (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 Isc. We aimed to evaluate whether morphological changes in the jejunal crypts contribute to the rescue of Isc.
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 Isc are therefore likely not attributed to changes in intestinal crypt morphology.

Keywords:
genistein, intestine epithelia, jejunum, optical clearing, diabetes

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