Analysis of jejunal crypt morphology from diabetic mice fed genistein diet using two methods: traditional cryostat and novel optical clearing

Abstract

Gastrointestinal dysfunctions are commonly associated with obesity and diabetes. We have previously shown a significant decrease intransepithelial short circuit current (Isc) in jejunal segments from ob/ob diabetic mice, compared to lean controls. Moreover, we recently demonstrated a significant increase/rescue in Isc in ob/ob mice following chronic consumption(4‐weeks) of a genistein‐containing diet, 600 mg genistein/kg food (600G). Therefore, the current study is evaluating whether or not morphological changes in jejunal crypts of ob/ob mice are responsible, at least in part, for the rescue of Isc. Male ob/ob mice were fed either a genistein‐free diet orgenistein‐containing diet (600G), and comparisons made to lean controls. Assessments of crypt morphology were performed via two methodologies:traditional cryostat sections in optimal cutting temperature compound (OCT), and a novel 3‐D optical clearing method. At completion of the 4‐week diet study, mice were given an injection of EdU (5‐ethynyl‐2′‐deoxyuridine) to tag for actively dividing DNA prior to euthanizing. Freshly isolated segments of jejunum were used. Cryostat sections of OCT embedded jejunum (8 μm) were stained with an EdU Click‐iT assay, imaged at a 20× objective with an inverted microscope (15 measures/mouse, n=3/group). Additional jejunum segments underwent the clearing process, fixed with 4% PFA, stained with an EdU Click‐iT assay and imaged at a 40× objective with a confocal microscope using 100–160 μm total thickness of jejunum, with reconstructions of 0.44 μm z‐stacks (12 measures/mouse, n=3/group). The following measures were taken per image: crypt depth and width and number of EdU+ cells. Interestingly, each method gave differing results. Using the cryostat, there was a significant decrease in EdU+ cells in the ob/ob mice fed genistein‐free diet (7.03±0.506, P<0.05) compared to leans (8.93±0.544) and genistein diet significantly reversed this (8.911±1.09, P<0.05). With cryostat technique, we noted no differences in crypt depth or width. In contrast, analysis of the optically cleared samples demonstrated a significant decrease in crypt depth (CD) and crypt width (CW) in ob/ob genistein‐free fed mice (CD=58.02±2.92μm and CW=31.28±2.30 μm, P<0.05) compared to leans (CD=71.75±3.08 μm and CW=41.30±3.08 μm) and genistein rescued this (CD=94.33±4.75 μm and CW=41.08±2.35 μm, P<0.05). In addition, we will determine measures of crypt volume utilizing the 3‐D optical clearing method. Additional measures with a further four mice/group will determine if our measures and statistical evidence are consistent with a larger sample size. The data suggest that caution should be used with cryostat methodology, whereby one ‘sees a snapshot’ of the jejunum versus the optical clearing methodology, which enables visualization of 3‐D crypts in their entirety, thus providing a virtual reconstruction of the intact tissue. The optical clearing methodology demonstrates a more thorough view of whole tissue samples, thereby generating a more accurate account of the tissue, and specific morphometrics under investigation. Thus, we conclude that given the data shown above, increased cellular proliferation within the jejunal crypts does not appear to account for the genistein‐mediated increase in Isc in ob/ob mice fed 600G.Support or Funding InformationMidwestern University Department of Biomedical Sciences (NS‐S and SR), DAREF (LA), Midwestern Intramural funds (LA and JK)