Background Medical students experience high levels of stress during training due to demanding course loads which often leaves less time for self-care. This study combines the self-care technique of yoga with learning anatomical locations, innervations, actions, and functions of the muscles and organs to determine if anatomy tests scores are improved and whether students’ stress levels attenuate from participating in yoga. Methods In this randomized controlled study, 64 student volunteers were randomized into either a yoga intervention group or wait list control group throughout the M1 anatomy course. The yoga group (n = 32) participated in 8 yoga sessions synced with the anatomy topics they were learning in lecture. The wait list group (n = 32) went through their normal anatomy curriculum but had an option to participate in the same yoga sessions after the anatomy course. The primary research purpose was to determine whether yoga improved anatomy exam performance by comparing four anatomy exam scores between the two groups. The secondary research purposes included the following: to determine whether yoga classes including anatomy teaching still conferred acute and long-lasting stress relief by, respectively, comparing a students’ own pre- and post-yoga stress level and self-perceived stress levels between the two groups; and to determine if a student’s confidence in anatomy was improved after a yoga session. Results There was no significant difference in anatomy exam performance between students who received yoga and those on the waitlist (all p > 0.05). For students who received yoga, their average self-reported stress levels decreased after each yoga session, their average DASS (Depression, Anxiety and Stress Scale) score decreased after a yoga session, but they were not significantly less stressed than their waitlist peers prior to an exam, and their self-reported confidence in anatomy material related to the back, upper extremity, head and neck, and abdomen/pelvis increased. Conclusion With this sample, there was no evidence that yoga sessions paired with anatomy lecture material improved overall anatomy exam performance, as opposed to only the musculoskeletal portion which other studies have looked at. However, yoga acutely reduced stress levels, and subjective feelings of knowledge improvement were noted by participants. Both of these can provide benefits to medical students.

New methods on optical clearing provide a valuable alternative to traditional physical section histology. Optical clearing allows investigation of relatively large tissue samples at histological resolution while maintaining the three-dimensional architecture of the intact system. There is significant potential for applying optical clearing to gastrointestinal tissues. In particular, intestinal crypts contain high concentrations of stem cells, making these structures especially important for research on cellular proliferation in the intestinal epithelium. The objective of our study is to demonstrate an optical clearing method that is easy to implement and is compatible with mitotic fluorescent labeling. The optical clearing method we present utilizes a Triton/DMSO delipidization step followed by refractive index matching, rendering the tissue nearly transparent. We use EdU click chemistry to fluorescently label mitotic cell nuclei. Our results demonstrate successful clearing of jejunal samples with readily visible EdU staining by means of confocal microscopy. Three-dimensional reconstruction of labeled samples reveals preservation of intestinal cytoarchitecture including muscular, submucosal, and mucosal layers. Additionally, the morphology of intestinal crypts and individual EdU-positive mitotic nuclei are visible in sharp detail within their intact three-dimensional organization. In summary, we present an optical clearing method that is easy to implement and has the potential to provide more accurate assessment of cellular proliferation within the gastrointestinal tract in both healthy and disease states.

IntroductionDiabetes 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.MethodsMale 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.ResultsWe 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.ConclusionThus, 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.

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)