Digital Holographic Microscopy for Assessment of Experimental Colitis and Monitoring of Intestinal Wound Healing in vitro

Abstract

Purpose: Intestinal epithelial barrier function is crucial for pathogenesis of inflammatory bowel disease and for evaluation of potential anti-inflammatory drugs in vitro. Digital holographic microscopy (DHM) enables marker-free quantitative phase contrast imaging and provides assessment of cell thickness and tissue density by measuring optical path length delay (OPD). Aim of this study was to evaluate DHM for assessment of experimental colitis in vivo and wound healing in vitro. Methods: Experimental colitis was induced by dextran sodium sulfate in CL57/B6 WT mice. Cryostat colonic sections (5 μm) of healthy (n=7) and colitic mice (n=9) were examined via DHM. To quantify tissue density, segmental OPD analysis of colonic wall (75 measurements/animal) was performed according to anatomic structure (epithelium (e), submucosa (sm) and stroma (st)). DHM was used for time lapse monitoring of wound healing in scratch assay of human intestinal fibroblasts and intestinal epithelial cells (Caco-2 cells) by automated measurements over time (digital holograms were recorded every 3 min). Results: Average optical OPD varied significantly within the different intestinal wall-layers in all mice (e: 1.50 ± 0.30, sm: 1.22 ± 0.59, st: 1.80 ± 0.60 rad; p < 0.001). In contrast, OPD in colitic mice (n=9) was significantly decreased reflecting a significant loss to density in all parts of the colonic wall compared to healthy mice (e: 1.62 ± 0.27 vs. 1.41 ± 0.29; p < 0.001, sm: 1.77 ± 0.42 vs. 0.80 ± 0.29; p < 0.001, st: 1.84 ± 0,53 vs. 1.76 ± 0.66 rad; p < 0.05). Wound assays via DHM from living Caco-2-cells and human intestinal fibroblasts allow quantification of proliferation over time (Caco-2: T1(12h)=34.2%, T2(24h)=53.2% and fibroblasts: T1=(24h)=15.0%, T2=(48h)=30.4% growth in plane axis) with simultaneous morphology imaging (Figure 1).Figure: [1622] Figure 1 Wound assay: time-dependent quantitative DHM phase contrast images of living Caco-2-cells (a-c) and fibroblasts (d-f) coded to 256 gray levels. The wound borders are marked with arrows at different time intervals (T0,T1,T2).Conclusion: DHM allows differentiation between healthy and inflamed intestines and correctly detects density changes of colonic wall during experimental colitis, reflecting loss of epithelial barrier function. In addition, DHM may serve as a valuable tool for quantitative live monitoring of wound healing in vitro for evaluation of novel potential drugs in different cell types.