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Quantitative Ex Vivo Imaging of Tissue O 2 Reveals Effects of Inflammation, Mitochondrial Respiration and ROS Production on O 2 Levels in Colonic Mucosa
Author(s) -
Zhdanov Alexander V,
Okkelman I. A.,
Golubeva A. V.,
Aviello G.,
Melgar S.,
Hyland N. P.,
Knaus U. G.,
Cryan J. F.,
Papkovsky D. B.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.742.14
Subject(s) - ex vivo , epithelium , pathology , barrier function , intestinal mucosa , intestinal epithelium , inflammation , in vivo , chemistry , biology , microbiology and biotechnology , immunology , medicine
Mucosal epithelium is a multifunctional barrier tissue protecting the organism against bacterial invasion from intestinal lumen. Loss of mucosal barrier function leads to the development of intestinal inflammation, which is associated with re‐arrangement of tissue metabolism and oxygenation. However, data on the oxygenation of healthy and inflamed intestinal mucosa are controversial. In healthy gut, mucosal epithelium sustains deep hypoxia with periodic fluctuations of O 2 levels; however the borderline between normal and pathological tissue oxygenation is hard to define. O 2 availability reciprocally regulates the composition and activity of colonising bacteria, absorption and permeability of the epithelium and gene expression. Hence, knowledge on local O 2 levels is of paramount importance. Here, we present quantitative ex vivo imaging of mucosal oxygenation using high‐resolution phosphorescence lifetime imaging microscopy (PLIM) in different models of intestinal dysfunction. Methods 10 week old C57BL/6 mice were given 3% dextran sulphate sodium (DSS group, 7 mice) or water (control group, 7 mice) for 4 days, and then euthanized. The distal colon was dissected for phosphorescent staining, O 2 imaging and RNA extraction followed by gene expression analysis. Mucosal epithelium was stained ex vivo with a cell‐penetrating O 2 sensitive probe, Pt‐Glc and then imaged on a confocal PLIM system equipped with a time‐correlated single photon counting (TCSPC) module (405 nm excitation and 635–675 nm emission). Results Pt‐Glc brightly stained mostly epithelial cells within the upper 50–60 μm layer of the tissue. In healthy mucosa O 2 levels were 20±15 μM, tending to decrease in deeper tissue areas. Four‐day treatment with DSS induced colonic inflammation, as reported by elevated local IL‐6 and mKC gene expression, and significantly increased mucosal oxygenation, suggesting reduced O 2 consumption. Treatment with dimethyloxalylglycine (DMOG), known to protect the tissue against DSS‐induced injury, inhibited mitochondrial respiration and increased mucosal O 2 . This demonstrates the potential of DMOG to relieve metabolic stress imposed by hypoxia on the inflamed colon in vivo. Analysing the impact of non‐mitochondrial O 2 consumption, we found that mucosal oxygenation was affected by NADPH oxidase deficiency. The imaging of human colon biopsies is being pursued, but currently the technique will require refinement to allow discrimination between healthy and inflamed tissue samples. Conclusions PLIM‐TCSPC technique shows high applicability to study oxygenation of the colonic mucosa and sufficient sensitivity to detect changes in O 2 levels in different animal and disease models. Support or Funding Information This work was supported by Enterprise Ireland (CF/2012/2346) and Science Foundation Ireland (12/RC/2273)