A44 NOVEL AND HIGHLY SENSITIVE FLOW CYTOMETRIC BASED METHOD FOR CONTINUOUS TRACKING OF INTESTINAL PERMEABILTIY

Abstract

Abstract Background Inflammatory bowel diseases (IBD) are chronic, inflammatory conditions of the intestinal tract. In addition to a complex mixture of genetic, and environmental factors, increased intestinal permeability is also thought to be involved. Despite its diagnostic importance, there is no reliable yet minimally invasive way to measure intestinal permeability in patients and animal models. Currently, most assays involve the detection of orally given sugar molecules in either the urine (MLR method) or the plasma (FITC-dextran). These methods offer only limited accuracy, and do not allow continual tracking of intestinal permeability within the same animal due to the requirement of euthanization. Herein, we describe a novel cytometric-based method using an ingested dietary protein (ovalbumin (OVA)) and flow cytometry. We show our method is reliable, highly sensitive, minimally invasive and allows for the continuous tracking of intestinal permeability within the same individual using small blood volumes. Aims To compare the utility of our bead-based method of intestinal permeability measurement to the FITC-Dextran method. Methods Wildtype (WT) C57BL/6 mice, and mucin 2-deficient (Muc2-/-) mice (known to suffer a leaky gut) at baseline, or mice given chemical or infection-induced colitis were gavaged with a solution containing OVA protein and FITC-Dextran. After 6 hours, mice were euthanized to collect plasma for spectrophotometry (FITC-Dextran), or for flow cytometry (bead-based ELISA) analyses. Results We found that both OVA and FITC-dextran were detectable in plasma samples of the Muc2-/- mice, but not the WT mice, 6 hours after oral co-administration, however the bead-based method produced more consistent readings. Immunostaining of tissue sections with the same antibodies also showed that OVA can readily diffuse through the gut epithelium of Muc2-/- mice but not the WT mice, validating the specificity of the antibodies. We also tested our assay on WT mice undergoing dextran sodium sulfate (DSS) colitis, or infected with Citrobacter rodentium. We found that our method could detect statically significant changes in intestinal permeability 2 to 3 days earlier than the FITC-dextran method, while providing greatly reduced variability between technical repeats. More importantly, we were able to use a small volume (5 μL) of whole blood collected via tail poke, to measure intestinal permeability without requiring euthanization of the mice. This allowed the continuous tracking of permeability changes within the same animal. Conclusions We conclude that the bead-based method is more sensitive and reliable than the FITC-Dextran method as tested in several murine colitis models. More importantly, we showed that the bead-based method allows continual tracking of intestinal permeability within the same animal, enabling time course measurements. Funding Agencies CAG, CCC, CIHR, NRCBC Children’s Research Institute