In vivo fluorescence microscopic imaging for dynamic quantitative assessment of intestinal mucosa permeability in...

Herein, we introduce a novel intravital microscopic method to quantitatively assess mucosal permeability in mouse intestine. To this end, the time course of changes in the accumulation of fluorescent markers (sodium fluorescein [MW 376] and fluorescent dextran [FD4000, MW 4000; Merck, Darmstadt, Germany]) was examined in the perivascular interstitium of the mesentery of sham-operated animals and animals undergoing 30 min intestinal ischemia and 90 min postischemic reperfusion. An increased permeation of intraluminally installed sodium fluorescein was found only in the late reperfusion period, while permeability was enhanced during the entire postischemic examination period when renal excretion of the fluorescent marker was excluded by kidney ligation. Similarly, the late reperfusion phase was associated with enhanced tissue fluorescence to FD 4000 when the kidneys were ligated. Fluorescence values in the plasma, as measured by standard fluorimetry, showed a significant correlation with tissue fluorescence determined by intravital microscopy. Further, villus tip denudation at 90 min reperfusion correlated with increased permeability of sodium fluorescein in the early (10-20 min) and FD 4000 in the late reperfusion phase (90 min). Thus, intravital microscopic measurement of mesenteric perivascular fluorescence provides a simple and easily applicable on-line method to quantitatively assess epithelial permeability, allowing to detect altered mucosal barrier function and to evaluate salvage therapies which target mucosal integrity.