Real‐time in vivo imaging of ischemia‐reperfusion damage in mouse small intestine

Abstract

The intestinal mucosa is easily injured by ischemia and reperfusion (IR), but detailed insight into the sequence of cellular events during IR is lacking, so we developed in vivo microscopy systems to measure IR damage. Jejunal villi of anesthetized male mice were imaged during 30 min ischemia (vascular ligation; remote inflatable clamp) and 60 min reperfusion (deflated clamp). Within 5 min of ischemia, microvasculature dilated (stereo microscopy), blood cells in villus tips immobilized (confocal reflectance) and some villus tip cells were injured (uptake of Lucifer Yellow, cell swelling; confocal fluorescence). By 20 min ischemia, these events had spread to ~50% of villus tips. After 5 min of reperfusion, injury expanded (including shedding of villus tip cells), and cell metabolism decreased (decreased intensity of NAD(P)H autofluorescence). Damage was widespread (>95% villi) after 20 min reperfusion. Release of the vascular clamp rapidly restored blood flow, but there was heterogeneity in response. Vessels on the mesenteric side returned to normal diameter promptly, but remained dilated for >20 min on the anti-mesenteric side. In tissue regions with restored blood flow, only 70% of villi had consistent villus tip blood flow during most of the reperfusion interval. The heterogeneity of villi response may help explain confusion about the timed sequence of events in response to IR damage.