Abstract
The early events in an intestinal ischemic episode have been difficult to evaluate. Using in vivo microscopy we have analyzed in real-time the effects of short (15 min) and long (40-50 min) ischemia with subsequent reperfusion (IR), evaluating structure, integrity, and functioning of the mouse jejunal mucosa while monitoring blood flow by confocal microscopy. IR was imposed by inflation/deflation of a vascular occluder, and blood flow was monitored and confirmed with scanning confocal imaging. After short ischemia, villus tip cells revealed a rapid increase (23%) in the intracellular NAD(P)H concentration (confocal autofluorescence microscopy), and the pH-sensitive probe BCECF showed a biphasic response of the intracellular pH (pH(i)), quickly alkalinizing from the resting value of 6.8 +/- 0.1 to 7.1 +/- 0.1 but then strongly acidifying to 6.3 +/- 0.1. Upon reperfusion, values returned toward control. In contrast, results were heterogeneous after long IR. During long ischemia, one-third of the epithelial cells remained viable with reversible changes upon reperfusion, but remaining cells lost membrane integrity (Lucifer Yellow uptake, LY) and had membrane blebs during ischemia. These effects became more pronounced as the reperfusion interval progressed when cells exhibited more severely affected NAD(P)H and pH(i) values, larger blebs, and more LY uptake and eventually were shed from the villus. Results from stereo microscopy suggest that these irreversible effects of IR may have occurred as a result of incomplete restorations of local blood flow, especially at the antimesenteric side of the intestine. We conclude that the adverse effects of short ischemia on the jejunum epithelium are fully reversible during the reperfusion interval. However, after long ischemia, reperfusion cannot restore normal structure and functioning of a majority of cells, which deteriorate further. Our results provide a basis for defining the cellular events that cause tissue to transit from reversible to irreversible damage during IR.