Objective: To study the temporal progression of injury in skeletal muscle after ischemia-reperfusion insult by means of intravital videomicroscopy and nuclear fluorescent dyes.
Design: A controlled study in an animal model.
Setting: A vascular research laboratory at a university-affiliated hospital.
Subjects: Eight male Wistar rats, for each of which the extensor digitorum longus muscle of the hind limb was exposed and prepared.
Interventions: Two hours of complete no-flow ischemia followed by 90 minutes of reperfusion in five of the rats; the other three rats acted as controls and underwent the same surgical procedure but not ischemia. During the reperfusion period the fluorescent vital dyes bisbenzimide, which permeates all cells, and ethidium bromide, which permeates cells with damaged membranes, were applied. Recordings to videotape were made with the intravital microscope very 15 minutes during the reperfusion period.
Main outcome measures: The number of perfused capillaries crossing three straight lines on the video monitor were counted as a measure of microvascular dysfunction. An index of tissue injury was calculated as the ratio of the number of nuclei stained by ethidium bromide to the number stained by bisbenzimide (E/B). The number of stuck and rolling leukocytes and the velocity of the rolling leukocytes were determined in postcapillary venules.
Results: The mean number of perfused capillaries (and standard error of the mean) fell from 20.71 (1.64)/mm before ischemia to 11.69 (1.18)/mm during reperfusion in the experimental group but remained constant in the control group. In the experimental group E/B progressed from 0.43 (0.05) at the onset of reperfusion to 0.87 (0.03) at the end of reperfusion, the number of rolling leukocytes increased from a preischemia mean of 4.00 (1.90) to 14.80 (1.30)/1000 microns2, and the number of stuck leukocytes increased from 1.42 (0.20) to 9.20 (0.70)/1000 microns2. The velocity of the rolling leukocytes did not differ between the control and the experimental groups.
Conclusions: Although microvascular perfusion decreased quickly to a constant level after 2 hours of noflow ischemia plus reperfusion, a progressive increase in tissue injury occurred, which may correlate with the number of stuck leukocytes.