MEASUREMENT OF TISSUE VIABILITY USING INTRAVITAL MICROSCOPY AND FLUORESCENT NUCLEAR DYES

Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injur y following disease processes. However, this assumes that prolonged ex posure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (ED L) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbit al (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbe nzimide (5 mu g/ml; labels nuclei of all cells) and ethidium bromide ( 5 mu g/ml; labels nuclei of injured cells) or had dyes topically appli ed 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. N oxious stimuli (i.e., hypoxia:FiO(2) of 8-10% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were us ed to test the ability of ethidium bromide, when used in conjunction w ith intravital microscopy, to differentiate injured tissue. Video reco rdings at the surface of the EDL muscle were made every 30 min for 5 h r from which the number of perfused capillaries was counted (NCper). T he numbers of bisbenzimide- and ethidium bromide-labeled nuclei were c ounted at the surface of the muscle and at two to three additional loc ations within the muscle (to a maximum depth of approximately 120-160 mu m). The average NCper (19.05 +/- 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 +/- 125. The average number of ethidium bromide-labeled nuclei (380.4 +/- 36.8) remained stable for at least 80-90 min, after which the number of labeled nuclei increased linearly. The number of ethidium bromide-labeled nuclei increased com pared to control (P less than or equal to 0.05) following all noxious stimuli. Thus, within the first 80-90 min of exposure, use of fluoresc ent nuclear dyes in conjunction with intravital microscopy provides a means of evaluating spatial and temporal changes in tissue viability. (C) 1995 Academic Press,Inc.