A dual label fluorescence technique for measuring macromolecular clearance in the mouse

Abstract

We modified the dual isotope method of Renkin et al (Am J Physiol 255: H458, 1988) to use fluorescently labeled macromolecules to measure tissue clearance as an index of vascular permeability. With this approach measurements of macromolecular permeability should be possible using the same tracer in individual capillaries or whole organs in both rats and genetically modified mice. Bovine serum albumin (BSA) was labeled with AlexaFluor 647 (BSA-647) for the tracer and with AlexaFluor 555 (BSA-555) for the vascular reference. In mice (C57Bl6/J, 25–35 g) anesthetized with pentobarbital, BSA-647 (40 μl) was injected through a jugular cannula followed by a control infusion of Ringer (1 μl/min). BSA-555 (40 μl) was injected 30 min later. After a further 5 min, blood samples were collected and the animals euthanized with KCl. Lower back skin, quadriceps and hamstring muscles were harvested postmortem and the amount of labeled BSA extracted from minced tissue was measured by comparison with fluorescent-BSA standards in tissue extract. Apparent volumes of distribution of tracer (Vt) and vascular reference (Vr) were calculated as amount of labeled BSA/plasma concentration, and tissue clearances calculated as Vt-Vr/time. Tissue clearances (expressed as μl/g dry/30 min ± SEM) were: skin 10.3±0.8 (n=12); quadriceps 10.5±1.6 (n=14); and hamstring 13.3±1.8 (n=14). Results are similar to those in rats using the dual isotope method (Renkin et al Am J Physiol 257:H525, 1989). These data form the basis for further investigations of tissue clearance in mouse models of normal physiology and of inflammatory disease.