Application of a Fluorescent Multiple Indicator Method to Study Changes in Cardiac Permeability with Chemotherapy

Abstract

Classical measurements of tissue permeability use radioactive indicators, which are very accurate but also limited by radiation exposure hazards. We have developed a fluorescent multiple indicator dilution method using Texas Red-conjugated Dextran (TR, MW 70,000 Da) as the reference dye, and sodium fluorescein (NaFL, MW 376 Da) as the diffusible dye. The characteristic excitation/emission wavelengths are 485/515 nm for NaFL, and 590/630 nm for TR. By comparing the output profiles of the two dyes versus time using a spectrofluorometer, we can estimate the permeability-surface-area-product (PSP) of the capillary network. We have studied changes in cardiac capillary permeability after treatment with doxorubicin (DOX) using our method. Our hypothesis is that DOX treatment will cause endothelial damage and lead to increased cardiac capillary permeability. Sprague-Dawley rats were randomly assigned to a control (n= 4) or DOX group (n = 5). Rats were injected intraperitoneally with 3 mg/kg of either saline solution or DOX on days 1, 3, 5, 7, 9, and 11. On day 12, we performed the isolated heart experiment. In three replicates, we injected 25 μL of the fluorescent mixture above the aortic cannula (12.5 μL each of 1.56 μg/mL TR, 1.56 ng/mL NaFL), and collected output samples for 45 seconds. We measured the samples using a Fluorolog-3® spectrofluorometer. The results showed that the control group had a lower average PSP (0.04±0.01 cm³/s) than the DOX group (0.09±0.01 cm³/s), p<0.05. This indicates that DOX treatment causes an increase in cardiac capillary permeability related to its cardiotoxic effects. The results also show that our fluorescent indicator dilution method is able to sensitively detect changes in cardiac capillary permeability. The applicability of this method is not limited to the isolated heart setup, and the protocol can be adapted for use in other studies of organ or tissue permeability.