Green Fluorescent Protein as an Indicator of Cryoinjury in Tissues

The fluorescence intensity of Green Fluorescent Protein (GFP) has previously been demonstrated to be an accurate indicator of cellular viability following cryoinsult in individual GFP-transfected cells. In an attempt to ascertain whether GFP fluorescence intensity may also be used as a viability indicator following cryogenic insults in whole tissues, this study examines the transient fluorescence intensity of GFP-transfected mouse hepatic tissue ex vivo following cryoinsult. The observed trends are compared with diffusion-based models. It was observed that the fluorescence intensity of the exposed tissues exhibited slow exponential decay, while the solution in which the tissues were placed inversely gained fluorescence. This slow decay (similar to 3 h) is in contrast to the rapidly diminished fluorescence intensity (seconds) seen in GFP-cell cultures following cryoinsult. These trends suggest that mass diffusion of GFP in the interstitial space, and ultimately into the surrounding medium, is the primary mechanism which determines the fluorescence loss in cryoinjured tissues. These results suggest GFP-transfected tissues may be effectively used as indicators of cryoinjury, and hence viability, following hypothermal insult provided that a sufficiently long incubation is held before observation. It was found that a meaningful observation (15% reduction in fluorescence) could be made three hours subsequent to cryoinjury for the tissues used in this study.