INJURY AND PROTECTION IN SPLIT-THICKNESS SKIN AFTER VERY RAPID COOLING AND WARMING

The ability of low glycerol concentrations and high cooling and warmin g rates to optimize the survival of frozen/thawed split-thickness porc ine skin was investigated. LH nuclear magnetic resonance spectroscopy was used to measure the diffusion kinetics of glycerol in skin at 4, 1 2, and 22 degrees C. Equilibrium concentrations were 44 to 69% of the external bathing medium. Rate constants for glycerol diffusion (D/I-2) were calculated from the uptake data using a plane sheet model and a least squares method and were independent of external glycerol concent rations: D/I-2 = 3.84 x 10(-4) s(-1) at 4 degrees C with an activation energy of 11.2 +/- 4.3 kcal/mol. Skin was cooled rapidly (-5100 degre es C/min) after different times of glycerol permeation at 4 or 22 degr ees C, and survival was assessed after warming (+5400 degrees C/min) b y an oxygen consumption assay. Recovery of aerobic activity increased in a concentration-dependent manner, and reached 100% after a 10-min e xposure to 2 M glycerol at 4 degrees C or 3 min at 22 degrees, for an uptake of 1.1 M glycerol. Light micrographs of freeze-substituted skin showed a glycerol-dependent decrease in the nucleation and growth of ice in the dermis and epidermis after rapid cooling. A 5-min exposure to 2 M glycerol at 22 degrees C resulted in the elimination of all obs ervable epidermal ice, except for extremely small ice crystals (less t han or equal to 0.5 mu m diameter) in the intercellular spaces and in few nuclei, and complete preservation of the fibrous structure of derm al collagen bundles. This cryoprotective mechanism has the potential t o offer complete protection of both dermal and viable epidermal target s of freeze/thaw injury and may be applicable to other thin, membranou s tissues. (C) 1997 Academic Press.