Injury and protection in split-thickness skin after very rapid cooling and warming

The ability of low glycerol concentrations and high cooling and warming rates to optimize the survival of frozen/thawed split-thickness porcine skin was investigated. 1H nuclear magnetic resonance spectroscopy was used to measure the diffusion kinetics of glycerol in skin at 4, 12, and 22 degrees C. Equilibrium concentrations were 44 to 69% of the external bathing medium. Rate constants for glycerol diffusion (D/l2) were calculated from the uptake data using a plane sheet model and a least squares method and were independent of external glycerol concentrations: D/l2 = 3.84 x 10(-4) 8-1 at 4 degrees C with an activation energy of 11.2 +/- 4.3 kcal/mol. Skin was cooled rapidly (-5100 degrees C/min) after different times of glycerol permeation at 4 or 22 degrees C, and survival was assessed after warming (+5400 degrees C/min) by an oxygen consumption assay. Recovery of aerobic activity increased in a concentration-dependent manner, and reached 100% after a 10-min exposure to 2 M glycerol at 4 degrees C or 3 min at 22 degrees C, 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-mm exposure to 2 M glycerol at 22 degrees C resulted in the elimination of all observable epidermal ice, except for extremely small ice crystals (< or = 0.5 micron diameter) in the intercellular spaces and in few nuclei, and complete preservation of the fibrous structure of dermal collagen bundles. This cryoprotective mechanism has the potential to offer complete protection of both dermal and viable epidermal targets of freeze/thaw injury and may be applicable to other thin, membranous tissues.