Freeze tolerance in turtles: visual analysis by microscopy and magnetic resonance imaging

Two visual techniques were used to analyze the patterns of natural freezing and thawing in freeze-tolerant hatchling painted turtles Chrysemys picta marginata. Directional solidification plus light microscopy of liver, heart, and skeletal muscle slices was used to compare freezing at -4 degrees C (a survivable temperature in vivo) and -20 degrees C (not survivable). At -4 degrees C tissues showed large amounts of ice in expanded extracellular and vascular spaces, occupying 36% (liver) and 61% (muscle) of total tissue volume. Cells at -4 degrees C were shrunken, but intracellular water remained; at -20 degrees C, however, cells showed little evidence of free water. Liver micrographs showed novel spherical shells of water associated with intracellular particles (apparently glycogen granules) suggesting that a noncolligative method of cell water retention was employed. Proton magnetic resonance imaging was used for noninvasive analysis of freezing and thawing in the intact animal. Images showed that freezing propagated in a directional manner through the body with ice formed first in extraorgan spaces (e.g., abdominal cavity, brain ventricles). However, thawing occurred uniformly throughout the body core, and organs melted more rapidly than the extraorgan ice surrounding them.