Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog

The technique of directional solidification coupled with low-temperature scanning electron microscopy was applied to analyze the freezing of liver slices from the freeze-tolerant frog Rana sylvatica. Micrographs of liver slices from 5 degrees C-acclimated frogs frozen on the directional stage to -7 degrees C showed continuous ice formed along an expanded vasculature with hepatocytes that were shrunken and virtually dehydrated. However, when frogs were given a survivable freezing exposure at -4 degrees C for 24 h, liver slices subsequently frozen in vitro at -7 degrees C were much less shrunken and the presence of intracellular ice crystals (formed when samples were plunged into liquid N2 before microscopy) demonstrated that ample free water remained in these hepatocytes at -7 degrees C. This reduced level of cell dehydration was correlated with the buildup of 280 +/- 61 mumol/g wet wt glucose as a cryoprotectant in liver during the -4 degrees C exposure in vivo. The study provides the first direct cytological analysis of the freezing process in an organ of a freeze-tolerant vertebrate and the first confirmation of the relationship between maintenance of a critical minimum cell volume and freezing survival by these animals.