Biophysics of freezing in liver of the freeze-tolerant wood frog, R. sylvatica.

This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog Rana sylvatica. Experiments were performed using both low temperature microscopy and a differential scanning calorimeter (DSC). Tissue samples were cooled at 2 and 5 degree C/min by a "two-step" freezing technique to end temperatures of -4, -6, -8, -10, and -20 degrees C, followed by a slam cooling (> 1000 degrees C/min) step. Stereological analysis of the low temperature microscopy results leads to the conclusions that 74% of the control tissue is cellular (26% vascular), Vb (osmotically inactive cell volume) is 0.4 Vo and the Krogh cylinder dimensions are: distance between adjacent sinusoid centers, delta X = 64 microns, original sinusoid (vascular) radius, rvo = 18.4 microns and length of the Krogh cylinder, L = 0.71 microns (assuming a single isolated hepatocyte cell diameter of 16 microns). A parallel study was also done using the DSC at 2 and 5 degrees C/min, and the measured heat releases from the tissue were used to calculate water transport data. Both techniques confirmed that tissue cooled at 5 degrees C/min does not dehydrate completely, but does so when cooled at 2 degrees C/min. By curve fitting a model to 5 degrees C/min water transport data from both techniques the biophysical parameters of water transport were obtained: Lpg = 1.76 microns/min-atm and ELp = 75.5 Kcal/mol. A modified Krogh model was used to account for the fact that approximately 24% of the hepatocytes were found not to be in direct contact with the vasculature. This model was then used to explain the experimentally measured water retention in some cells on the basis of different volumetric responses to dehydration of cells directly adjacent to vascular spaces and cells at least one cell removed from the vascular spaces.