Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose I. Experimental measurements

In this study, two methods are used to assess the equilibrium and dynamic c ell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a "two-step" low-te mperature microscopy (equilibrium and dynamic) freezing method and the seco nd is a differential scanning calorimeter (DSC) technique. These two techni ques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; ( ii) the "equilibrium" (infinitely slow) cooling behavior and the osmoticall y inactive cell volume (V-b) of R. sylvatica liver cells; and (iii) the dyn amic water transport response of R. sylvatica liver cells in the presence a nd absence of the CPA (glucose) at a cooling rate of 5 degrees C/min. Stere ological analysis of the slam frozen (>1000 degrees C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cell ular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Mod el 1) yielded distance between adjacent sinusoid centers, Delta X = 64 mu m ; original sinusoid (vascular) radius, r(vo) = 18.4 mu m; and length of the Krogh cylinder, L = 0.71 mu m (based on an isolated frog hepatocyte cell d iameter of 16 mu m). A significant observation was that similar to 24% of t he frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modifie d Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2 ) a second radius, r(2) = 28.7 mu m, was defined tin addition to the origin al sinusoid radius, r(vo) = 18.4 mu m, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle-van't Hoff plot, the osmotically in active cell volume, V-b was obtained as 0.4 . V-o (where V-o, is the isoton ic cell volume). The two-step dynamic micrographs and the hear release meas urements from the DSC were used to obtain water transport data during freez ing. The DSC technique confirmed that R. sylvatica cells in control liver t issue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min. (C) 1999 Academic Press.