In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a 'two-step' low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques 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 osmotically inactive cell volume (V(b)) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5°C/min. Stereological analysis of the slam frozen (> 1000°C/min) micrographs led to the datermination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model I) yielded distance between adjacent sinusoid centers, ΔX = 64 μm; original sinusoid (vascular) radius, r(vo) = 18.4 μm; and length of the Krogh cylinder, L = 0.71 μm (based on an isolated frog hepatocyte cell diameter of 16 μm). A significant observation was that ~24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r2 = 28.7 μm, was defined (in addition to the original sinusoid radius, r(vo) = 18.4 μm, 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 inactive cell volume, V(b) was obtained as 0.4 · V(o) (where V(o) is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min.
Bibliographical noteFunding Information:
This work was supported by a grant from the National Science Foundation (NSF-BES 9703326) and a grant from the Whitaker Foundation to J.C.B.
- Differential scanning calorimetry
- Directional solidification stage
- Krogh cylinder
- Liver tissue
- Low temperature microscopy
- R. sylvatica