INTRODUCTION

The extreme winter climate in some areas of the world requires animals to develop freeze tolerance in order to survive. This mechanism allows the wood frog, R. sylvatica, to cool to temperatures of -8°C with up to 65% of the frog's water in the ice phase, without incurring freezing injury over many weeks^1-2. Current technology for the cryopreservation of mammalian tissues does not allow for successful storage of organs under the same conditions. If the mechanism whereby R. sylvatica survives freezing could be more fully understood and exploited, it could have tremendous impact in the storage and banking of frozen/viable mammalian tissues. Two new experimental methods, one using freeze substitution low temperature microscopy³ and another using a differential scanning calorimeter (DSC)⁴ have been developed which allow more accurate quantification of the processes that occur during freezing in tissue systems. In this work, these methods will be used to quantify water transport in the liver of the freeze tolerant wood frog R. sylvatica, in the hopes of learning more about how these frogs freeze and thus perhaps suggest how freeze tolerance is achieved.

THEORETICAL BACKGROUND

The driving force for water transport across the cell membrane is the difference in chemical potential of water between the unfrozen cell interior and the partially frozen extracellular solution⁵. The water flux between the cell and vascular/extracellular spaces has been modeled using a Krogh cylinder approach, where the permeability of the membrane separating the cellular and vascular/extracellular spaces, Lp is given by⁶:

This permeability depends on the water transport parameters: L_pg the permeability of the cell membrane to water at the reference temperature T_R (273.15 K), and E_Lp which is the activation energy for the process, T is the absolute temperature and R is the universal gas constant. The scarcity of space precludes further description of the model, which is described in detail elsewhere^3,6.

MATERIALS AND METHODS

Freshly isolated liver samples (1mm³) of wood frogs (R. sylvatica) were frozen using a directional solidification stage⁷ by one of three methods: (a) slam freezing (> 1000°C/min), (b) two-step equilibrium (2°C/min) freezing, or (c) two-step dynamic freezing at 5°C/min³. The frozen tissue samples were freeze substituted, embedded in resin, sectioned and imaged under a light microscope fitted with a digitizing system^3,8. Image analysis was then performed with NIH Image software™ (NIH, Bethesda, MD), to obtain the required cellular and vascular/extracellular volumes during freezing³.

The DSC protocol developed to extrapolate water transport from measured latent heat releases during freezing was used on frog liver tissue, as previously described for a cell suspension system⁴. DSC experiments were conducted at cooling rates of 2 and 5°C/min on frog liver tissue (1.0 to 1.5 mg of tissue slices in 8 to 10 mg of PBS with 0.3 to 0.5 mg of a natural ice nucleator Pseudomonas syringae) and the measured heat releases were then translated to water transport data, as described elsewhere⁴.

RESULTS AND DISCUSSION

The slam tissue images (Fig. 1A) were analyzed using NIH Image software™ (NIH, Bethesda, MD) to obtain the following Krogh model dimensions: average distance between the sinusoids, DX=64µm; initial sinusoid radius, r_vo=18.4µm; and the length of the Krogh cylinder, L=0.71µm (assuming that an isolated frog hepatocyte has a diameter of 16µm). Comparable dimensions in rat liver tissue are DX=22µm, r_vo=3.8µm, and L=11.4µm³. In addition, a higher magnification analysis of slam tissue showed that up to 24% of the frog hepatocyte cells were bounded by other hepatocyte cells and the remaining 76% are next to vasculature, as compared to mammalian (rat) liver tissue where 100% of the hepatocyte cells are next to vascular spaces.

A Boyle-van't Hoff (BVH) plot was constructed by examining freeze dehydrated tissue slices which were allowed to come to "equilibrium" with the extracellular ice at temperatures of -4, -6, -8, -10 and -20°C. By extrapolating the BVH plot to infinite osmolality (Osm = DT/1.858, where DT=273.15 - T, K), the osmotically inactive cell volume, V_b = 0.4V_o, was obtained (data not shown).

The frog liver response to dynamic freezing at 5°C/min is shown in Fig, 1. As freezing begins the cells dehydrate and water moves out of the cells and into the vasculature (Figs. 1B, C, and D). Cellular dehydration (and therefore water transport from the cells) appears to cease at -10°C. In Figs. 1B, C, and D, white spaces that were equal to or less than the size of the hepatocyte cell diameter (16 µm) were assumed to be intracellular ice. These spaces were included as cellular spaces in the analysis of end volume represented in Fig. 2. This observation was supported by the 5°C/min DSC data, which showed a secondary heat release at -14 to -16°C that translates to ~20% of the total intracellular water volume. The 5°C/min dynamic water transport data from both the techniques is shown in Fig. 2. Assuming a two compartment Krogh model, a nonlinear curve fitting technique⁹ was used to predict the best fit biophysical parameters of water transport: L_pg = 1.76 µm/min-atm, and E_Lp = 75.5 Kcal/mol. These are comparable to other mammalian (rat) liver tissue parameters: L_pg = 1.86 µm/min-atm, and E_Lp = 69.3 Kcal/mol, estimated using a similar technique and model³.

CONCLUSIONS

This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog R. sylvatica using low temperature microscopy and differential scanning calorimetry. Stereological analysis of the tissue micrographs showed: 74% of the control tissue is cellular space (26% is vascular space) and V_b = 0.4V_o. The biophysical parameters of water transport obtained in this study for frog liver tissue; L_pg=1.76 µm/min-atm, and E_Lp=75.5 Kcal/mol, are comparable to those obtained in rat liver tissue³. Both the techniques confirmed that frog liver tissues do not dehydrate completely at 5°C/min but do so when cooled at 2°C/min, as opposed to the rat liver tissue which dehydrates completely at both these cooling rates³. The reason for water retention at 5°C/min in the frog liver tissue appears to be, primarily due to the differences in the morphological architecture of the frog liver vs. rat liver (i.e. differences in the Krogh model dimensions, DX, r_vo, L; and the fact that 24% of frog hepatocyte cells are not in contact with the vasculature) and not due to altered membrane permeabilities (L_pg and E_Lp).

REFERENCES

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