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"Cold-blooded Solutions
to Warm-blooded Problems"
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A series Magnetic Resonance Images of a wood frog in the process of freezing:
The dark region is where ice crystals have formed.
Freezing progresses toward the cluster of vital organs.

The V-shaped liver is the last to freeze. The liver produces the glucose which lowers the frog's freezing point and protects cells.

MRI's courtesy Dr. Boris Rubinsky.

Dr. Rubinsky explains how the pattern of freezing in tissue works and how it impacts his method.

When liver tissue was frozen to -7°C, the individual cells were partially dehydrated, and shrunken to as little as 50 percent of their original size. Blood vessels were expanded beyond their normal size to accommodate the large amounts of ice. Despite these changes, the tissue remained intact. Liver tissue frozen at -20°C, however, did not fare so well. The cells were completely dehydrated, and their membranes shriveled to the point of collapse. Blood vessels were ruptured by expanding ice crystals.

These observations have led Rubinsky and Storey to understand cell dehydration in terms of the osmotic balance between a cell and its surroundings. As ice forms outside a cell, water is drawn from the interior of the cell into the cell's surroundings in order to compensate.

"In order to freeze tissues and still preserve them," concludes Rubinsky, "you cannot lose more than a certain amount of water. That is what we have learned from the frogs."

The critical threshold lies around 60 percent. If the cells in the wood frog's body lose up to 60 percent of their water, they can still rehydrate and regain their original shape when thawing occurs. However, if the cells dehydrate beyond this point, the process becomes irreversible--the cell membranes become so compressed and folded upon themselves that they cannot regain their original shape.

By binding strongly to water molecules within the cells, cryoprotectants like glucose inhibit dehydration, but even very high levels of glucose cannot lower the temperature at which catastrophic dehydration occurs by more than a few degrees. Seeing that thousands of years of evolution had not allowed the wood frog to survive temperatures colder than -8°C or so, Rubinsky abandoned science's long-cherished goal of preserving organs in liquid nitrogen at -196°C.

Once Rubinsky and Storey had identified dehydration as the factor which determined whether or not individual cells survived, they sought to understand how the frog coped with freezing on a larger scale. This time, as frogs were frozen and thawed, they watched what happened inside of them using MRI -- the same technology that allows physicians to see cross-sectional views of their patients.

Rubinsky and Storey found that as the wood frog's internal organs froze, they underwent increasing dehydration. As in the earlier experiments, water accumulated as ice in blood vessels; but a much larger volume of water was transported completely out of the organs and into the frog's major inter-organ spaces -- the abdominal and thoracic cavities -- where it accumulated as large pieces of ice which eventually surrounded the central organs. Some organs, such as the liver, lost 50 percent of their volume as water was transported into these spaces.

Rubinsky speculates that the accumulation of ice in the thoracic and abdominal cavities may allow the ice more room to expand without rupturing such delicate structures as blood vessels.


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