series Magnetic Resonance Images of a wood frog in the process
dark region is where ice crystals have formed.
progresses toward the cluster of vital organs.
V-shaped liver is the last to freeze. The liver produces
the glucose which lowers the frog's freezing point and protects
courtesy Dr. Boris Rubinsky.
Dr. Rubinsky explains how the pattern
of freezing in tissue works and how it impacts his method.
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.
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.
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.