Frozen Alive

11.03.2011 | Health, Research
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A frog with a remarkable ability to survive freezing temperatures may hold the key to giving human organs scheduled for transplant a longer shelf life.

A team of University of Dayton and Wright State University researchers has developed an innovative method for understanding how Cope's gray treefrogs freeze themselves to survive the winter.

Keeping people alive in a frozen state — depicted in popular movies like Austin Powers: International Man of Mystery and Avatar — is still the realm of science fiction, but the team's research could one day help doctors save lives.

"Right now, we can preserve an organ for transplant for just a few hours, putting doctors and transplant patients on short notice, creating a small window of time for transportation and surgery," said University of Dayton biology professor Carissa Krane. "But if we can replicate the process these frogs use, we may be able to viably freeze organs and bank them for extended periods of time until they are needed."

The National Science Foundation in September awarded a joint, three-year grant of $562,000 to the University of Dayton and Wright State University to investigate the role of a specific group of proteins in the freezing process of Cope's gray treefrogs. The researchers have already published results of their ongoing research in several scientific journals.

The key to cryopreservation is to prevent damage to cells from ice forming inside a body, Krane said. As the temperature around a cold-blooded animal drops, the water inside the body begins to freeze, forming jagged ice crystals that could damage the surrounding cells' critical components. But that doesn't happen to these frogs. They have a method of moving water inside their bodies that allows them to survive — frozen — in temperatures below 32 degrees Fahrenheit.

Glycerol is the critical ingredient in this process — the cryoprotectant. Glycerol creates an "osmotic balance" that draws water to the outside of the cells, where ice will cause less damage, and helping to stabilize the structures of critical molecules inside cells. As a result, the frogs can survive more than half of their body water turning to solid ice.

Scientists have known about the cryoprotectant properties of glycerol for years, but no one has been able to explain how the glycerol gets in to the cells, said Krane, who has conducted research on cryoprotection since 2001. She and her collaborators believe "aquaporins" are the answer and have developed a method for testing their theory on the cellular level.

Aquaporins, a class of proteins first discovered in the early 1990s, exist in all animals, including humans. These "water channels" earned their name because they allow water to pass in and out of a cell through the cell membrane, though further research has shown that a subset also allow the transport of ions, gases and small organic materials, including glycerol.

"We know there is a correlation between an increase in aquaporins and cold-acclimation, but we need to show causation," Krane said. "If we can remove and manipulate the aquaporins, we should be able to determine their function. We want to know, if you don't have aquaporins, can you still cryoprotect?"

In 2007, a graduate student in Krane's lab identified an aquaporin in the gray treefrog's cells known as an aquaglyceroporin, which allows the transfer of both water and glycerol. Earlier this year, doctoral student Venkateshwar Mutyam developed a method for creating a lab culture that can isolate and "knock down" the ability of this protein to function. BioTechniques published this method in its May issue.

"This discovery is an important development, because it allows us to experiment with the frog's cells in different conditions with and without the aquaglyceroporin to determine its function," said Mutyam, who received the American Physiological Society's annual Scholander award in April for his findings.

Krane and Mutyam have already used the method to test the effect of summer-like temperatures and fall-like temperatures on the regulation of the important protein at the cellular level.

Their results, published in the August issue of the Journal of Experimental Zoology, showed cells in cold weather had higher expressions of the aquaglyceroporin than cells in warm weather. The tests also confirmed the new method is viable for future experiments.

Krane also intends to examine the cellular process that leads to the insertion of aquaporins into a cell membrane and their stability.

Researchers at Wright State University, led by biology professor and department chair David Goldstein, are conducting similar tests on the frogs themselves in cold and warm weather as well as varying periods of light and dark.

The researchers at both universities are collaborating with the Dayton Regional Science, Technology, Engineering and Math (STEM) school to engage high school students in the project and contribute to the development of the strategic STEM pipeline in the region.

For more information, contact Cameron Fullam, assistant director of media relations, at 937-229-3256 or fullam@udayton.edu.
For more information, contact Cameron Fullam, assistant director of media relations, at 937-229-3256 or fullam@udayton.edu.