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Of poplar twigs and organ transplants.

Why is a scientist with the American Red Cross visiting an Agricultural Research Service laboratory to study frozen twigs from poplar tree?

"Very few higher plants survive cold temperature as well as popular trees do," says Allen G. Hirsh of the American Red Cross Transplantation Laboratory in Rockville, Maryland.

"We think we can learn a lot from the cells of poplar trees," he says, "that will help us freeze and preserve human kidneys and possibly other donated organs for future transplant operations."

Successful techniques for preserving major human organs in a frozen state have yet to be developed. according to Hirsh because ice crystals invariably form within the tissue and destroy it. Antifreeze solutions are being tested, but so far those strong enough to block the ice formation are toxic to tissue.

"It tragic," he says, "but many organs donated for transplant are never used, simply because they can't be stored long enough for matching to a suitable recipient."

To investigate the superior cold tolerance capabilities of poplar tree cells, Hirsh - one of two plant physiologist in the Transplantation Laboratory - is working informally with scientist at the ARS Electron Microscopy Laboratory (EML) in Beltsville, Maryland.

William P. Wergin, head of the EML, says that electron microscopy imaging techniques developed by ARS are giving Hirsh and other scientists a better look at the structural details of plant cells and other living tissue. [See Agricultural Research, November 1990, pp. 18-21.]

"Preparing frozen twigs for imaging by an electron microscope is no simple matter," Wergin says, "and one of our scientific staff, Eric Erbe, has done some remarkable work in this area."

Hirsh agrees but takes it further "I doubt that any other scientist in the United States has the skill or experience to get the kind of images of frozen woody tissue that we need," he says. "The techniques mastered by Eric Erbe have made it possible for us to make some highly important discoveries."

Hirsh found, for example, that ice crystals can form inside the cell of actively growing woody tissue - even when it's been cooled and frozen very slowly.

The prevailing view holds that under such conditions, ice in woody tissue would form only in cell walls or intercellular air spaces between the walls. However, images from the EML indicate the formation of tiny ice crystals in cells's cytoplasm, beneath the plasma membrane that lies inside the cell wall, even at reduction rates of less than 2 [degrees] F per hour.

"I think our evidence is pretty conclusive," says Hirsh as he displays an electron micrograph of the plasma membrane of a slowly frozen poplar cell. The holes and ruptures caused by ice formed during freezing of the cytoplasm are clearly visible.

"If this happens to cells from poplar trees," he says. "imagine what it could mean for animal and human tissue in which the cells have no walls and are simply stuck to one another. It certainly strengthens the case that ice could form inside kidney cells and destroy them - regardless of how slowly the tissue is cooled."

Some scientists maintain that small amounts of ice in human organs might be tolerable during long-term storage, notes Hirsh, and that research in cryogenic preservation need not strive to entirely eliminate ice formation.

"Quite frankly, I think they're badly mistaken," he says. "Judging from the ice damage we've seen in poplar cells, I doubt that allowing ice to form at any level in human tissue is going to be part of the answer."

Is there an answer - yet?

"One of the critical factors in organ preservation is likely to be temperature stability," says Hirsh. "It's not going to be the whole answer, of course, but keeping things steady might add some storage time Maybe a lot of time."

That hopeful observation stems from a discovery by Hirsh that sugars in cell fluids of poplar tissue will crystallize when cold-storage temperature fluctuate over a wide range, even if the change occurs slowly.

Such crystallization contradicts the assumption that extremely hardy, dormant tissue like that of poplar twigs is fully resistant to slow, subzero temperature changes - regardless of the range of fluctuation.

Experiments by Hirsh show that taking poplar twigs from 0 [degrees] C to minus 70 [degrees] C and back again several times at only 3 [degrees] C per hour will cause sugar within the poplar cells to crystallize.

"No one had even suspected that this would occur," says Hirsh,"and without the EML images we might never have seen the evidences that loss of protective sugars leads to cellular membrane damage."

Sugars in cell fluids normally protect the cells against freeze injury, Hirsh explains. But the crystallization of sugars has the effect of removing them from the fluids. The result is a craterlike appearance formed by redistributed MAP's (membrane-associated particles, mostly lipids and proteins) on various internal membranes. This has been seen for the first time in Erbe's micrographs.

"Many scientists believe that sugars can effectively replace water on the internal membranes of cells dehydrated by freezing," Hirsh says. "In fact, recent theories on membrane protection depend on natural or artificially added sugars for that reason. Now we can see that it doesn't always happen that way if cell temperatures fluctuate."

Eric Erbe's electron micrographs also enabled Hirsh to see which membranes within the cells were, most affected by temperature fluctuations.

"This kind of evidence is crucial to understanding the entire process of tissue destruction due to freezing," he says. "It allows us to directly see the damage in a living system, and it show us where the damage is most likely to occur."

By keeping temperatures stable, Hirsh was able to store twigs from poplar trees at -4 [degrees] F for 23 months without killing the cells. the previous record at the temperature was about 8 months.

"The implications for preserving human tissue are exciting," he says. "It's clear from electron micrographs that sugars can play a vital role in protecting cell membranes if storage temperatures are stable. But a lot more research is needed, and that means more imagery from the ARS Electron Microscopy Laboratory."

Electron Microscopy Technology

Electron microscopy had advanced considerably since coming into general use nearly 50 years ago. This applies to the preparation of biological specimens for examination as well as to the technology itself. Without certain innovations in tissue preparation, researchers in the life sciences could not have taken full advantage of the increasingly powerful and versatile electron microscopes available to them.

Perhaps foremost among the many developments in tissue preparation was the invention of freeze etching by the late Russell L. Steere, a botanist and internationally recognized authority on plant virology and biophysics who retired from ARS in 1984.

Freeze etching, the technique used by Eric Erbe to prepare frozen poplar tissue for examination by an electron microscope, allows scientists to see thin cross sections of frozen cells in great detail.

After a frozen cell is cracked open and partially dehydrated through a freeze-drying process, Erbe explains, ice inside the cell is etched away to expose internal particles and structures. Ultra-thin films of metal and carbon are then deposited on the fractured cell like a microscopic cast. The actual cell material is dissolved with acid, leaving an exact replica of the fractured cell for placement in an electron microscope.

Steere pioneered freeze etching in the mid-1950's as a scientist with the University of California at Berkeley. His use of the technique to obtain detailed electron micrographs of tobacco mosaic virus (TMV) crystals stands as a landmark in electron microscopy. And it's one of the things that brought him to ARS in 1959 to help establish the agency's Plant Virology Laboratory in Beltsville.

As head of the laboratory, Steere initially concentrated on the isolation and purification of TMV and other plant viruses for research purposes. By the late 1960's, however, electron microscopy had become a critical tool in the lab's mission. So Steere returned to his labor of love: the perfection of freeze etching procedures and techniques.

The next 15 years saw a series of variations, refinements, and improvements to freeze etching that paralleled a dramatic growth in electron microscope technology. Much of this research on freeze etching was done by Steere in collaboration with Eric Erbe, who came to work in Steere's lab in 1970 while a student at the University of Maryland.

"Russell Steere was the single most influential person in my life," says Erbe, who as a high school junior firs met the scientist in a school science club that Steere had organized.

"He did a lot of things like that for young people who were interested in science," Erbe says. "He was our friend, as well as our mentor, and I know I'm not the only one he inspired to pursue a scientific career."

Throughout the 1970's, and well into the 1980's, Erbe and Steere co-authored 34 publications on electron microscopy, especially as it involved freeze etching if cellular tissue. Even after Steere's retirement, the two frequently met to review Erbe's latest work and to discuss new developments in the field.

"Yes, he always treated me like a full colleague and partner," says Erbe. "But make no mistake about it - he was the teacher. He introduced me to the whole field of electron microscopy and guided me every step of the way. What I know, I learned from Him."
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Title Annotation:cell research for organ preservation; includes related article on freeze-etching research of Russell L. Steere
Author:Miller, Stephen Carl
Publication:Agricultural Research
Date:Aug 1, 1992
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