In Cold Lazarus, the last play written by British television playwright Dennis Potter just before his death from cancer in 1994, Daniel Feeld awakens after 400 years into a world ruled by media moguls. Feeld, who was the central character of Potter's previous and linked play Karaoke, has died of cancer but his head has been frozen. He is revived by a media baron who turns Feeld's memories of twentieth-century life into a profitable nightly entertainment. The parallels with Potter's own situation are obvious and both Karaoke and Cold Lazarus are partly concerned with the relationship between playwrights and their audience. The literary device that Potter used in Cold Lazarus - the use of suspended animation, in this case by freezing, to propel a character into some sort of imagined future - is an old one in science fiction. However, it finds some sort of present reality in the cryogenics movement. For a mere US$28000-$120000, you can have your body, or for a lesser amount just your head, frozen in the hope that at some point in the future it will be possible for you to be revived and resume your existence - which only goes to show that you can take it with you! There are presently over 70 bodies and heads held in cryonic suspension by the various organisations in the USA that provide these facilities.
It might be worth considering what problems would have to be overcome for these bodies to be successfully revived. Not only would whatever killed them in the first place have to be remedied, the damage caused by the freezing process itself would need to be repaired. During freezing, the formation of millions of ice crystals in the body's tissues pushes their cells apart and ruptures blood vessels, which means that, when thawed, the tissues turn to mush. If the heart and lungs had not stopped already, freezing would cause them to cease, starving the tissues of oxygen and food. The formation of ice in the body fluids concentrates the salts in the water that remains unfrozen as water is sequestered into the growing ice crystals. This creates an osmotic gradient which results in the withdrawal of water from cells, resulting in their collapse and, if dehydration is sufficient, destruction (Figure 5.1). The cryogenic technicians attempt to mitigate these effects by keeping heart and lung functions going after death, by slow freezing and by perfusing the bodies with various chemicals. Despite these precautions, considerable damage must occur.
Although we may treat the claims of the cryogenecists with some scepticism, there are, remarkably, a number of organisms that have solved these problems and are able to survive the freezing of a substantial proportion of their water. These include a number of animals -some nematodes, molluscs, earthworms, insects, other arthropods and even frogs and turtles.
Cryobiology is the study of the response of organisms, cells and tissues to cold, especially extreme cold and freezing. There have been two main strands to this field. The first is concerned with attempts to preserve biological materials by freezing (cryopreservation). This includes the storage of human tissues and organs for medical purposes, preserving sperm and eggs of domestic animals for breeding, banking of seeds and other material for conservation, and the preservation of food. Cryopreservation often involves exposing the material to freezing conditions that the organism, or the organism from which it originated, does not naturally survive and may not even experience under natural conditions. The other strand in cryobiology has been the study of organisms that have a natural ability to survive freezing conditions. These two strands overlap. Cryopreservation techniques are often developed largely by a method of trial and error. The techniques that prove successful are often those by which some organisms naturally survive freezing. This includes the control of the rate at which freezing and thawing occurs and the use of sugars or polyols (sugar alcohols, such as glycerol) as cryoprotectants. If we can further understand how some organisms survive freezing naturally, we can use that knowledge
152 LIFE AT THE LIMITS
Extracellular water containing solutes
ICE FORMATION BEGINS
Ice formation removes water and concentrates the solutes in the remaining liquid water.
ICE FORMATION CONTINUES
The increase in osmotic concentration outside the cells draws water out of the cells iigure 5.1 The freeze concentration effect.
The increase in osmotic concentration outside the cells draws water out of the cells
iigure 5.1 The freeze concentration effect.
to improve our techniques for the cryopreservation of material that does not naturally survive. This chapter focusses on the ability of some organisms to survive exposure to subzero temperatures that they experience as a normal feature of their environment.
One of the first descriptions of a whole organism surviving freezing was made by Henry Power in 1663. He took a jar of vinegar that was infested with 'minute eels' (probably the vinegar eelworm Turbatrix aceti, a nematode) and immersed it in a freezing mixture of salt and ice. When the vinegar was thawed out 2-3 hours later, the little animals 'danced and frisked about as lively as ever'. Shortly after this (in 1683), the physicist Robert Boyle published his observations on the physical, chemical and biological effects of cold. Boyle's biological work had been inspired by the observation that bodies which had been buried in the frozen soil of Greenland were preserved for 30 years or more without any signs of putrefaction. He showed that low temperatures could help preserve eggs, meat, fruit and a variety of other biological materials, but that their texture was changed if they froze and then were thawed. He tried freezing frogs and small fish (gudgeons) in jars of water. They could revive after being encased in ice for a short period, but fish that had been frozen for three days died. The French scientist and entomologist René-Antoine Ferchault de Réaumur was the first to describe freezing experiments on insects (in 1736). He used accurate thermometers to measure the temperature of the insects, using the temperature scale which he invented (the Réaumur scale which takes the freezing point of water as zero and its boiling point as 80 °R). He found that an unnamed species of caterpillar could survive freezing to —20°C ( - 17°R) and that the blood (haemolymph) of different caterpillars froze at different temperatures. He equated this to brandies of different strengths, since a weak brandy will freeze at a higher temperature than would a stronger brandy. This was the first suggestion that the resistance of animals to cold might depend on the physical and chemical properties of their blood.
Nineteenth-century travellers to the Arctic often returned with tales of frozen animals. Sir John Ross, who, together with his nephew
James Clark Ross, located the magnetic north pole, found caterpillars that had survived temperatures between —11 °C and —47°C. Sir John Franklin, who died attempting to locate the Northwest Passage, recorded that a fish (a carp), which had been frozen for a day and a half, thawed out and leapt about enthusiastically. One explorer even described how Alaskan blackfish (Dallia pectoralis) kept frozen in blocks of ice for weeks were fed to his dogs. The fish apparently thawed out and revived in the warmth of the dogs' stomachs, causing them to vomit up the live fish. Unfortunately, this report has never been substantiated. These accounts, however, encouraged the belief that almost any animal could survive freezing, an idea supported by laboratory experiments which have reported the survival of snails after freezing to — 130°C, frogs to —28°C and goldfish to — 15°C. Other reports, however, have disagreed, finding that the same species of snail could not survive —5°C and that frogs died at — 1.8°C.
The reason for the discrepancies between these reports is that it can take a remarkable length of time for an animal to freeze. If an animal is transferred from room temperature (say 20 °C) to a freezer (at —20 °C), it will take many hours for it to cool by the 40°C required to reach the temperature of its new surroundings. If the animal is an endotherm (such as a mammal), it can generate its own heat which, together with its insulation, delays cooling even more. Hence the reports, which have appeared in newspapers, of families' pet hamsters emerging unscathed from the freezer having been accidentally trapped there overnight. An animal could be encased in ice for some time and yet its body remain above its freezing point. Even if the animal starts to freeze, ice formation will occur in its surface layers and it may take many more hours before fatal freezing of the deeper tissues occurs. It was many years (not until 1982) before it was conclusively demonstrated that a few species of terrestrial vertebrates (reptiles and amphibians) could indeed survive freezing, with ice formation in their bodies going to completion. There have been no convincing experiments that have shown freezing survival by a mammal, bird or fish. There are, however, many invertebrates that can tolerate the freezing of their bodies.
As we saw in Chapter 2, there are a number of environments in which organisms are exposed to temperatures below 0 °C and thus the risk of freezing. In polar regions, terrestrial organisms are exposed to freezing temperatures for most of the year. In more temperate regions, they have to tolerate several months of winter, when subzero temperatures may persist for long periods of time. High mountains are another place where there is permanent snow and ice, even on the equator. Exposure to subzero temperatures may occur on a daily and/or seasonal basis.
Endothermic animals (birds and mammals) can stop their bodies from freezing by generating their own heat. They retain heat because of the insulation provided by feathers or fur, and the layer of fat beneath the skin. Other heat conservation measures include huddling together and recovering heat from exhaled breath and from the blood circulating to the extremities of the body. The Emperor penguin is perhaps the most spectacular example of these methods for survival in the cold (see Chapter 2). Endotherms can remain active in the cold if they can find enough food, or they can reduce their metabolism and lie dormant until warmer conditions return. Although air temperatures may be low, the temperature beneath an insulating layer of snow, under the ground or at the bottom of a lake may remain above 0°C. Organisms may avoid the cold altogether by migrating somewhere warmer during the winter. Most organisms, however, cannot avoid the freezing temperatures and, for them, the choice is to survive ice formation within their bodies or to prevent their bodies from freezing.
To freeze or not to freeze?
Organisms run the risk of freezing at temperatures that are below the melting point of their body and cell fluids. There are two main responses: they can either survive ice forming within them (they are freezing tolerant) or they have mechanisms which ensure that their fluids remain liquid at temperatures that are below the freezing point of water and the melting point of their body fluids (they are freeze avoiding). The strategy that an organism uses depends on the structure and physiology it has developed during its evolutionary history and on the particular demands of its environment. If the organism is living in a wet or damp environment, ice is likely to make contact with its surface when its surroundings freeze. This may cause its body fluids to freeze by the ice travelling across the cell or body wall, or through body openings such as the mouth or anus - a process known as inoculative freezing. Most organisms surviving low temperatures in such environments are thus likely to be freezing tolerant, since inoculative freezing will cause their bodies to freeze. Some, however, may have a structure such as a cuticle, eggshell, cocoon or sheath which allows them to prevent inoculative freezing by acting as a barrier to the spread of ice into their bodies. This allows them to maintain their body or cell fluids as a liquid, despite their surfaces being in contact with external ice, and enables them to avoid inoculative freezing. In a situation where the organism is likely to be exposed to subzero temperatures with little or no water in contact with its surface (many terrestrial insects for example), it does not have the problem of inoculative freezing and it is perhaps easier for it to maintain its body fluids in a liquid state at low temperatures and thus survive by avoiding freezing.
The two strategies of cold survival are, however, not always mutually exclusive. There have been a few reports of insects which were apparently freezing tolerant switching to being freeze avoiding. The overwintering larvae of a beetle (Dendroides canadensis) from northern Indiana, when studied by John Duman and Kathy Horwarth from the University of Notre Dame in the winters of 1977-1978 and 1978-1979, froze at -8°C to -12°C but survived down to -28°C. When examined again in 1982, however, they froze and were killed at - 26 °C, apparently switching from a freezing-tolerant to a freeze-avoiding strategy during the intervening years. There are adaptations in common between freeze-avoiding and freezing-tolerant insects which may make it easy to switch between the two strategies. It must be said, however, there has been only one other report of an insect (another beetle, Cucujus clavipes) displaying a shift in strategy of this sort. The Antarctic nematode Panagrolaimus davidi is freezing toler ant when immersed in water, but, when free of surface water, there is, of course, no inoculative freezing and it can survive by avoiding freezing. The cold tolerance strategy displayed thus depends on the particular characteristics of the animal's microenvironment. The situation where the nematode is free of surface water but not desiccated is, however, likely to be a temporary one and, should water loss continue, the nematode will survive anhydrobiotically.
Many organisms cannot survive subzero temperatures at all and are killed by the lethal effects of low temperatures which are not the result of freezing. These organisms are referred to as being chilling intolerant and as suffering prefreeze mortality. The lethal effects of low temperatures include disruption of the structure of membrane and proteins, and the production of fatal imbalances between metabolic pathways. This sort of damage may only manifest itself after the organism has been exposed to low temperatures for some time. Care must therefore be taken to determine that an organism does not suffer prefreeze mortality before concluding that it can survive subzero temperatures by a freeze-avoiding strategy.
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