GOAL: HUMAN CRYO-ANABIOSIS

by Armand Karow, Jr.

Part I – SHORT TERM PRESERVATION BY HYPOTHERMIA

Deep cooling (without actual freezing) an organism has been discussed in CRYONICS REPORTS as a temporary alternative to freeze preservation to avoid the problems of ice crystal formation until a truly effective freezing and thawing method is developed. The determination of how long tissues can be kept at near freezing temperatures without irreversible chemical changes taking place is of clinical and surgical interest.

Cooling a body from 37ºC (98.6ºF) to 29ºC (84.2ºF) decreases the brain’s need for oxygen by 50%. At 37ºC it can survive without oxygen for 4 minutes before damage begins. The time limit at 29ºC is 8 minutes. By lowering the temperature to 4ºC (39ºF) the cellular requirement for oxygen is reduced by approximately 95%.

These time-temperature relationships are fairly constant for a particular kind of tissue, but vary widely from one tissue to another. Using present methods, blood cells can be kept in a hypothermic state successfully, longer than any other type of cell studied. They can be stored at 4ºC for three weeks and still be used for transfusion. Human corneas are kept at 4ºC for only four days if they are to be suitable for transplantation. Bone marrow cells (producers of blood cells) can be kept in a special solution for two days before measureable deterioration begins.

Isolated dog kidneys can be successfully transplanted after refrigeration to 0ºC if perfused with oxygenated blood during a period of hypothermia lasting no longer than 24 hours. Kidneys and hearts which are not perfused constantly in the manner will survive only eight hours at 0ºC. During heart surgery, human beings are probably never maintained at 15ºC (59ºF) for more than two hours.

Evidently, the act of merely cooling a body to temperatures near freezing does not sufficiently reduce metabolism to prevent tissue deterioration for a long enough period. To extend the tissue storage time at these relatively high temperatures, surgeons under the leadership of Drs. W.G. Manax and R.C. Lillehei experimented with the idea of using a combination of hypothermia and high oxygen pressures at the University of Minnesota Medical School. They found that dog hearts, kidneys, and small bowel segments could be stored successfully in a pressure cylinder containing three atmospheres of oxygen at 2ºC (35.6ºF) up to 24 hours. When the oxygen pressure was increased to 15 atmospheres, the organs were successfully preserved for 72 hours. Unfortunately, lung and brain tissue are very sensitive to excess oxygen. High oxygen pressures (greater than three atmospheres) result in irreversible damage to these vital organs.

No one really knows how the combination of hyperbaric oxygen (as it is called) and hypothermia act to prolong the storage period. The Minnesota group has suggested that the toxicity of the oxygen inhibits metabolism by literally poisoning the processes which maintain life to allow prolonged cold storage. If this hypothesis is correct, metabolic inhibitors (chemicals which arrest metabolism) should promote the prolongation of storage time.

Metabolic inhibitors having irreversible effects are called poisons. Many chemicals fall into this category. For successful preservation, reversible inhibition must be achieved.

Discovery of reversible metabolic inhibitors is only beginning. A combination of sodium fluoride and adrenochrome acts as a reversible cardiac metabolic inhibitor, as do high concentrations of magnesium ions. The administration of either the fluoride-adrenochrome combination or excess magnesium will cause a cessation of cardiac contractions for prolonged periods, even at normal body temperature. Rat hearts maintained at 37ºC in an oxygen-free solution for 30 minutes soon stop beating and cannot be made to resume contractions even when subsequently perfused with a well oxygenated solution. However, in one study in which all contractions ceased when perfusion was begun with a fluoride-adrenochrome solution deprived of oxygen, more than 30% of the hearts resumed coordinated beating four hours later, when perfused with an oxygenated solution containing magnesium and glutathione. Good contractions were observed in 30% of hearts arrested with high concentrations of magnesium and an oxygen-free solution, after the magnesium concentration was reduced to normal six hours later.

There is much to be learned about metabolic inhibitors, and an important area for study is the response of organs with different metabolic processes to a particular inhibitor. Further progress in combining the techniques of metabolic inhibition and hypothermia might be a fruitful approach for scientists concerned with tissue and organ preservation at low temperatures. Someday such techniques may prove to be clinically practical for use on human beings.

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Part II – TISSUE PERFUSION

In living animals blood continuously supplies the tissues with nutrients and oxygen and carries away waste products. If the organism is to function effectively, the blood supply to the tissues must be constantly maintained.

Blood is composed of cells and salt water. Most of the cells in blood are red cells (erythrocytes), which carry oxygen to the tissues. Nutrients and chemical wastes must be dissolved in the salt water.

Blood salt water is called plasma. It contains a relatively large amount of sodium chloride (table salt), as well as lesser amounts of potassium chloride, calcium chloride, and bicarbonate.

The concentration of salts in plasma is similar to the concentration in the fluid surrounding tissues. If the salt concentration of tissue fluid is altered beyond relatively narrow limits, the cells cease to function normally and soon die. It is very important for the salt concentration in plasma to remain constant, because the salt water of blood and tissue mix freely.

Blood freshly pumped from the heart is under the influence of relatively high pressure. As a result some of the salt water portion is forced out of the microscopic vessels (capillaries) and into the space between the cells. Thus, blood pressure is a major factor in the transfer of nutrients and oxygen from the vessels and into the tissue fluid.

Another important component of plasma is protein. Proteins are too large to go through the pores of the capillaries and so remain in these vessels. As the blood continues to travel the pressure falls. The presence of protein in the plasma causes some of the tissue fluid to return to the vessels by a process called osmosis. Without plasma proteins the tissues would soon swell abnormally with excess salt water.

Scientists can manufacture a blood plasma substitute called a perfusate. The proper concentration of salts can be added to pure water. As it is impractical to add proteins to the perfusate, a sugar called glucose is frequently used an an osmotic substitute. On other occasions the use of very large molecules of dextran is more effective than glucose.

Oxygen also has to be added. Oxygen dissolves in water. If it did not, fish and other aquatic animals would be unable to survive. However, water at human body temperature (37ºC, 98.6ºF), will hold only about one-twentieth of the oxygen carried by red cells. As there are no red cells in a synthetic perfusate, other methods must be used to increase the oxygen concentration.

One method is to decrease the temperature. This not only increases the absolute amount of oxygen in solution, but also decreases the cellular need for oxygen. It has been discovered that heart tissue with an artificial perfusate functions best if the solution is kept at 30ºC (86ºF). In this way enough oxygen to sustain tissue life can be placed into solution.

Recently, vessel pulsation has been shown to be an important factor in tissue perfusion. In the body, vessels pulsate in response to each heart beat; but in an artificial, laboratory situation, a special pump must be constructed to produce a pulsatile perfusate flow.

A pulsatile flow allows one to accomplish tissue perfusion with lower pressure, improve kidney blood flow, reduce damaging tissue swelling, and prolong and extend the perfusion period without noticeable detriment to the tissues.

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Part III – HEAT TRANSFER RATES

There are at least five interrelated factors involved in attempting to maintain viability when freezing tissues. They are:

1) Biologic variation

2) Variation of biochemical reaction rates

3) Thermal shock

4) Solute concentration

5) Ice crystal formation

These factors can be controlled to some degree by the modification of heat-transfer rates (cooling & warming), as well as the addition of a cryoprotective agent.

Biologic variation means that different types of cells vary in their reaction to the freezing process. This variation refers both to tissues within a particular organism and in different species. For example, glycerol penetrates the red cell membrane in a human being 50 times faster than in a dog, and 170 times faster than in a sheep. Glycerol is a good cryoprotective agent for human blood, but is almost worthless for beef blood.

If frozen rapidly, a relatively large proportion of red cells and some tumor cells can survive the freezing process without a cryoprotective agent.

When freezing large tissue masses such as organs or animals biologic variation must be taken into account. It is a major problem.

Biochemical reactions are the essence of metabolic processes. Usually, they slow down as temperature is lowered. However, freezing alone does not produce suspended animation (the cessation of biochemical reactions). Some enzymes, which are involved in the regulation of metabolism, operate more efficiently at temperatures below 0ºC than at body temperature.

Rates of enzymatic change vary. In fact, it has recently been shown that cooling some biochemical mixtures to temperatures slightly below freezing greatly increases the reaction rate. Apparently, the myriad biochemical reactions which maintain normal body functions are synchronous only at body temperature. As the temperature is lowered, some reactions slow while others speed up. This variation should even be taken into account when administering relatively mild hypothermia during a surgical operation.

At the temperature of liquid nitrogen all biochemical reactions cease. Therefore, one method of minimizing this problem is to freeze the tissue to liquid nitrogen temperature.

Thermal shock refers to the injury incurred due to the fall in temperature, when cells are cooled rapidly. It is also thought to be related to a derangement of biochemical reactions.

It is often difficult to determine whether a specific type of cellular injury is the result of thermal shock, yet recent experiments with sperm seem to substantiate the phenomenon. Sperm are especially sensitive to the sudden lowering of temperature. Perhaps other tissues also experience this kind of injury.

When tissues begin to freeze, water is extracted from solution to form ice crystals. As the temperature is lowered and water continues to change from liquid to solid, the other substances in solution (solutes) increase in concentration. This tends to speed up chemical processes and denature (destroy) proteins. Enzymes are proteins, and, of course, are subject to the denaturation process.

Finally, the formation of ice crystals inside the cell is deleterious. If cells are frozen rapidly, crystallization tends to occur within the cell. This causes disruption of the minute cellular structures that are vital to the life processes. Ice crystals which form outside the cell are apparently much less damaging. Cooling cells slowly promotes the formation of extracellular ice.

In summary, a table listing the factors involved in freezing tissue and the proposed remedies is presented.

FACTOR                                                              REMEDY

1. Biochemical rate changes                             Very fast freezing
2. Thermal shock                                              Slow cooling
3. Solute concentrations                                   Fast freezing
4. Ice crystal formation                                     Very fast freezing
5. Biologic variation                                          Optimal freezing rate as determined for a given tissue

The various factors are interrelated in a most complex fashion. Increased sophistication in the control of heat-transfer rates is needed in order to fully perfect the freezing process. At present, the extent of the damage caused by a particular factor, and the cells’ ability to withstand a particular kind of damage must be taken into consideration before deciding upon a freezing rate.

Attention has been focused upon the cooling process, because less is known about what happens to tissues during rewarming. The problems associated with warming and thawing are at least as complex. It is generally believed they can be minimized by using a fast thawing procedure.

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Part IV – NEED FOR CRYOPROTECTIVE AGENTS

Enzymes are large, complex proteins known as macromolecules that regulate and control biochemical reactions. Life processes depend upon them.

They are composed of amino acid building blocks. To function properly, it is necessary for them to be folded into special shapes. Sometimes, parts of a chain are hooked together. One type of hook that locks chains together is known as a disulfide bridge. A thin covering of water molecules seems to be very important in maintaining the proper shape of proteins.

DNA, the chemical responsible for heredity, along with a similar chemical, RNA, is capable of making enzymes. DNA and RNA, although not proteins, are also considered macromolecules because of their size and complexity. Water also helps these essential substances to maintain their proper shape.

When a protein macromolecule loses its shape, it is said to have been denatured. As an analogy, an unravelled sweater is no longer capable of functioning as such, yet its components — yarn, dye, and buttons — remain unchanged. One might say that the sweater had been denatured.

Many macromolecules are denatured during the freezing process, as a result of exposure to high salt concentrations after crystallization. Changes in the acid content of cells can also cause denaturation. Perhaps the most significant cause is loss of the thin covering of water molecules.

Chemicals such as glycerol and DMSO are called cryoprotective agents. They help protect against protein denaturation. Several theories concerning the manner in which these substances operate will be discussed in a subsequent column.

There are two different types of cryoprotective agent; those that penetrate cells (intracellular), and those that cannot (extracellular). Glycerol, DMSO, and propylene glycol are intracellular cryoprotective agents. Sucrose, dextran, and PVP are extraceullar agents.

No single cryoprotective agent is equally effective for all tissues. Prof. Suda’s report indicates that glycerol is a good agent for the cat brain. Other cryobiologists have discovered that glycerol is lethal to heart tissue, even in concentrations as low as 15 per cent.

One approach is to use a combination of cryoprotective agents. Theoretically such a mixture might contain a particular agent for every major kind of tissue in the body. A mixture might provide better cryoprotection for a given tissue than any single agent. This has been found to be the case for some of the cells in blood. Sucrose and DMSO gives excellent protection for platelets, the white blood cells responsible for clotting. The mixtures that have been tried for kidney preservation, however, have failed to produce better results than single agents. Certain mixtures used in heart perservation, such as DMSO and dextran, have been found to be far less beneficial than a single agent.

The search for new cryoprotective agents and mixtures needs to be intensified. It is a vital factor in the freezing process.

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Part V – NEED FOR RESEARCH

Dr. James H. Bedford has been frozen with the use of cryoprotective agents by two medical doctors assisted by a team. Dying of cancer, he desired and provided for his cryonic suspension. Showing even greater foresight, he went one step further, and endowed a foundation to support research in cryobiology. Whether it will be possible to restore Dr. Bedford to life cannot be predicted. However, by his act, he has definitely improved the chances of those who will be frozen in the future.

Only through scientific research can the enormous problems involved in the freeze-preservation of human beings be overcome. The problems include:

1) Vital organs must be protected against irreversible damage during the pre-freezing and post-thawing periods. This can be achieved by the use of proper techniques of perfusion, with the possible aid of metabolic inhibitors.

2) Suitable cryoprotective agents must be carefully chosen. A balance between cryoprotective ability and inherent toxicity must be achieved. Agents will have to be administered with full knowledge that irreparable damage may result in certain parts of the body. In the future, perhaps an organ judged to be replaceable will have to be sacrificed so that the brain may be better protected.

3) Cooling and freezing rates (the rate of ice crystal formation) must be controlled carefully. The effects of various cooling rates must be explored and understood thoroughly.

4) Warming rates are perhaps as important as cooling rates. The achievement of rapid thawing of large tissue masses is technically much more difficult, than controlled cooling and freezing. Thawing presents certain engineering problems which have not yet been solved.

With imperfect techniques, freezing before death is socially unacceptable. Dr. Bedford, however, provided funds to subsidize research to discover how to freeze living human beings with reasonable assurance of future reanimation. Indeed, efforts in viable freeze preservation are focused entirely upon living cells, tissues, organs, and animals. Dr. Bedford’s wisdom will be recognized by all when a living human being is frozen, thawed, and successfully resuscitated.

Part I: Cryonics Reports, Vol. 2, No. 2, February 1967

Part II: Cryonics Reports, Vol. 2, No.3, March 1967

Part III: Cryonics Reports, Vol. 2, No. 4, April 1967

Part IV: Cryonics Reports, Vol. 2, No. 5, May 1967

Part V: Cryonics Reports, Vol. 2, No. 6, June 1967