X-Message-Number: 3817 From: Date: Wed, 8 Feb 1995 11:01:22 -0800 Subject: TRANS TIMES newsletter SCI.CRYONICS The following is extracted from THE TRANS TIMES, Volume 4 Number 1, February 1995. Published by Trans Time, Inc. RESEARCH UPDATE by Hal Sternberg, Ph.D. We are conducting research to improve techniques for low temperature storage of organs, tissues and whole organisms. In the latest series of experiments, we: (1) Compared the outcomes using different approaches to aggressively thaw cryoprotected hamsters from liquid nitrogen temperatures without excessive damage (microvascular, cellular, and gross morphologic); (2) Examined the relevance of freezing rate to -196 deg. C of cryoprotected hamsters, upon microvascular reperfusion, leakage, and myocyte contractile activity. Although we performed considerable research to develop a sophisticated radiowave rewarming device, we have not succeeded in constructing a prototype that works effectively. That project is currently on hold. Thus we have been thawing animals using a microwave oven, while varying both the settings and pulsation schedules. We have made progress in optimizing these conditions to provide fast and even thawing. Interestingly, upon using an inappropriate setting and pulsation schedule, it is possible to destroy the normal texture and connectivity of the brain. This extent of destruction prevents any reperfusion to the brain as well. The poor outcome can be avoided upon using an appropriate setting which is substantially less aggressive. Freezing rate We have also been testing the effect of the freezing rate of cryoprotected hamsters. Some hamsters were frozen directly to liquid nitrogen temperature, while others were frozen in a stepwise fashion to liquid nitrogen. Based on some preliminary observations, it appears that maintenance of frozen animals at temperatures warmer than -80 deg. C for extended periods is not advisable. We rarely see cracking in our frozen/thawed hamsters, except occasionally in the brain and liver. Freezing abruptly to liquid nitrogen temperatures increases the probability of cracking in the brain, from about 1 or 2% to about 5% of cases. We have never seen more than a single crack in an animal however. The cracks typically will be in a particular location on one hemisphere of the cortex and protrude through the cortex but not lower. Histology We are fortunate to have the cooperation of a medical pathologist-histologist to provide us additional information of the integrity of tissues following cryoprotection, freezing, thawing and reperfusion. We have examined the tissues of some frozen/thawed hamsters histologically. It is evident that while tissue appears generally intact, there is some separation that is apparent between structures, particularly within cardiac tissue. Also, there is clear damage to the cells lining capillaries, as nuclei shape is rounded. Some myocytes also undergo morphologic change, as nuclei become less oval and more condensed and darkened. We hope to test whether some of these changes are reversible or repairable. New equipment Lastly, to further investigate the effects of freezing rate and storage temperature on tissue and organ viability, we have purchased a -152 deg. C freezer which can steadily hold temperatures ranging from -100 deg. C to -152 deg. C. The interior volume is approximately a 20" cube, with rated capacity 128 liters. We are very optimistic that this instrument will further our knowledge in cryobiology. Additionally, we are equipped with apparatus that can precisely lower temperature in a programmed fashion (using a cooling bath and computer) from 5oC to -30 deg. C, a freezer that operates at -40 deg. C, a freezer that operates at -80 deg. C, and liquid nitrogen storage. SPACE MEDICINE Here is the abstract of a talk submitted for presentation to the 16th Annual Gravitational Physiology Meeting, March 19-24, 1995, Reno, Nevada, U.S.A. LOW TEMPERATURE PRESERVATION AND SPACE MEDICINE __SEGALL, P.E., STERNBERG, H., AND WAITZ, H.D.__ BioTime, Inc., Berkeley, California 94710, U.S.A. The treatment of sick or injured astronauts presents a problem of significant magnitude. Prolonged maintenance of compromised individuals at reduced temperature may permit extraterrestrial stabilization of patients until return travel is possible. Also, sick or injured patients may not survive gravitational forces during reentry. Rodents, dogs and primates can revive and survive long-term after complete blood substitution and maintenance at near-freezing temperatures. Blood-substituted baboons have been kept below 5 deg. C for more than 1 hr., dogs more than 3 hrs., and hamsters for 5 hrs., with full revival to consciousness. Studies are ongoing to extend these times further. Blood-substituted cryoprotected hamsters have been frozen overnight at temperatures ranging from -1.5 to -4.0 deg. C and then thawed with microwaves and infused with donor blood. These animals have displayed beating hearts and an active EKG, but have not yet been successfully revived. Full-thickness skin samples several centimeters in diameter were removed from the belly of blood-substituted cryoprotected rats after shaving and then frozen in liquid nitrogen for days to weeks. These grafts were then rotated 180 degrees and transplanted to the backs of recipient rats. The grafts survived, and hair grew back in the opposite direction, proving that the grafts were viable. When cryoprotected blood-substituted hamsters were frozen to liquid nitrogen temperatures and then thawed, warmed and perfused with donor blood, their hearts did not beat but did show microscopic quivering indicative of survival of some portions of the myocardium. The ability of whole animals to survive profound blood-substituted hypothermia, and of heartbeat and skin and heart tissue to survive various degrees of freezing, suggests that the low temperature maintenance of sick or injured astronauts to allow their return to terrestrial hospitals is a reasonable goal. TAKE IT WITH YOU! In the August 1994 TRANS TIMES, we reported on the potential benefits of establishing a Charitable Remainder Unitrust (CRU) as part of arranging for your suspension. In summary, the advantages of making a living donation to a CRU are as follows: 1. You avoid capital gains tax on appreciated assets. 2. You obtain an income tax deduction for the charitable contribution. 3. You avoid any further taxes on the capital gains and income from the asset during your life, except on the 5% per year that you may have directed to yourself. 4. You will later get a charitable deduction from your estate tax for the value of the remainder interest that passes to your charitable trust for cryobiological research that funds your suspension. 5. You protect these assets from lawsuits or other creditor s judgments against you. We have recently completed a model Charitable Remainder Unitrust, for use by our clients. Two independent attorneys have reviewed this model trust, so we have confidence that it meets IRS requirements. You can use our model trust by simply filling in the blanks. However, this area of the law is complicated enough that we recommend you set up such a trust in consultation with an attorney experienced in establishing charitable trusts. We now include this model Charitable Remainder Trust as part of the package of legal forms we send to our Emergency Responsibility clients. This service is available for $96.00 per year from us. Call or write us for an application. 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