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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