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Date: Tue, 20 Feb 1996 00:54:12 -0800 (PST)
Subject: Trans Time Newsletter
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THE TRANS TIMES
Life Extension through Cryonic Suspension
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Volume 5 Number 1 February 1996
Low Temperature Preservation and Space Medicine
Paul E. Segall, Hal Sternberg and Harold D. Waitz
BioTime, Inc., Berkeley, CA 94710
Reprinted from the *Journal of Gravitational Physiology*,
2:1, 143-144 (1995).
Introduction
Cold maintenance may be an option for compromised space-borne
astronauts. Contemporary aneurysm surgery (1) can involve cooling
below 20 degrees C for nearly one hour. Dogs (2) and baboons (3)
have survived blood-substituted hypothermia for 1-3 hours.
Hamsters (4) have recovered from partial-freezing below 1
degrees C, and supercooling at -5 degrees C. Laboratory frogs
have survived partial-freezing from -9 degrees C, while in
nature, frogs may overwinter in these states (5). While some
invertebrates can tolerate freezing to cryogenic temperatures
(6), no vertebrate has survived complete freezing. The following
studies were conducted to explore low temperature preservation of
rodents, dogs and baboons.
Methods
Hypothermia
Hamsters, dogs and baboons were cooled and blood substituted for
various time periods, then warmed, transfused and revived.
Six female hamsters (45-65 g) were anesthetized i.m. with
ketamine (4-6 mg/100 g), chilled to rectal temperatures of 12-15
degrees C and their right femoral arteries cannulated. They were
ventilated with oxygen and cooled to 5 degrees C. Hextend
(trademark), a fully synthetic artificial plasma solution (BioTime,
Berkeley, CA) was infused intraarterially to replace blood, their
right jugular veins punctured, and the venous effluent removed.
Their hearts were stopped after 30-40 mEQ/ml KCl was added to the
Hextend infusion and the ice-cold hamsters were maintained in
respiratory, circulatory and cardiac arrest until Hextend
infusion, followed by donated blood, was resumed. Ventilation
with oxygen was begun until breathing resumed and the animals
revived.
Six male dogs (25-38 kg) were anesthetized i.v. with pentothal or
pentabarbital (20-35 mg/kg and p.r.n.), instrumented for EKG,
central and pulmonary arterial pressures, esophageal and rectal
temperatures, and ventilated with 100% oxygen. They were placed
on cardiopulmonary bypass and subjected to core and surface
cooling. The bypass circuit was primed with Hextend, which was
continuously oxygenated and circulated during each procedure.
Hextend was used to completely replace blood at 22-25 degrees C.
The dogs were maintained blood-substituted and below 10 degrees C
for 4-4.5 hours. Minimum esophageal temperatures ranged from 0.8-
3.9 degrees C. During warming, Hextend was gradually replaced
with autologous and donated blood beginning at 10-18 degrees C.
Warming was continued, ventilation with 100% oxygen resumed, and
heartbeat re-established by defibrillation. The animals were
weaned from bypass, their catheters and cannulas were removed,
and they were allowed to recover.
Four male baboons (24-28 kg) were anesthetized with pentothal (8-
12 mg/kg/hr), ventilated with 30-40% oxygen, instrumented for
EKG, EEG, somatosensory evoked potentials, intracranial and
central arterial pressures, brain, esophageal and rectal
temperatures, placed on cardio-pulmonary bypass using Plasmalyte
(registered, Baxter, Glendale, CA) as a priming fluid,
and subjected to surface and core cooling. When esophageal
temperatures fell below 17 degrees C, Plasmalyte - mannitol was
substituted for circulating blood, followed by ice-cold Hextend.
Hextend was then circulated, oxygenated and cooled throughout all
or most of each procedure prior to warming. The baboons were kept
blood-substituted below 15 degrees C for 1/2 to 3 hours, with
minimum temperatures ranging from 2.8 9.9 degrees C. Upon warming
to 17 degrees C, Plasmalyte-mannitol replaced Hextend, which was
then replaced with autologous and donated blood. Ventilation with
30-70% oxygen was initiated, and the animals were warmed further.
When body temperature approached normal, their hearts spontaneously
defibrillated and heartbeat resumed. The baboons were weaned from
bypass, their catheters and cannulas removed, and recovery begun.
Sub-Zero Experiments
Additional experiments in which hamsters and rats were chilled
and blood-substituted with Hextend, and then blood substituted
with Hextend to which glycerol and other cryoprotectants were
added, were conducted. Where hamsters were used, the animals were
frozen to -2 to -4 degrees C, maintained at this temperature
overnight, thawed using a commercial microwave oven, and perfused
first with Hextend, then with blood, and warmed and ventilated
with 100% oxygen. Alternatively, they were frozen in liquid
nitrogen for 24 hours.
In the experiments in which rats (Sprague Dawley) were employed,
full thickness skin grafts approximately 5 cm in diameter were
removed from the abdomen following whole body perfusion of the
blood-substituted rat with cryoprotective solution, and frozen to
liquid nitrogen temperatures. After storage for 1-4 weeks, these
samples were thawed and sutured in place onto the backs of host
rats of the same strain from which a similar-sized patch was
removed. Using another group, hind-limb joints with the
connecting pedicle containing artery and vein were removed from
each of the cryo-protected rats, and frozen in liquid nitrogen
for 2-3 weeks. These were thawed, and the vessels were
anastomosed to host rats.
Results
Hypothermia
All 6 hamsters revived to consciousness and reactivity, despite
circulatory arrest for 3-5 hours and cardiac arrest for 3.5-6
hours. All 6 dogs survived to recover consciousness, but one died
2 days later from accidental asphyxiation. The others made a full
recovery, except for one dog with impaired vision secondary to a
hemorrhage following a hypertensive episode during revival.
All of the baboons survived. The last baboon studied was blood
substituted for 3 hours with its deep esophageal temperature
below 15 degrees C. The baboon's body temperature was reduced to
below 3 degrees C, and its deep esophageal temperature was then
maintained without flow for 45 minutes prior to warming. This
animal was alive and well the following day, could assume normal
posture and was eating solid food. Motor function, coordination,
behavior and cognitive skills were assessed in each baboon using
a 160-point evaluation. The first baboon, cooled only to 9.9
degrees C, lost 30 points for low aggression. The others scored
160/160.
Sub-Zero Experiments
Hamsters partially frozen overnight from -2 to -4 degrees C
produced EKG waves and coordinated heartbeat after thawing and
warming. There were no signs of brain activity, such as
responsiveness or respiration. Heartbeat was not observed in
hamsters thawed after storage in liquid nitrogen, and there was
no sign of brain function. However, examination under high power
stereo-microscopy revealed micro-scopic quivering of the surface
of the heart, and microscopic examination following infusion of a
fine suspension of carbon particles into their carotid arteries
revealed that much of the brain micro-circulation remained
intact.
Each of four skin flaps thawed after storage in liquid nitrogen
and grafted onto the backs of host rats survived, and grew hair.
Since the flaps had been rotated 180 degrees prior to grafting,
the hair that grew back pointed to the head of the animal, rather
than its tail, indicating that both skin and hair cells remained
viable. Of the knee joints transplanted, the best results were
noted in 2 procedures employing steroids and anticoagulants.
After one week post-transplantation, both joints were
vascularized with a patent pedicle. Histologically, bone and
cartilage matrix were intact with reduced number of viable cells.
Bone marrow was virtually absent. The pedicle artery was patent
and exhibited moderate intimal hyperplasia and partial necrosis
of the media (7).
Discussion
The results indicate that higher mammals, such as dogs and
baboons, can fully recover after hours of complete blood
substitution and near-freezing body temperatures, and are
consistent with the potential use of this technology in areas of
medicine such as neuro and cardiac surgery, organ
transplantation, critical care and others. Hamsters can also
recover after similar treatment, and provide an inexpensive and
convenient model for studying revival from lower temperatures and
for longer time periods. Experiments in which rodents are reduced
to sub-zero temperatures following blood substitution demonstrate
that although full recovery is not yet possible, partial
viability of tissues and even organs can be achieved after
freezing.
1. Spetzler, R.F., M.N. Hadley, D. Rigamonti, L.P. Carter, P.A.
Raudzens, S.A. Shedd and E. Wilkinson. Aneurysms of the basilar
artery treated with circulatory arrest, hypothermia, and
barbiturate cerebral protection. *J. Neurosurg*. 68: 868-879,
1988.
2. Bailes, J.E., M.L. Leavitt, E. Teeple, J.C. Maroon, S.-R.
Shih, M. Marquardt, A.E. Rifal, and L. Manack. Ultraprofound
hypothermia with complete blood substitution in a canine model.
*J. Neurosurg*. 74: 781-788,1991.
3. Segall, P.E., H.D. Waitz, H. Sternberg, J.M. Zabramski, and
W.D. Bichard. Baboons revived after 1-3 hours of bloodless cold.
*FASEB J*. (in press).
4. Smith, A.U. Studies on golden hamsters during cooling to and
rewarming from body temperature below O degrees C. *Proc. R.
Soc*. 145: 391- 442,1956.
5. Schmid, W.D. Survival of frogs in low temperature. *Science*
215: 697-698,1982.
6. Asahina, E. Prefreezing as a method enabling animals to
survive freezing at an extremely low temperature. *Nature* 184
(4691):1003-1004,1959.
7. Meuli-Simmen, C., S. Kehrer, T. Eiman, R. Schiestl, S.
Griffey, O. Placik, K.M. Hoffman, and H.J. Buncke. Microvascular
transplantation of cryoprotected knee joints. *Annals of Plast.
Surg*. (in press).
TRANS TIME Investments II
by Art Quaife, Ph.D.
Financially 1995 was terrible for TRANS TIME; we lost $169,000.
However, there is one bright spot: our equity portfolio continues
to do spectacularly well. Our rate of return for just the year
1995 was 38.20%. Our rate of return for all stock investments we
have ever made is a world-class 74.45% annual return, compounded
yearly! You can find much more detail on our investments and
investment policies in my previous article in the April 1994
*TRANS TIMES*.
I calculate dollar-weighted returns, using the same formulas as
are used to determine the internal rate of return on an
investment project. Our returns would be even *higher* if we
calculated time-weighted returns, as mutual fund companies do.
(Both methods give the same result if there is just one initial
cash investment and no withdrawals; the methods differ in how
they treat subsequent investments and withdrawals.)
Our most spectacular returns have been from early investment in
startup cryonics-related companies. We have also done very well
letting Warren Buffett manage most of our money (through
Berkshire Hathaway); it could hardly be in better hands. Thanks
again to Ed Thorp for pointing out this marvelous investment to
me 9 years ago. Warning: the stock is currently selling at a
historically high ratio to book value.
These figures do not include the value of the Sunnyvale building
we partially own, which has certainly turned out to be the worst
investment we ever made. *No* thanks again to the person who is
mostly responsible for turning that purchase into a disaster, and
for disrupting Northern California cryonics.
Steve Seinberg: New Employee
TRANS TIME is proud to welcome aboard our newest employee, Steve
Seinberg. Steve's main responsibilities will be maintaining our
Peachtree accounting system and handling our Paradox database, as
well as utilizing several other software packages.
Steve is a graduate of San Jose State University, where he
majored in Business Administration with a minor in Biological
Sciences. He is also a graduate of Hastings College of the Law
and has passed the California Bar Exam. Steve has expressed an
interest in branching out at Trans Time and adding some legal
responsibilities to his accounting and other office tasks.
Prior to working at TRANS TIME, Steve spent a year working at
several different law firms in the Bay Area, but found the
atmosphere a bit stifling. "One of the things I like about TRANS
TIME," he told us, "is that the subject matter being dealt with
here is anything but mundane. Most people are familiar with
things like insurance and employment disputes and all the other
issues I was working on at my firms. Cryonics, on the other hand,
is really new territory to just about everyone. It raises some
serious questions about our notions of life and death, and I ve
already had a few arguments about the morality of cryonics. It s
thought-provoking, and I think I need that in a job. Not to
mention the fact that the work being done here could yield
incredible benefits to the whole race, something I couldn t have
said at a law firm."
Steve also writes fiction in his spare time--he is contracted to
a Bay Area comic book publisher, and is working on a horror
novel--and until recently, played bass and sang in a rock band.
He s hoping to start up another band when his schedule permits.
"One of the fringe benefits of working here is that it might give
me some ideas for my writing. Have you ever been to TRANS TIME?
There s probably dozens of screenplays and stories floating
around this place just waiting to be written . . ."
Good luck, Steve, and welcome aboard!
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