X-Message-Number: 5858
From: "Pichugin" <>
Date: Fri, 1 Mar 1996 13:19:39 +0200 (EET)
Subject: SCI.CRYONICS ukrainian research
Date: 01 Mar 96
FROM DR. YURI PICHUGIN--
SUMMARY OF RECENT COMMUNICATIONS
Abridged, edited and made notes [ ] by R.C.W. Ettinger. Accuracy not
guaranteed. This is his response to my request to summarize his recent
activity and outlook. Dr. Pichugin's e-mail address is
<>
R.C.W. Ettinger
------------
2 Feb. 1996
Research at the Institute of Cryobiology [Ukraine, Kharkov] has been
virtually paralyzed since June of 1995 because of delay of funding by the
Ukrainian government; but the work for the Cryonics Institute and Immortalist
Society proceeds, slowly and with difficulty.
Recently I tested a new freezing chamber for work with sheep heads. Two
untreated heads (not glycerolized) were frozen and thawed at a rate between
10 and 20 deg C per hour. One was frozen to - 80 C, the other to - 196 C.
There were no visible cracks in the first, but many visible cracks in the
second. Samples of tissue were fixed, but histological preparations have not
yet been made. In February we expect to continue the work with glycerolized
sheep heads and brain slices.
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5 Feb. 1996
After cryopreservation of sheep heads last year by the Cryonics Institute
method, and discussion of results among myself, Prof. Zhegunov, and M. Darwin
about Darwin's evaluation of our histological and electron microscope
results, I got the idea of using brain slice cultures with the aim of
reanimation of cryopreserved brain tissues, using measurement of BEA
[bioelectric activity] as a functional, physiological method of examination
of viability of the brain tissues [since there was considerable disagreement
on evaluation of the micrographs, with Darwin's evaluation much more
pessimistic than Pichugin's/Zhegunov's].
With the help of electrophysiologists [colleagues] I successfully completed a
primary stage of this idea in 1995. Results have been published in The
Immortalist. Rabbit brain pieces were frozen to - 196 C; after thawing,
washout of glycerol, and reanimation in brain slice cultures, the tissue
showed both spontaneous and induced coordinated bioelectrical activity (BEA).
Because of the difficulty of obtaining here complete cryobiological
literature since 1986, we are not sure whether our work is the first of its
kind. A paper by Scott & Lew, Exp. Cell Res. v. 162, p. 566, 1986, studied
effects of freezing on electric membrane properties of neural cell cultures
of adult mouse dorsal root ganglia; but that study did not show the state of
synapses and integral BEA of neural networks.
[Dr. Pichugin had an exchange with Greg Fahy to follow up this question of
possible prior
similar work, and the conclusion was that probably no other work has been
done on BEA of cryopreserved brain tissue in culture after 1986. Thus it
appears that Pichugin's work in this area is new, and that it goes well
beyond the work of Suda, Kito and Adachi in the sixties.]
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12 Feb. 1996
Getting back to the similarities and differences between our work and that of
Scott and of Suda:
Scott's work, like ours, froze neural cells of adult animals to - 196 C.
(Success with adult cells and tissues is much more difficult than with fetal
ones.) With various procedures and
cryoprotectants (glycerol and DMSO), nerve cells showed a high level of
cryopreservation,viz. the presence of BEA, although altered.
Our work differed in that (1) we used neocortex of rabbit brains; this more
directly reflects
cryonics interest than does study of mouse dorsal root ganglia; (2) they used
in essence cell suspensions after preliminary culturing, and it is known that
success with such is easier than with native material in the form of tissue
or uncultured suspensions; (3) the main difference is that they investigated
only electrical membrane properties of individual neurons, which gives no
information about state of synapses; while we measured integrative BEA of
brain slices that reveals information about preservation of synapses and
function of neuron networks; slices from various regions of a brain may be
an approximate model of the whole brain--closer to the interests of cryonics.
Suda's work, similarities: Freezing of adult mammalian brain tissue with
glycerol protection and use of registration BEA to indicate function.
DIFFERENCES: Suda et al obtained their main positive results after storage of
cat heads for 777 days at - 20 C (also at - 60 C for weeks, but results were
maybe slightly worse and were not published). More significant damage was
found in the cat brain after storage at - 20 C for 7.25 years; hence this
temperature is not acceptable for cryonics without complete blockage of
degenerative processes. Freezing the cat heads to - 90 C did not result in
integrative BEA, although single neurons had function.
Now some brief remarks on the state of cryobiology and cryonics and the
importance of the brain slice culture work:
1. In cryobiological work, usually there is ischemia for not more than 5
minutes; in cryonics often hours of ischemia and even postmortem changes over
one or two days.
2. In cryobiology there is much better control of conditions and procedures,
and often focus on just one or a few types of cells or tissues.
3. Cryobiology usually judges success by restoration of vital functions and
spontaneous
reanimation. Revival after cryonics may need, and cryonics may allow,
artificial or forced or
guided reanimation, possibly with deep reconstruction of the whole biological
system.
4. Ascertaining of viability (or potential viability) may use direct
(physiological) methods or
indirect, morphological methods including histology and electron microscopy.
[Either one can be deceptive. E.g. a very small and potentially repairable
defect may completely inhibit function; on the other hand, a substance such
as glycerol, which allows reanimation of some tissues, may even in small
concentrations be totally toxic to a whole organism.]
My work at our Institute of Cryobiology from 1978-1993 involved study of
cryoprotectants;
results were summarized in my article, "Results and perspectives in searching
for new
endocellular cryoprotectants," published in our Institute's Problems of
Cryobiology 1993, #2. I demonstrated and can show in detail that searching
for new endocellular hydrophilic
cryoprotectants is not promising.
Maybe, a human organism would have been preserved on 99,9% using even the
modern achievements of cryobiology. However, modern science is not able (in
the present!) reanimation frozen patients because that 0.1% is very
essential specific bonds which create the highest level of the human
biological system.
New types of cryoprotectant are needed for the protection of the essential
and weak bonds, or thin and weak places in the biological system where it
"breaks." (An example of the importance of "thin places". Glycerol,
ethylene glycol, methanol, and many other compounds that lack specific
toxicity at the cellular level may have even strong toxicity at the level of
the organism. Even 1% of glycerol in a human may be fatal. Also, 1% of
glycerol depressed brain BEA, according to researchers at BioTime [Paul
Segall and Hal Sternberg]. But exposure of individual neurons to 20%
glycerol only somewhat and non-specifically altered their BEA, according to
data of the Institute of Cell Biophysics in Pushchino, Moscow region.)
Therefore the new types of cryoprotectant must have other than hydrophilic
structure, and the types or effects must be as various as the "thin places."
I think solution of this problem is far in the future, since it demands
thorough understanding of the organism's biological organization.
------------
14 Feb. 1996
I repeat that further significant progress in cryobiology may take a long
time. There has been relatively little progress since the milestone discovery
of glycerol as cryoprotectant in 1949. Many processes in nature, society, and
science develop by leaps forward, and cryobiology has had its leap. Now
cryonics ought to make its leap forward in search of the optimum method of
cryopreservation of patients.
At present we mostly do not even know the types and degrees of damage.
Possibly the present damage is not too bad, if we consider the possibility of
perfected future methods of guided deep reanimation. To obtain more detailed
information, I decided to try for the first time (research priority) to do
first steps on a new level, more improved conditions for spontaneous
reanimation of cryopreserved brain tissues using tissue (slice) cultures and
functional methods of assessing neuron networks by measuring integrative BEA.
To the great surprise of my colleagues, the very first step was successful.
[See above.] They had not hoped or expected that much BEA of cryopreserved
brain tissues would be found after deep freezing to - 90 C, let alone to -
196 C as I did.
I think this is an important step for cryonics because it opens up the
possibility for not only morphological studies of results but also
physiological methods. The demonstration of preservation of some capacity for
function of neuron networks after rewarming from - 196 C may give cryonicists
greater confidence and attract new members to the cryonics organizations. I
hope it will at least serve as stimulus for further research in this
direction.
It is advisable, at first and under conditions existing here, to use only cat
brain pieces. My further suggestions are as follows.
1. Develop sufficiently perfected culture methods for cryopreserved brain
tissues.
2. Compare time of active life of slices of cryopreserved brain tissues with
intact control slices.
3. if the first slices show a stationary period of function, then more
detailed study can better compare procedures of cryopreservation.
3 a. This point must be pursued in parallel with the previous. The question
of cracking of soft tissues, especially brains, is very important for
cryonics. Thus, before elaboration of the optimum method with brain slice
cultures and functional tests, it is necessary to determine "limits of
cracking," i.e. regions of concentration of glycerol and maximum rates and
modes (variation of rates in temperature zones) of freeze-thawing where
cracking is observed or not.
Because human brains are so much larger than those of experimental animals,
only human (cadaver) brains can give reliable results on cracking. Human
cadaver brain studies should follow those on sheep. Ukrainian law requires a
24 hour wait after legal death to use cadaver brains for experiment, and to
obtain permission for cryonics use may be very difficult, but I'll make an
attempt. If even after 24 hour cold ischemia of a human brain it showed no
cracking after use of the optimum method for sheep heads, we could
confidently use this method on patients.
After that come the next phases of the program, using the regions of glycerol
concentration and freeze-thaw modes which insure a brain against cracking.
4. Next we can vary conditions of cryopreservation for choice of optimum
method using only brain pieces.
5. After that, we can verify the selected optimum method using whole cat
heads, with study of functions of slices from various regions of the brain to
make corrections or improvements.
6. Then it is very desirable to verify the method by trying for spontaneous
reanimation of cat heads, analogous to Suda's experiments. But in current
Ukrainian conditions this cannot be done.
The broader program can include working out the optimum method using other
cryoprotective agents (DMSO; 1,2 propanediol, plus some extracellular
agents). Usually, the main parameters of the method (concentration of
cryoprotectant and modes of freeze-thaw) are developed for each agent because
comparison at conditions optimal for glycerol is often deceptive, as they can
give higher survival of tissues at other parameters of cryopreservation.
Later I will have comments on ways to improve the CI procedure to reduce
osmotic stress.
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