X-Message-Number: 15310
From:
Date: Wed, 10 Jan 2001 10:01:03 EST
Subject: microphotos
This is Instalment 6 of my vitrification series. (On our web site the
arrangement is different.)
It is important for newcomers especially to understand why Alcor's recent
claims for its "vitrification" procedure are not to be taken at face value.
As I have said several times, the evidence for efficacy is only partial,
indirect, and not confirmed by independent investigators. Confirmation is not
even possible at this point, in fact, since the procedures are secret, even
though they are believed based on procedures developed at 21CM that have been
disclosed at least in part through patents and professional publications.
I might interject at this point something I omitted in previous comments
about Fred Chamberlain's article ("Vitrification Arrives!") in the current
issue of the Alcor publication CRYONICS. Fred adduces as partial evidence of
efficacy of Alcor's current procedure the results of microscopy of vitrified
rabbit kidneys and of blood vessels. But on our web site we have extensive
quotations from one of the lead scientists at 21CM, who in 1988 wrote in part
as follows:
" if we wish to understand what happens to the brain when it is frozen, we
cannot argue on the basis of results obtained with kidneys or plant cells or
granulocytes, but must, instead, focus specifically on the brain."
But today I want to look at fixation and staining. These are topics of which
the average reader has only a vague notion, yet there is considerable
relevance to questions at issue.
Doug Skrecky and others have commented recently on chemical fixation as a
possible alternative-or adjunct-to cryopreservation. As a crude first
approximation, one can say that chemical fixation (akin to embalming) has the
potential to preserve structure well-perhaps better than
cryopreservation--but is so highly toxic that (to my knowledge) no tissue so
treated has ever been revived. What some readers may not appreciate is that
evaluation of cryopreserved specimens by microscopy involves fixation, and
usually staining as well.
Fixation before microscopy has several purposes--to arrest deterioration
during handling and preparation, to differentiate the solid phase of the
protoplasm from the aqueous phase, to make cell parts insoluble during
subsequent treatment, and to protect cells from distortion and shrinkage when
subjected to fluids such as paraffin and alcohol that may be needed for
further stabilization or/and better visualization. Stains may additionally be
used to improve contrast.
An ideal fixative should penetrate rapidly; coagulate cell contents into
insoluble substances; protect tissue against shrinkage and distortion during
dehydration, embedding, and sectioning; allow cell parts to be made
selectively visible; and prepare tissues for staining. As one can easily
appreciate, because of the different requirements of different tissues and of
different conditions, fixation and staining techniques constitute an art
based on experience, and not a mathematical science.
Think about this a little. The variables involved in evaluation of
cryopreservation include differences in the techniques and even the personal
skills of the technicians, as well as variability in specimens. This is one
of the many reasons why we cannot blindly rely on the reports of others in
deciding what techniques to use on our patients.
I also remind readers at this point that BOTH fixation and freezing preserve
structure so well, in many cases, that BANKS of fixed or/and frozen tissues
and organs are kept for the use of students and pathologists to compare
normal material with diseased or damaged material. (There are both frozen
brain banks and pickled brain banks, or there were the last time I looked.)
Think about this a little. If freezing or fixation preserves microscopic
structure well enough for use as a standard, that surely tells us
something-namely, that while both freezing and fixation inflict damage, both
also confer a great deal of preservation.
Besides frozen banks, pathologists also use freezing in diagnostic clinical
medicine, applied to tissue from patients. One text (Humason's, 1997) on
tissue techniques says that, "The freezing technique for preparing specimens
is unsurpassed in certain situations." Clearly, then, freezing (WITHOUT
cryoprotection) saves structure of small specimens sufficiently well to allow
differentiation of normal and diseased tissue, at least in some cases.
At this point also we realize that a microphoto of a piece of formerly frozen
or vitrified brain does not show us what the material looked like while
frozen or vitrified. It shows us what it looked like after warming, fixing,
and staining.
And there is the additional complication of the CPA. While frozen or
vitrified, the tissue is not in its normal condition; it has foreign material
(the CPA) and less water. After warming, one can make the microphotos either
with or without first washing out the CPA. In either case you will get
something different than existed during the frozen or vitrified condition.
As far as I know, there is not yet any method of doing microscopy on frozen
or vitrified material. There is a so-called "freeze substitution" technique
which for example allows one to use cold alcohol to dissolve ice out of the
sample before warming it. There are methods of fixing in cold acetone, but
detail is not well preserved.
There exist cryostats and associated equipment (microtomes or "paring knives"
etc.) allowing the removal of thin sections of frozen tissue, and it can be
done quickly. But the temperature of the cryostat is ordinarily far above
cryopreservation temperatures, and the microscope stage itself is never, to
my knowledge, at cryogenic temperature.
Reprise:
The import of all this is twofold:
1. Cryopreservation (freezing or vitrification) sometimes preserves structure
relatively well, and so does chemical fixation.
2. Everything you see in a microphoto of a formerly frozen or vitrified
specimen must be interpreted and not taken at face value, because you are
seeing the results of many manipulations and not the true appearance of the
specimen while it was cryopresereved.
Microphotos are valuable, but not definitive, even if no deviation from
normality is evident. For reasonably reliable evidence of complete success,
we need at minimum good results from microscopy, from chemical tests of
physiology, and from electromagnetic tests of neural nets.
Tests done on sheep brains and on rabbit brain pieces respectively, perfused
with glycerol and frozen to liquid nitrogen temperature and rewarmed, show
relatively good results (compared to unperfused controls) by microscopy and
by tests of electrophysiology. See the CI web site.
Tests done recently, as reported in part by 21CM, show very good results by
microscopy, when applied to brain tissue cooled only to - 80 C and rewarmed;
and about 53% viability in rat hippocampal slices after perfusion with a less
concentrated (hence presumably less toxic) solution than Alcor's. This is
also on the CI web site.
Robert Ettinger
Cryonics Institute
Immortalist Society
http://www.cryonics.org
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