X-Message-Number: 15478
Date: Tue, 30 Jan 2001 02:50:02 -0500
From: Paul Antonik Wakfer <>
Subject: More Cryobiology Comments - #15403, 15409, 15420

>CryoNet #15403
>From: 
>Date: Sun, 21 Jan 2001 12:01:13 EST
>Subject: CI procedures again

[SNIP]

>It seems pretty clear that newcomers, by and large, find our disclosures and 
>explanations satisfactory. People tend to "vote with their feet," and CI 
>growth has been faster than anyone else's in the last couple of years. No 
>amount of fulminating on Cryonet can change that.

Here Mr Ettinger is confusing science with politics.
Describing work that it purported to be "scientific" in less than full
detail may influence novice layman who understand little of the demands
of real science and may even win their votes and allegiance, but as
science it is still worthless. Those laymen who are rational will in
time listen to credible scientists who care enough to point out that
worthlessness and will vote against the original fraudulent behavior.

>Nevertheless, there may be some who don't get an impression of clarity, and 
>we always try to improve, so we will shortly modify the web site pages to 
>include a clearer and more succinct summary of our procedures. 

Since, as far as I know, CI did not take many of the most simple and
important measurements (such as timings, temperatures, effluent glycerol
concentrations, etc) necessary to record what actually happened (and
therefore to allow some kind of assessability and reproducibility), I
don't think that it will be able to make things "clearer and more
succinct" by any addition of summarizing verbiage.

>But let me emphasize again--exactly what is it that Thomas, or anyone else, 
>would gain from ever more detailed explanations of our procedures?

If CI had recorded and measured these in proper scientific fashion, they
would know sufficient detail of the procedure and of what measurements
were obtained to enable reproducibility. These intermediate dynamic
measurements often tell much more than a few electron micrographs (which
after all are of an insignificant amount of the total tissue which was
processed).

>Would you compare that information with reports in the cryobiological 
>literature and make a theoretical guess as to whether our methods conform to 
>"accepted" views of what is best for brains?

There are no accepted views of what preservation "methods" are best for
brains, since "best" implies a specific purpose, and the purposes of
some uses of brains are not at all the same as other uses. Finally,
"best" is
by its very nature not an objective of science which is the
investigation of reality and the discovery of the facts thereof. "Best"
is a purely subjective term of valuation and as such is totally
non-scientific. However, even in subjective practical terms, no one in
cryobiology or any other branch of science (except perhaps those at INC
and 21CM) currently has the purpose of restoring life as their purpose
for preserving brains!
In addition, "theoretical guesses" are practically useless in complex
areas of biology as countless guesses made by the most experienced
scientist have shown over and over again in such fields as medicine and
nutrition when their "best guesses" were disproved by their own
experiments.

>Would you actually go to the trouble and expense of replicating our
>work, or paying someone else to do it, and then paying for its
>evaluation by independent professionals? 

No, because CI has never provided sufficient details of its dynamic
procedures and sufficient dynamic measurements (ie time history of all
important I/O parameters) to allow such replication. To attempt
replication without this is a matter of guessing and largely a waste of
time. 

>Is it not obvious that the bottom line is RESULTS?

Though once again not a scientifically meaningful statement, this
nonetheless might be correct *if* the description and specification of
the "results" were detailed and multidimensional enough to be relevant
and meaningful.

>Two sets of independent professionals have evaluated our work,

Please state their professional qualifications.
Ie. what have they done which directly relates to what CI had them do?

Electron micrographs are about 100 microns on a side. The field of view
may be far less depending on the magnification used. Even if they
examined in detail 100 such micrographs, the total examined tissue would
be insignificant compared to the size of the tissue which was
cryopreserved.
Since Mr Ettinger has admitted elsewhere that the cryoprotectant did not
equilibrate (implying that a uniform (stable) and potentially
reproducible state was never reached), from which portions of the tissue
were the micrographs taken? Were these from the highly glycerolized
portions near the vasculature, the intermediate concentration portions
further away, or the nearly untouched portions farthest from any
glycerol perfusate?   

>Is there anyone--any newcomer especially, but also anyone else,
>including Thomas--who would rather bet on his own theoretical
>guesses than on actual professional evaluation of results?

The evaluation of science experiments is not equivalent or even related
to "betting on guesses" by anyone, either theorists or professionals.
Paid professional evaluation can and will only go so far as it can from
the information given to it, especially if the professionals have no
intrinsic interest in what they are evaluating or in the implications of
that evaluation. One has to have detailed knowledge of the whole
experiment and its purposes, in order to conduct a worthwhile
evaluation. Mike Darwin and Jerry Leaf found this out many years ago
when they first began trying to get electron micrographs done. As with
any complex field there are many, many ways to do things. Which of these
methods can be applied to give the most useful information for
cryobiology-related purposes is not clear to begin with and is not
easily decidable by a non-cryobiologist no matter how competent a
professional he may be. 

>Less defensible is Fred Chamberlain's over-reaching in his article in 
>CRYONICS and on their web site, "Vitrification arrives!" The decision 
>itself--to use the new procedures--is defensible, and it may have been
>the right decision. But we don't know, whereas the thrust of the article
>is to convey the impression that we do know.

*If* "the thrust of the article was to convey the impression that we do
know" then that is wrong. We do not know what is best and we *will not*
know what is best until an animal brain is restored with full
functionality in all reasonably measurable parameters.

>As I have said, critics of CI in most cases, asking for more details, 
>probably just want to score debating points, not to test our methods for 
>themselves.

As I have addressed above and elsewhere, the problem is that CI did not
even *record* the necessary details to allow replication. Ie CI did not,
in fact, do any science!

>We on the other hand, in desiring more information about the 
>Alcor methods, truly do want to test their methods ourselves,

That would be a valuable provision of validation of other results (INC
would love to have its results so validated, and I bet 21CM would too),
*if* CI had ever shown that it knows how to conduct a scientific
experiment in the first place!

>and we will pay 
>for professional evaluation of those tests, if those methods are still 
>relevant when we get the information.

If one doesn't know enough to evaluate one's own results, then one does
not know enough to be doing the experiment! To separate the evaluation
of results from the conduct of the experiment which produced them is not
feasible scientifically. 

>Cryonet #15409
>From: "Jeff Grimes" <>
>Date: Sun, 21 Jan 2001 15:08:51 -0700
>Subject: Misleading?

[SNIP]

A quote from the CI website.

>     "Also, and more importantly, the cryoprotectants used in
>vitrification are toxic. Thus, vitrification kills cells: it
>poisons and injures them to the point of actually disintegrating
>cell membranes in some cases. Indeed, the damage done by
>vitrification has been so immense and so much worse than
>conventional suspension treatments that, on balance, every last
>cryonics organization has throughout cryonics history opted for
>the less destructive option of conventional glycerol suspension
>and cooling and liquid nitrogen storage."

Elements of this are completely false by any reasonable criteria and
others are so grossly distorted that I am appalled to read that it
appears on CI's website. This is especially inconsistent with Mr
Ettinger's continued attack on a far less distorted article about
vitrification which appears on Alcor's website. 

>I am also really surprised by the last part of the quote stating
>that all other organizations have "opted for" conventional glycerol.
>Did they have a choice? From previous posts it seems that only one
>organization (Alcor) has been allowed a license to use the
>vitrification system, which was only developed last year anyway.
>But "throughout cryonics history" suggests vitrification has been
>an option for years. It hasn't, has it? This seems intentionally
>misleading.

Actually, as I have stated before, vitrification has always been
available for cryonics purposes. However, the cryoprotectants and
concentrations needed to vitrify were so toxic (as alluded to above)
before recent 21CM discoveries and 21CM/INC research verification, that
a correct decision was made by all cryonics organizations to use
glycerol at lower concentrations. However, with the above statement CI
is guilty (whether accidentally or on purpose, I do not know) of grossly
distorting the present picture by associating the damage done by the
potential vitrification methods of 20 years ago with that done by the
vitrification methods proposed for use today.

>Here's another part that bothered me:
>
>"What does 'viable' mean? It does not mean that half the cells
>were cooled to a temperature that forestalled all decay and were
>then restored to healthy life. It means that, in two experiments,
>about half the cells managed to retain a potassium/sodium ratio
>that cells require to be alive. About half did not, and were
>irrecoverably dead (by present methods)."
>
>But we saw a post here recently that absolutely contradicted this
>and stated that the percentage viability measures the overall
>ability of cells to do what they normally do, which is like saying
>an athlete can only run 53% as fast as he used to (but he's still
>running, not lying on the ground, with half of him paralyzed). How
>did Cryonics Institute get this so wrong?

Actually, CI's statement is worse than Jeff has said.
53% of the normal sodium/potassium ratio does not at all mean 53% *of
what a cell requires to be alive*! Lots of cells in the body have for
brief periods of time or during major traumatic situations
sodium/potassium ratios below the optimum normal levels (and likely
sometimes even above that level, which afterall is only a measured
"norm", not a rigorous standard - this is not black and white Newtonian
physics here). However, these cells are still alive and within this
supportive, healing environment of the body will then proceed to
increase their health back to more normal levels. In fact, that is what
was meant when it was stated elsewhere that 66% of a normal
sodium/potassium ratio for kidneys has been enough in the past to allow
full recovery of the kidney after transplantation (ie within the
nutrient supplying, waste product removing body environment).
Certainly, 53% does not in any manner imply that 47% of the cells were
irrecoverably dead! It is even possible that *none* of them would be
irrecoverably dead if they could be put back exactly to where they had
come. Although this is unlikely given that the 53% is an average of the
ratio of the whole slice, and thus some cells might be close to zero and
definitely irrecoverable (while other cells were much better than 53%,
of course).

>CryoNet #15420
>From: 
>Date: Mon, 22 Jan 2001 11:30:49 EST
>Subject: Donaldson & "viability"
>
>I believe that Dr. Thomas Donaldson is a member of the Board of Directors of 
>the INC (Institute for Neural Cryobiology), which is one of the organizations 
>involved with the rat hippocampal slice cryopreservation project. He is also 
>a self-taught scholar in neuroscience and to some degree at least in 
>cryobiology. 

While Thomas is, in fact, the founder of INC and did all the original
work to get US and California charitable status for it, which has been
so important to its current success, he has not been involved in any
current decisions regarding its research and contracts with outside
organizations, nor been given any special updates about its results. If
there is any *blame* for this, it rests entirely on my shoulders as
President of INC for the past 4 years. Between my unpaid and unassisted
(until recently) involvement in INC, my need to earn a living, my
burgeoning interest and practice of life-extension apart from cryonics,
and my need to often attempt to explain and enlighten on CryoNet and
sci.cryonics, I have not had sufficient time to send special updates to
either the INC directors or even the many INC donors.

>There appears to be disagreement about how to interpret the "viability" of 
>rat brain slices vitrified and then warmed, washed out, cultured or 
>incubated, and tested for viability by the potassium/sodium ratio criterion. 

There *is* no disagreement except by *Mr Ettinger* and his associates.

>One interpretation is that, in a sample containing many cells, the average 
>K/Na ratio was 66% of normal, with the variance unknown or unreported.

This is the *only* correct interpretation. When the experiments are
written up and published the full conditions of the experiment
sufficient for reproducibility, the number of samples used, and the
ratio found for each will be reported.
>From a scientific pov, *none* of this information should have been
released since from the currently accepted pov of proper scientific
methods, until an experiment is published in a peer-reviewed journal it
is not complete, ie it is still in progress. Like many research efforts
however, and probably moreso than most, INC has to get its results 
before the "public" as early as possible if it is to have any hope of
securing the funding necessary to conduct more research.

>That 
>is, it is unknown how many were dead or close to it by this criterion, and 
>how many were normal or nearly so.

This is correct.

>The other interpretation is that every individual cell was functioning at 66% 
>normal, or very close to it, by this criterion. This latter seems very 
>unlikely just as a matter of general experience with populations in 
>statistical studies. Variance is hardly ever close to zero.

Mr Ettinger has been told several times that this is an incorrect
interpretation, and as he says, it is even logically untenable, so why
does he keep bringing it up?

>Next, the K/Na criterion is said to be a good one for gauging overall cell 
>life or function-if the K/Na ratio is near 100%, then most or all cell 
>functions are close to normal. Is this really correct? How is that known?

Because many, many basic cellular functions, which in turn are necessary
for many other cellular end products such as protein production and
mitosis (cellular division), must be functioning to maintain the extra-
to intracellular sodium/potassium ratio. Among these functions are:
mitochondrial energy (ATP) production (which itself requires fully
functional mitochondria and a large amount of functional cellular
machinery outside the mitochondria), ATP flowing from the mitochondria
to the cellular membrane (which means it is also available to all
intracellular machinery including the protein producing ribosomes, and
the existence of functional sodium/potassium ATP (the sodium potassium
pump) within the intact cell membrane.
However, once again, why does Mr Ettinger continue to question this
when fully credible biological scientists have stated that it *is*
a good test of overall cellular function (viability)?

Here is an old medline abstract which compares the value of various
viability criteria.

Hoppe Seylers Z Physiol Chem 1975 Jun;356(6):827-38
Criteria of viability of isolated liver cells.
Baur H, Kasperek S, Pfaff E

Various cellular parameters were measured with regard to their
usefulness as criteria of viability of isolated cells. Stainability
by trypan blue and release of lactate dehydrogenase indicate only
severe irreversible damage of cells. Neither endogenous respiration
nor even the ATP/ADP ratio is a sensitive criterion of viability.
On aging of cells, the ATP/ADP ratio remains high, even though the
membrane potential, the intracellular K concentration and the
content of adenine nucleotides decrease considerably. A sensitive,
easily performed test is the stimulation of cellular respiration by
1mM succinate. Only a damaged plasma membrane allows succinate
permeation of a rate sufficient to stimulate respiration. The
membrane potential and the intracellular Na and K concentrations
are the most sensitive criteria of viability, since they indicate
the earliest changes on aging. (For freshly isolated cells, we found
a membrane potential of 36.4 +/- 3.4 mv [n = 5], an intracellular K
concentration of 109.0 +/- 9.1 mM, and an intracellular Na
concentration of 47.0 +/- 13.4mM.) The incorporation of [14C]uridine
also sensitively reflects cellular damage.
PMID: 241693, UI: 76044920

>I remind readers that Suda in the Sixties got relatively good corticograms 
>from glycerolized cat brains rewarmed after relatively short times at  
>relatively high sub-zero temperatures.

That was essentially formatted EEG readings from whole brains, which is
not what can be tested on slices.

>A couple of years ago Pichugin 
>rewarmed frozen (not vitrified) glycerolized rabbit brain pieces from liquid 
>nitrogen and obtained coordinated electrical activity in networks of neurons. 
>This is surely an important marker of "viability" from the cryonics 
>standpoint, and yet, as I understand it, the K/Na  test would not have given 
>good results.

First, I wish to point out that this use of the term "piece" is a very
unscientific and undefined term, whereas brain slice is a well
characterized and often used term. If Mr Ettinger uses the term "piece"
again, would he please specify what they are, with enough precision to
give a reproducible method of obtaining similar "pieces".

Second, the answer to Mr Ettinger's point above was something of which I
was not aware, but is quite interesting and important. You see, it turns
out that neuronal synapses can keep firing 100's of thousands of times
after the cell to which they are related is completely dead! This is
because the amount of energy and operational cellular machinery needed
for this purpose is extremely minimal. Therefore, neuronal activity
alone is *not* a good criterion of viability. In fact, it is only useful
to measure *after* cellular viability has been well established by other
means. Here is a quote on this from one of the involved scientists.

"The propagation of action potentials does not require cells to be alive
or properly functioning.  Action potentials propagate via the action of
simple voltage gated ion channels in membranes.  These channels are
triggered merely by the presence of a voltage difference across the
membrane. Guyton's Textbook of Medical Physiology states that 100,000 to
500,000 impulses can be transmitted by nerve fibers until the
transmembrane ion concentration gradients require reestablishment by
active ATP-powered ion pumping.  Thus simple electrophysiology
experiments tell us nothing about the comprehensive state of a cell's
molecular machinery."


-- Paul --

The Institute for Neural Cryobiology - http://neurocryo.org
A California charitable corporation funding research to
perfect cryopreservation of central nervous system tissue
for neuroscience research & medical repair of the brain.
Voice-mail: 416-968-6291  Fax: 559-663-5511

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