X-Message-Number: 5050 Date: 24 Oct 95 19:17:19 EDT From: Mike Darwin <> Subject: SCI.CRYONICS: On Turbulence I have in small measure as I've aged (I'm 40 now) learned a valuable lesson and that is to sometimes WAIT AND SEE even when inclined to jump into the fray. Sometimes I want to jump in because I see an idea I care about being attacked, sometimes because someone or something is being unfairly attacked, sometimes because of ego, and sometimes just to correct a simple misunderstanding or provide clarification that will end a debate and save others time and trouble "flaming" over an issue that is irrelevant. Sometimes it *is* wise to hop right in. But other times it can be richly rewarding to simply wait and watch. Now, we come to the heart of the matter. I am not formally educated, and in particular, I suffer from dysnumeria which is just as crippling as dyslexia, but as not as noticeable since humans are mostly story creatures who deal with the day-to-day with imprecision and with fuzzy things called words. In the realm of science, however, these defects can be devastating. It is odd that I have chosen science for a career. But, when one examines what "science" (cryonics) I chose perhaps it is closer to the truth to say that I chose what I *first* wanted as an occupation as a very young child: religion. Perhaps with more irony, I wante to be Catholic priest as a small child, even though, as far as I can tell I have never believed in God. (For the unitiated in the nuances of human behavior not believing in God, or believing as the case may be, has almost nothing to do with choosing the priesthood as a career and further, belief in God is absolutely contraindicated if you are ever to be a cardinal or pope.) I offer this digression to put into perspective what I am about to say. At the time I raised the issue of "turbulence" occurring during freezing I used the word in a nonrigorous sense. By this I mean that I had absolutely no idea of not only the *rigorous* use of the word employed by Merkle, Donaldson, Harris and others, but did not understand the underlying concept and still do not specficially, although I think I do in a fuzzy sort of way generally. I have set time aside to be tutored (in a nonmathematical way!) further about my deficient understanding of the concept of turbulence inside physics, and I feel that, unlike some other areas of physics, I will be able to understand it conceptually fairly well. Rigorous use of words is NOT being mocked here, and this post is not intended as sarcasm. In fact, in biology the same sort of rigor is demanded and it can be a devastating blow to someone's credibility if they misuse a word or use it with imprecision. An example from biology would be someone who claims to be a biologist using the word "wall" to discuss the cell membrane of say mammalian cells. Plants have cell walls (cell membranes too) but animals have only cell membranes. Even within a discipline misuse of a word can tell you about someone's degree of sophistication or lack thereof: a cryobiologist who uses the word osmolarity as opposed to osmolality is betraying a poor education or sloppiness in word useage which should put the listener or reader on alert. Note, I did not say reject out of hand, I said be on alert. And here Donaldson's comments are very well taken. Sometimes out of the mouth of babes come great things; neither Edison or Marconi understood the physics underlying their work worth diddly squat, in fact, IMHO I understand the physics of electromagnetic radiation better than Marconi did. Lucky thing I wasn't trying to develop transcontinental radio communication. So, what was I talking about when I used the word turbulence? Was my use of the word correct? The answeer to the second question seems to be unclear, but likely that I used the word without rigor or good understanding and thus incorrectly. This is no great tragedy; I once took a very public drubbing by Bob Ettinger for misusing the word "gradient" and while it was a little embarrassing, it was well worth the correction because it improved my understanding of physics a little and I never made the same mistake again!). It has thus amused me to see all this heated debate go on while I waited and watched instead of simply piping up and saying, whoaaa guys, what EXACTLY do you mean by this word turbulence :). The wait has been rewarding. In any event, there is still to dispose of the question of what I meant by the word. Here I will simply have no recourse but to describe what I have seen in my own experience, on videotape of others' work, and what I know or think I know about the probable/possible mechanics of cryoinjury at the cellular and molecular level. OBSERVATIONS: When cells with intact membranes are frozen at very slow rates (well below 1 C min and more on the order of 4-5 C per hour) ice forms outside the cells and concentrates in front of the growing ice mass the dissolved solutes that are excluded from the crystals of pure (semi pure) water that are being formed as freezing proceeds. Since freezing does not occur everywhere and at once, there is a state where the crystals grow and where there are pools or lakes of liquid between them, and when no more water can be extracted from the pools or lakes, what you have is large masses of ice with the tissue compressed into very small channels of concentrated solute and/or cryoprotectant if cryoprotectant is present. The more cryoprotectant, the larger the channels. I do not know enough about the physics of strucures on this scale to know if the flow of water out of the cells and the wave of material being pushed in front of the advancing ice is "turbulent" or not. What I have seen is that as the ice grows and the concentration of dissolved solids and cryoprotectant rises not only does water come streaming out of the cells but the cells themslves shrink and experience enormous stress on their membranes. One result of this is that cells will sometimes bud off spheres of membrane material and that certain structures stuck onto or studded in the membrane will come off of or out of the membrane and into the unfrozen solution. Further, in tissues as opposed to suspensions of individual cells, the water leakage is not uniform from the cell mass: some cell of material being pushed in front of the advancing ice is "turbulent" or not. What I have seen is that as the ice grows and the concentration of dissolved solids and cryoprotectant rises not only does water come streaming out of the cells but the cells themslves shrink and experience enormous stress on their membranes. One resultrain tissue in the frozen state we see that axons and cell bodies occupy about 25% of the space that they did before freezing, and that not only are they shunken, but that they stretched out like taffy in some case: the best analogy is silly putty where you have "picked up" an image from a newspaper and then stretched it out, sort of disorting it like a funhouse mirror can. You also see rips or tears and pieces (very small 1 micon or so) ) of material that look very far removed from where they should be. One way I have checked for this is to infiltrate gold fish tails with small particlls of carbon black, and then very slowly freeze them. You get carbon particles moved over (on the ultramicroscopic scale) vast distances: pushed around by the ice front, "hydraualically squeezed" by ice building up "pressure" on tissue or adajacent liquid areas that are not frozen, and so on. Further, if you look in these channels of fluid as freezing very slowly progresses you can see the that particles of carbon are bouncing off each other and giving the apperance of being stirred (presumably so-called Brownian motion). Further, you will see a little pool containing fluid and particles remain undisturbed, start to change shape and then very rapidly get squeezed empty and its contents shift to some other space. In other words, what seems to be happening is that the water and tissue in front of the advancing ice are experiencing compressive forces, and sooner or later those forces cause some sort of change that result in either slow or rapid fluid shifts. Further, some of the tissues where the ice is growing from are dehydrated neatly, but the dehydration seems to be causing them to pull apart from structures that are not yet dehydrated (shrunken) or which are do not respond in the same way to which they are attached. Finally, as the ice crystals get larger and larger areas of tissue become trapped beweeen moving ice fronts with liquid flowing through any path between the cells and multicellular structure and non cellular tissue matrix structure as well as between the ice crystals. Some of these trapped tissues are just squeezed to the point that they tear. These tears occur at about 30-60 micron intervals throughout the tissues and appear as raggeed cuts or cavities on EM in the frozen state or after thawing. There appears to be more tearing in the brain on examination both post thaw and by freeze substitution EM (i.e., in the frozen state at -79C) in the white matter than in the grey, and very substainial tearing and big discontinuities in the pattern and size of ice crystals in the fairly sharply demarcated zone between gray and white matter. Determinations of "turbulence" in the sense I think Merkle means it is beyond me. But I would point out that if the model is some isolated cell then the model is likely not to be very predictive of what happens in tissues freezing inhomogenously, losing water at different rates over scales of 10-20 microns, and interleaved with a mesh of fine structural proteins (which cannot be seen with light miroscopy) called the intercellular or extracellular matrix. I have wondered and even "thought" that the matrix, the tissue structures, the channels that open and the sudden "giving" or collapse of fluid pools or compression of tissues due to advancing ice would be the equivalent of "vortex mixing." As I understand it, vortex mixing is non laminar and the original or starting position of things distributed with such mixing is not easy to infer. CAVEATS AND QUESTIONS: 1) Now, as to whether vortex mixing can occur in solutions or pools of such solution in the presence of many complex and filamentous structures and with structures of widely varying geometery being pulled, stretched, torn and losing water from different areas of their surface at different rates) and ice pushing/squeezing hyperosmolar (dehydrating fluid) along tissue planes in front of it as it advances, and at the micon to millimeter scale at which events occur I do not know. 2) I do not know whether any of this flow or movement of fluid and particles in fluid is "turbulent" flow or non turbulent flow in the sense I *think* I understand Merkle to mean it. 3) I do not know whether loss of membrane material, receptors, antigens or other things from cells undergoing hyperosmotic dehydration constitutes a loss of information or structure which will have any impact whatsoever on the ability to restore memory, mentation, and "identity" to a person whose brain is subjected to these effects. 4) I do not know if debris or eluted or extruded cell components moved around in such a situation have inferrable starting positions. 5) I do not know to what degree the original state of things (axons, cell bodies, aggreates of cells attached to each other, etc.) which have been squeezed under enoumous pressure to the point that they are cut or seperated will be inferrable given our current understanding of physical law. 6) I do not know to what extent cells torn in half or long process which are cut undergo spontaneous reordering into "new" or different structures like the soap bubble analogy Steve Harris occur, no do I know if these changes if they occur preclude inferrance of the undamaged or starting state, or even if they do, whether they constitute an identity-critical loss of information. WHAT I DO KNOW AND FEEL: 1) I know I do not like what I see in brain tissue during freezing, in the frozen state, and after thawing with or without cryoprotectant using existing current techniques. 2) I know that I do not want these things to happen to my brain, to my lover's brain, to my friends' brains, or to brains of any patient I cryopreserve, whether they be friend, or foe, or neither. 3) I FEEL very strongly about 1) and 2) above and I feel motivated to do something about them. 4) While I do not know the physics of all of these phenomenon, particularly where it involves questions of turbulence, inferrability of vortex mixing, interaction of Brownian motion etc., I am willing to learn as much as I can, and in the meantime I am unconvinced and take a *conservative* stance about the reversibility of this kind of injury (or to use a more neutral word change(s)). 5) Everyone is free to draw their own conclusions, but it seems to me far more profitable to put up guard rails than to spend years standing around the debris of an automobile that tumbled down a moutainside debating about whether you can find all the pieces, put them back together, repair them, or otherwise order them so that that the car can run again and the passengers inside can drive it. 6) While 5) above is my preferred position, I understand and NEED and WANT to know if the automobile can be put back together again given our current understanding of the damage and physical law. This is so because I have friends and loved ones who are or will be careening off the road without a guard rail and I am deeply concerned about them and their prognosis. Having said this, I would go onto say that it is someone of questionable judgment at best who watches a caravan of cars (of which he or she and his or her loved ones are peronally a part of) go careening off a cliff or into a chasm where there should be a well built bridge or guardrail and do nothing more than try to reasure everyone in the car with you and behind and ahead of you that there are sound reasons to believe that GOD will come around someday, or Nanotechnology, or even nanotechnology, and put us all back together again. The take home message: get your fucking ass in gear and put up a guardrail and/or build a bridge. Mike Darwin Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=5050