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 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=15310