X-Message-Number: 2676 Date: 03 Apr 94 23:58:48 EDT From: Mike Darwin <> Subject: SCI.CRYONICS fracturing At the risk of sounding like a broken record I wish to second Thomas Donaldson's observations on the need for more research. As regular readers of Cryonet and Sci.Cryonics will probably have noticed, Thomas and I seem to have a regular mutual admiration society going on this point! I also wish to respond to remarks made by Bob Ettinger regarding both cracking and Cryonics Institute (CI) and Immortalist Society (IS) Research. Bob remarks that CI does not observe cracking and that the comments about the existence of cracking are "probably as a result of old Alcor research." In this he is partly right; the work done by Darwin, Leaf and Hixon some years ago at Alcor was the first to point out the existence of this phenomenon to the cryonics community and the first to document it in cryopreserved humans. However, the fact that it is "old" research should have little to do with whether or not it is "good" research or "valid" research. Nor do Bob's statements about CI's results with sheep heads lay the cracking issue to rest. But first a little background. We were not the first to observe cracks either in bulk solutions or in organs loaded with multimolar concentrations of colligative cryoprotectant(s). This phenomenon is reasonably well documented and was first observed in bulk solutions by Kroener and Luyet in 1966 (Formation of cracks during the vitrification of glycerol solutions and disappearance of the cracks during rewarming. Biodynamica 10:47, 1966). While I do not have the reference handy Rapatz reported fracturing in frog hearts cooled to -196C during *rewarming* after they had been loaded with 13M ethylene glycol. Similarly, Fahy and Sauer et al., have reported extensively on fracture formation in concentrated cryoprotectant solutions cooled to below their glass transition points (Tg) (Cryobiology 27:492, 1990). This problem also occurs in heart valves cooled rapidly from -150C to -196C by immersion in LN2 (Adam, et al. Cryobiology 27:605, 1990). Work I conducted using cats with Leaf and Hixon at Alcor and work I did using rabbits for Soma, Inc. in Indianapolis, In. consistently demonstrated fracturing in many organ systems including the brain and spinal cord. Similarly, examination of humans perfused to a tissue concentration of 1M DMSO and 3M glycerol demonstrated a similar pattern of fracturing (although the brain was not examined in these patients) (Federowicz, et al., Cryonics 5:16,1984). Recent work by Fahy wherein rabbit kidneys were perfused with 15% VS4 and slowly cooled to and rewarmed from about 10C below Tg (to -135C) disclosed the presence of large fractures as assessed by reperfusion with plastic and corrosive dissolution of the organ to reveal a plastic vascular cast (Greg Fahy, personal communication). So how do we reconcile these results with Ettinger's seemingly contradictory results? Part of the problem is that Bob has never published any kind of detailed report on his work. In order to evaluate the CI results we need to know under what conditions the experiments were conducted. What was the approximate concentration of glycerol in the tissue as determined by measuring the glycerol concentration in the venous effluent (this is simply done with a hand-held refractometer)? What concentrations of agent were perfused in and what was exact composition of the vehicle solution? And very importantly, what were the cooling rates and warming rates? The CI model as I understand it from the incomplete reports Bob has written for the IMMORTALIST differs in some important ways from work by done Alcor and others -- ways which might explain why CI did not observe cracking. One important difference was the immediate perfusion of very high concentrations of glycerol (50% to 70%). This would result in severe cerebral dehydration pulling the brain away from the skull. Another potentially critical difference is the opening of a large window in the skull to facilitate observation of the brain during perfusion. Both Greg Fahy and Biopreservation have demonstrated that it is possible to cool brains to LN2 temperature (after vitrification) without cracking providing they are freed from any point of contact with the container they are in and that they are cooled slowly. However, this has little relevance to the model of human beings being cooled to below Tg with brains still in their heads and still connected to their spinal cords. Not knowing the terminal glycerol concentration in the CI brains is another serious problem in evaluating the research. Both work conducted here and at Alcor has demonstrated that animals NOT perfused with cryoprotectant develop very few fractures even after cooling to LN2 temperature. In fact, fracturing appears to get worse as the concentration of glass forming cryoprotectant increases. The conditions under which Alcor patients and the Alcor animal research were carried out have been exhaustively documented in both published case histories, autopsy reports and in animal research. In fact the text of the cat research paper was posted here (I believe nearly two years ago) in messages 1389 through 1392. Until we see a similar level of documentation from CI it is impossible to know what to make of their results. I would urge Bob to carefully document what was done. The fact that CI did NOT observe fracturing is potentially very important. But before we can know that, it will be necessary to reduplicate the work. And before we can do THAT we must know the conditions under which it was done. Currently we (Biopreservation) have two dogs at -90C which were perfused under conditions virtually identical to those employed by us (and Alcor) on human cryopreservation patients. These dogs were perfused to 6.5M glycerol under tightly controlled conditions (using a gradual addition system for introducing the glycerol). They were then cooled at a rate of about 4C/hr to -90C. We hope to take delivery on our whole body aluminum pods in the next few days and then begin cooling these dogs to -135C. After this we plan to rewarm the animals and reperfuse them initially with substrate bearing perfusate and finally with fixative so that we can do ultrastructural and histological evaluations. In other words, we plan to do exactly the kind of work Bon has commissioned in Ukraine. I applaud CI's decision to commission work in Ukraine and to attempt outside verification of their results. I would urge them to publish complete documentation once this work is done. While in no way trying to discourage the work CI is doing with Ukrainian scientists (I think such work is worthy and deserves support) I would point out that work can be done here quite cheaply too. I can get electron microscopy and histology done on an animal for $200. The advantage here is that I am working with a top flight technician whose shoulder I can look over as the results come in. I would also point out that work done at Creative and BPI is done for ZERO labor costs and done in an environment using state- of-the-art equipment and more to the point the exact techniques and personnel used to carry out human cryopreservations. Right now this work is not being supported at all by the cryonics community (unless you call me, Paul Wakfer, Saul Kent, Steve Harris and a few others *the cryonics community*!). I guess what I'm saying here is: by all means send CI money to support starving Ukrainian cryobiologists and get important cryonics research done. But by all means send us starving American cryobiologists some money too! Hopefully the next few BPI Tech Briefs will report on blood gas and cardiac output data obtained from the three dogs we have "transported" using Thumper support and then perfused with cryoprotectant and frozen. Once we have gross, histological, and ultrastructural data on these animals we will report it here as well. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=2676