X-Message-Number: 2523 Date: 07 Jan 94 17:33:38 EST From: Mike Darwin <> Subject: SCI.CRYONICS Re: Perfusing Sheep Brains Tim Freeman has asked some questions about the CI sheep head research and the apparently contradictory findings to the results obtained by Alcor and Biopreservation, Inc. (BPI). I will try to answer his questions. First, the short answer is that I cannot tell you what the differences were/are because CI has never published an appropriate, technically detailed description of what they did. If you wish to know the results and the conditions under which the Alcor/Cryovita research was carried out please consult the draft of the paper "The effects of cryopreservation on the cat" by Darwin, et al. This was posted on CryoNet some time ago. I can repost it to a special reference file if you did not or cannot obtain a copy. I hope to have this paper in print (with accompanying light and electron micrographs) in Thomas Donaldson's journal NEUROCRYOBIOLOGY (providing, of course it is accepted). To BRIEFLY summarize our results: we found macroscopic cracking of the brain, spinal cord, and other organs, as well as poor ultrastructural preservation. These results confirmed similar earlier results (with respect to fracturing) observed in humans. The human cases were reported on in great detail including preparation method (i.e., perfusion cooling, etc.) and conditions under which rewarming and autopsy were carried out. The relevant cites for this information are as follows: Federowicz, M.G., et al., Postmortem examinmation of three cryonic suspension patients. Cryonics 5:16;1984. Leaf, J.D., Case Study KVM suspension. Cryonics August, 1981 pp 8-18. Leaf, J.D., Case report: two consecutive suspensions, a comparative study in experimental suspended animation. Cryonics 6:11;1985. Alcor Foundation, Histological study of a temporarily cryopreserved human. Cryonics #52, November, 1984 pp 13-31. The protocol by which these patients (and by which animals) was treated consisted of perfusion of multimolar concentrations of glycerol followed by slow cooling to -196C (see papers for details). Additionally, these same effects, i.e., fracturing upon cooling to below the glass transition point, have been observed with bulk solutions by several diffrent investigators over a long period of time starting with the paper by Kroener and Luyet: Kroener, C., and Luyet, B.J., Formation of cracks during the vitrification of glycerol solutions and disappearance of the cracks during rewarming. Biodynamica 10:1966 pp. 47-52. Rapatz, et al. also found cracking to occur in frog hearts cooled to below Tg after impregnation with multimolar concentrations of ethylene glycol. I do not have this cite at my fingertips but can dig it up if you REALLY want it. Rubinsky et al, did a mathematical analysis of this phenomenon: Thermal stresses in frozen organs. Cryobiology 17:1980 pp.66-73. I also have a number of unpublished micrographs by Suda, et al. documenting cracking on a histological level in cat brains trated with 15 (v/v) glycerol and frozen to -79C. Suda also observed oozing of blood from the pial surface during reperfusion and even noted the presence of small visible cracks. In personal communications Dr. Greg Fahy of the Red Cross Blood Research Lab in Bathesda, MD has also observed fracturing in organs (primarily the kidney and brain) treated with multimolar concentrations of cryoprotectant (glycerol, or DMSO-propylene glycol-formamide mixtures). For this reason Dr. Fahy is pursuing cryopreservation through vitrification at slightly below Tg, not at liquid nitrogen temperatures. Again, from personal communications with those in the tissue bank industry, I can report that viably preserved human heart valves will develop fractures (ruining them) if they are cooled to liquid nitrogen temperature. For this reason valves are stored in liquid nitrogen vapor. I believe there have been published reports about this problem and I will do a literature search to see what I come up with. As to what protocols are currently in use for human cryopreservation, I have sent to Kevin Brown a SUMMARY OF THE CRYOPRESERVATION PROTOCOL TO BE EMPLOYED BY BIOPRESERVATION, INC. (BPI) TO PREPARE BPI CLIENTS FOR CRYOPRESERVATION which he can make available for downloading to interested parties (it is over 50K and so is too long for posting here). Now, as to SPECULATION as to why Ettinger and more recently BioTime researchers have not observed fractures. Visible fracturing does not occur in my experience in brains poorly loaded with cryoprotectant. In fact, about 8 years ago Jerry Leaf and I subjected neonatal lambs (which had been sacrified in another, unrelated study) to straight freezing to liquid nitrogen temperature. We then tried cutting them up with various types of saws. The purpose of this research was to develop a safe, reliable, and RAPID technique for converting already frozen whole-body patients to neuro (the need was urgent since three patients would have been otherwise thawed if this were not an option). One of the things we noticed was the absence of macroscopic (i.e., visible) fractures in these animals even after sawing! In our work with cats and rabbits we found that a general rule of thumb was that the higher the concentration of cryoprotectant (glass former) the more fractures. Fractures do occur in straight frozen animals, but are smaller. Anyone who has looked at an ice cube in a tray will understand about fracturing in ice, even at relatively high subzero temperatures. So, one reason fracturing may not have occured is that the glycerol concentration in the tissue may not have been very high. Again, without data on arterial vs. venous glycerol concentration as a minimum, it is hard to evaluate the results. On the other hand, CI observed shrinkage of the brains in response to glycerolization and that is a reasonably good indicator of gross adequacy of distribution of the agent. Another reason fracturing may have been absent is the cutting of window (in the CI studies) in the skull combined with brain skrinkage. Isolated brains freed from contact with a container during cooling CAN be cooled down to -196C without fracturing or with minimal fracturing. This is particularly true if they are not handled in any way during the storage interval and are both cooled and rewarmed very slowly (G. Fahy, personal communication). Again, we have no grapic or other data on the thermal protocols used by CI and thus have no way to determine what is going on. One factor which I have come to understand contributes to the degree of fracturing is how much the system is disturbed mechanically after cooling. The cats, rabbits, and humans in which I have described fracturing were all moved (transferred from dewar to dewar or moved around in the dewar). I believe this is a reasonable model since humans WILL be handled similarly over the time course of their storage and in fact have been so far. Now as to the issue of using multimolar concentrations of agent. Fahy and others have conducted studies of brains treated with 4M glycerol and frozen to dry ice temperature after which they were freeze substituted (i.e., ice removed by solvent and fixed at dry ice temperature). The results were not encouraging: there was massive ice formation with cutting and disruption of the brain tissue at intervals of 10 to 30 microns. The pictures are devastating, and speak for themselves. Keep in mind that the lower your concentration of cryoprotectant the MORE ice you will see. I have seen claims and heard claims from BioTime to the contrary, but have seen no credible evidence to support them. I will believe these claims when I see EM and light microscopy documenting them. Reperfusion of brains with dye after freezing is easy and they look quite impressive. I can easily get beautifully dye-filled brains (pial vessels) after glycerolzing, freezing to -79C, and thawing. But that does NOT tell the tale of what is happening to the fine structure of the brain. Of course, if I cool to -196C with the brain in the head, fracturing will occur and prevent good distribution of dye solution. I have a projection slide from Suda showing cinematography of cat brains treated by various methods being reperfused with carbon black. Brains treated with plain old glycerol did beautifully by this test. But the histological injury was still there. SUMMARY: Responsible scientists working under well controlled conditions uniformly report gross mechanical injury from ice and fracturing using techniques broadly similar, and in some cases identical, to those in use by cryonicists today. My own conclusion is that the degree of histological and ultrastructural preservation we are achieving is dismal using existing cryopreservation techniques. We currently have a study underway using whole dogs perfused with ca. 7M glycerol which will look at a variety of parameters including the presence/absence of fractures during cooling to -135C or thereabouts. There will be detailed documentation of the results of these studies published along with any claims made. [ You can retrieve the BPI cryopreservation protocol, which Mike Darwin mention above, by sending email to me with the Subject line: "CRYOMSG 0028". - KQB ] Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=2523