X-Message-Number: 1441 From: Ben Best <> Date: Sun, 13 Dec 1992 19:00:00 -0500 Subject: cryoprotectant issues ERRATA: In my previous posting concerning freezing damage I stated that DMSO toxicity varies inversely with temperature, when I meant that DMSO toxicity varies DIRECTLY with temperature. I am delighted that Mike Darwin has chosen to respond to my "Freezing Damage" posting in such detail. And I am delighted in anticipation that I can bounce my speculations off of his vast fund of knowledge and experience. I will therefore address my remarks to Mike. But because I hope my remarks are of interest to others, I will explain myself in greater detail than would be necessary if I really were only addressing Mike. Reminder of acronyms: CCP -- Cat CryoPreservation study by Darwin, Hixon and Leaf DCF -- December 1991 article in CRYONICS magazine by greg Fahy I agree that the vision of nanotechnology has lured too many people into a smug belief that massive autolytic and freezing damage can occur without destruction of critical structures. But even if Dora Kent's head had received perfect suspended animation, nanotechnology will be necessary (although perhaps not even sufficient) to reverse the effects of Alzheimer's Disease, to undo the effects of aging, and to construct a new body. If I die, I hope to be very old -- and I will not expect to be reanimated until the ravages of old age are reparable and reversible. I think that the Smith Criterion is more than a "useful first approximation" to acceptable levels of freezing damage. It was no accident that Greg Fahy selected 3.72 Molar (27.2%) glycerol concentration for his DCF rabbit brain experiment. I should think 3.72 Molar should be the theoretical MINIMUM acceptable glycerol concentration under ideal conditions (and CCP should be evaluated in the context of its 3 Molar target concentration, knowing this). I agree with Paul Wakfer that 3-decimal place accuracy is ludicrous under such messy conditions as perfusion of the brain. Greg's "3.72 Molar" is virtually a *codeword* for the Smith Criterion. I think 4 Molar would be a suitable target concentration to fulfill the Smith Criterion under the best conditions. But applying the Smith Criterion to glycerol implies that glycerol perfuses perfectly into cell bodies -- while the significant observed dehydration implies that glycerol is NOT perfusing into cells well -- and is, in fact, osmotically drawing water out of cells. An idea cryoprotectant for the brain will readily perfuse the blood-brain barrier as well as cell bodies in the grey and white matter. I think this implies a cryoprotectant that is both hydrophobic (lipophilic) and hydrophilic (lipophobic) and has a low molecular weight. This could mean a suspension (in the pharmaceutical sense) like milk or soapy water. It could also mean molecules having both hydrophilic and hydrophobic properties, like glycerol and (especially) DMSO. My advocacy of DMSO is not intended to preclude the use of glycerol. DMSO [(CH3)2SO] is known to perfuse better than glycerol [(CH2OH)2CHOH], possibly in part because of its smaller molecular weight (78 versus 92) and in part because of the lipophilic methyl groups. Nonetheless, DMSO is a much more reactive molecule. In fact, it is used as a reagent based on both its ability to transfer oxygen, and its ability to transfer a methyl group. At lower temperature, however, this reactivity is greatly lessened and its cryoprotectant properties can be exploited. For this reason, in freezing 8-celled human embryos, glycerol is used initially and DMSO is only introduced at low temperature. The value of DMSO as a cryoprotectant cannot be viewed independently of the temperature at which it is introduced. Similarly, rewarming tissues cryoprotected with DMSO for microscopic examination is likely to result in DMSO chemical activation unless the DMSO is carefully removed. Nonetheless, Greg Fahy included DMSO in the cocktail with which he perfused kidneys at above-freezing temperatures (glycerol excluded), with 100% viability. How can this be accounted-for in light of the other evidence? If DMSO causes interstitial edema in ischemically injured patients, then it shouldn't be used for such patients. But why deprive others of possible benefit? Shouldn't suspension protocol be tailored to the needs of the patient? Suda's reference in BRAIN RESEARCH 70 (1974), p527-531 is to a paper "In preparation" which (as far as I can tell by searching the CITATION INDEX) was never published. (I think Suda was at the end of his career -- maybe he even died before publication.) We know nothing of his methods in comparing DMSO with glycerol, and we know lots about his shortcomings in the experiments he did publish (relatively low (15%) glycerol concentrations, inadequate washing of glycerol on re-warming, failure to include nutrient in the perfusion fluid, etc.). Even if it is true that glycerol is superior to DMSO under all conditions, it might also be true that both glycerol and DMSO in combination is superior to either one alone. I don't mean to sound like I am stidently advocating the use of DMSO. My objective is clarification, more than advocacy. Please explain what you mean when you say that the blood-brain barrier (BBB) can be "osmotically opened". I noticed that CRYONICS magazine reported a 6.02 Molar glycerol concentration in the Glennie suspension. What was the glycerol concentration on the cerebral side of the BBB? If glycerol is not perfusing cells, isn't there a danger that such high glycerol concentrations are simply causing more dehydration damage? Greg's biochemical content was on DCF page 14, upper left corner. -- Ben Best (ben.best%) -- Canada Remote Systems - Toronto, Ontario World's Largest PCBOARD System - 416-629-7000/629-7044 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=1441