X-Message-Number: 1404 From: Ben Best <> Date: Sun, 6 Dec 1992 19:00:00 -0500 Subject: freezing damage FREEZING DAMAGE: TRAGEDY AND HOPE My reaction to Dr. Merkle's article on the technical feasibility of cryonics is very similar to that of Hal Finney: What good is nanotechnological repair capabilities if the structural basis of memory and identity is destroyed by cryonic suspension procedures? I wrote Dr. Merkle a letter to this effect, but now that Mike Darwin has posted his cat cryopreservation studies to the net, I want to let loose with some comments, some of which have been brewing for over a year. I shall designate my primary references by three TLAs (Three Letter Acronyms): CCP, the Cat CryoPreservation study by Leaf, Darwin and Hixon; DCF, the December 1991 article in Cryonics magazine by Greg Fahy; and FCR, the Funding Cryonics Research comments in CRYONET messages 484, 485, 486, 490 and 498. As background, people should understand that the "Smith Criterion" has been central to cryonic procedures for many years. In the 1950s, Audrey Smith froze hamsters at temperatures not far from 0*C and achieved complete recovery of hamsters whose brains had been 60% (but not more) ice. The explanation for this is that with slow freezing, ice initially forms in the extracellular spaces and initially causes no damage. A 68% concentration of glycerol in water will vitrify (harden like glass) rather than form ice crystals at all temperatures down to liquid nitrogen (and below). But this solution is far too viscous to be used for cryonic suspension purposes. But based on the "Smith Criterion" that 60% ice formation in the brain will do no neurological damage, a 27.2% (3.72 Molar) glycerol concentration is deemed adequate to keep brain ice below 60% right down to liquid nitrogen temperature. In theory, ice crystals would form extracellularly while cell bodies and synapses were vitrified. Given this concern with the "Smith Criterion", why did the CCP study only attempt to achieve a 3 Molar target glycerol concentration? In any case, the evidence seems clear that perfusion fell below even this level -- massive dehydration was observed. It has been known for years, as the CCP paper acknowledges, that glycerol does NOT perfuse cells well and causes fatal dehydration of the brain at room temperature. WHY, then, has glycerol been used as a cryoprotectant in cryonics rather than DMSO, which has better perfusion properties? I have asked this question repeatedly and the only answers I have heard is that DMSO is more expensive and more toxic. Yet DMSO could be introduced at lower temperatures, since its toxicity varies inversely with temperature (and this is the procedure used in freezing human 8-celled embryos ot liquid nitrogen temperature). Moreover, the mechanisms of cryoprotectant toxicity are unknown and may involve nothing more than enzyme denaturation. If DMSO produces no STUCTURAL damage, the effects of toxicity may be more easily dealt with by reanimation technology than the damage to structure seen with glycerol. But in FCR, Greg Fahy reports 100% recovery of kidneys loaded and unloaded with a DMSO, formamide and propylene glycol cocktail at above- freezing temperatures.. Greg also says that he could customize a cocktail for cryonics suspensions to "reduce ice growth enough to preclude structural damage". Can there be any higher priority to cryonics research than to find a suitable replacement for glycerol? But why isn't DMSO being used NOW, given present knowledge? Greg Fahy used 3.72 Molar glycerol in his DCF electron microscope study of rabbit brains. He refers to remarkable tissue re-organization and complete recovery of biochemical function upon thawing, which seems to contradict the evident ultrastructure damage. CCP acknowledges that thawing damage, sectioning damage and fixation damage may have contributed to the ultrastructure damage observed. (And we can add dehydration damage and damage due to an inadequate target concentration.) FCR makes reference to Greg Fahy also rewarming and fixing with osmium tetroxide. Which raises the question, how much of this observed damage is really FREEZING damage? And why should rewarming be necessary when the method of choice for neurohistological ultrastructure studies is frozen microtome slices rather than chemical fixation? Given the superior ultrastructure preservation observed for the kidney and heart in CCP, have studies been made or how well cryoprotectants cross the blood brain barrier? Sodium lauryl sulfate is used to improve formaldehyde perfusion, maybe it could assist cryoprotectants as well. The final question I want to address is: What is the structural basis of memory and identity in the brain? As Thomas Donaldson has repeatedly alluded-to, long-term memory seems to be similar to embryological differentiation. Long-term memory seems to be associated with modification of synaptic connections and (perhaps more importantly for cryonicists) with modification of the neuronal DNA that programs the synaptic connections. The implication of this "redundancy" is that damage to synapses might not matter if the information encoding those synaptic connections is preserved in neuronal DNA. Future nanotechnology could reconstruct a brain from neuronal DNA alone, if this is true. Neuronal DNA appears to be very robust -- many tissues (including neurons from aborted human foetus' used for Parkinson's patients) have been frozen in liquid nitrogen temperature without losing tissue-type differentiation. My understanding is that the damage Greg Fahy observed was EXTRACELLULAR damage, meaning that neuronal DNA may have been mostly preserved. Damage does not mean destruction. Even current cryonic procedures may not be destructive. And titanic efforts may not be necessary for significant improvement. -- 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=1404