X-Message-Number: 15693 Date: Mon, 19 Feb 2001 13:02:03 -0800 From: Hugh Hixon <> Subject: Terminal cryoprotectant concentration/Re: Message #15685 In Message #15685, David Pascal writes in part: "I've answered this already, but it's still worth examining, in that it's an excellent example of why CI people have not bothered to answer it. It is first of all, factually incorrect. 'On CryoNet it has been established that CI uses a more concentrated solution of glycerol than has been used by any other organization.' By whom? Jeff's 'mystery correspondents'? If (again) a person can expend the effort to type www.alcor.org (a mighty effort, I admit), one will notice that Alcor's web site states in a description of their procedures: "After the surgical access is completed, a cold (about 5oC, just above freezing) perfusate is recirculated through the patient while the cryoprotective agent (glycerol) is gradually increased from a 4% glycerol solution to 75%..." This used to be standard for all patients; I would imagine it's now standard for whole-body patients, though I've heard Alcor has yet another CPA (secret, of course, but that's OK - all secrets are fine, provided they're not CI's)." ------------------------------ Alcor apologises for a poorly written description. To be correct and somewhat more elaborate; Alcor's perfusion circuit incorporates a mixing reservoir, which provides perfusate to the arterial pump. 75% glycerol MHP-2 is added to the mixing reservoir as venous return from the patient is removed from the circulation in equal volume. The result is a cryoprotectant ramp from 4%v/v glycerol to a cutoff point of about 55%v/v glycerol (~7.5M). There are pauses and changes in the ramp rate to attempt to balance toxicity and time against cryoprotection. The concentrations of the components of MHP-2 (Alcor's organ preservation solution) is the same in both the 4% and the 75% glycerol perfusates to maintain electrolyte and substrate balances, and to prevent edema. The object of the ramp is to prevent the severe osmotic shrinkage of cells that occurs when tissues are suddenly exposed to high concentrations of osmotically active active agents such as glycerol (incidentally, this is how disinfectants such as alcohol and brine work, by sudden severe dehydration of cells). At the ramp rates that Alcor uses, the glycerol and water concentrations inside and outside the cells remain fairly close as the glycerol/water ratio is increased. That is, glycerol replaces water in the cells at near-equilibrium conditions. To simply hammer the tissues with 7.5M glycerol until the arterial and venous concentrations matched has always been considered a very bad idea. Because the brain is uniquely protected by the blood-brain barrier (BBB), cryoprotection of the brain is somewhat more complicated; the brain is progressively dehydated through the BBB until in a glycerol concentration range of about 1.8 - 2.2 M, cell shrinkage of the capillary lining physically tears apart the tight junctions between the cells and allows entry of the glycerol, after which cryoprotection of the brain proceeds as described above. This picture is complicated by the breakdown of the osmotic barrier of the capillary bed for verious reasons, particularly including long intervals between death and perfusion. Once the differential permeability of the capillary bed is lost, the result is severe tissue edema, and the brain swelling and pump pressure causes the brain to be extruded through the various openings in the brain case. Hugh Hixon Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=15693