X-Message-Number: 33156 From: Date: Tue, 28 Dec 2010 19:31:02 EST Subject: Scoring Cases Content-Language: en From: Gerald Monroe <_ (mailto:) > Date: Tue, 28 Dec 2010 06:12:54 -0600 Subject: Re: CryoNet #33145 - #33149 --0015175cba0689cf7004987760eb >>One quick addendum, Mike : the Arrhenius equation is half the story for the case of near 0 degree C transport. What about the rate of diffusion? In living cells, diffusion is VERY slow even at body temperature...it is not possible for protein sized molecules to get from the nucleus of a typical neuron down the axon to the synapse without help from powered microtubule transport. (diffusion is speeds are inversely proportional to the size and mass of the molecule that is diffusing, hence why ions and water can get around)>> Gerald, the example of the diffusion of skeletal proteins through the axoplasm to the synapses is not a good one, in that it is not representative of diffusion in the cytoplasm, and in the extracellular spaces. It's rather like a US general projecting the speed of movement of a Chinese mechanized division (equipped with all terrain vehicles) towards LA from San Diego based on the speed they could travel up the 5 Freeway at rush hour. Regular traffic is slowed to a crawl because it is constrained by the "tube' of the freeway, the requirement to obey certain traffic rules, and so on. Diffusion time for the lytic proteins is faster than you might expect, because many of them are small, and they are not constrained by the narrow tunnel of the axon. Take a look at the MW ranges for the small serine proteases, for example (90 kd). The matrix metalloproteinases (MMPs) are also quite mobile, and they are *widely distributed, but inactive.* But, apart from diffusional mobility, you must also consider the fact that many of the most destructive proteases and lipases are NOT confined to the lysosomes. In fact, they are distributed all over the cells and in the extracellular spaces but are INACTIVE. For instance, the MMPs are activated by sequential proteolysis of the propeptide blocking their active site, and this is brought about by cell-associated plasmin generation by urokinase-like plasminogen activator: both of which are produced in abundance in ischemia, and both of which are sufficiently active in hypothermia to cause injury; principally degradation of the basement membrane of the capillaries leading to increased edema during subsequent perfusion. In turn, the MMPs appear to be involved in the initiation of cascades of activation of gelatinase A, collagenase 3 and gelatinase B which are also implicated in basement membrane destruction. This just one example that comes to mind, there are many, many more and anyone who has actually perfused cold ischemic cryopatients - even ones stabilized ideally, but then transported on ice for 24 hours (and sometime less) will report that the amount of edema is dramatically different between such patients and those who arrested locally and were promptly perfused and subjected to deep cooling. The brain is one of the most sensitive (and dramatic) indicators of this cold ischemic injury. In a patient with very little or no cold ischemia the brain will shrink (dehydrate) dramatically during cryoprotective perfusion and it will STAY shrunken. In cold ischemic patients the initial volume reduction is followed by a rebound to normal volume and then swelling. This rebound is NOT equilibration of the cryoprotectants, rather it is EDEMA. You must also realize that white blood cells, particularly the neutrophils (PMNLs) will have been activated during the agonal period and/or by the disease process killing the patient - and if not then, then during ischemia - even very brief periods of ischemia of 10 or 15 minutes. These cells contain enormous reservoirs of hypohalous acids - principally sodium hypochlorite (household bleach). The MW of NaOCL- is ~74, and it diffuses quite rapidly at 0 deg C and is quite chemically reactive at that temperature, as well. What's more, the PMNL chlorinated oxidants destroy I 1- proteinases inhibitor activating the proteases. In effect, chlorinated oxidants create a zone of oxidized I 1-proteinase inhibitor that allows released elastase to attack and degrade endothelial cell membranes and cell-cell junctions. PMNL activation and degranulation are operational on a large scale in most slowly dying patients, and in patients who are not cooled very rapidly to ~5 deg C there will be continuing neutrophil degranulation with associated release of chlorinated (and, in the case of the eosinophils, brominated oxidantants). These highly destructive molecules are small, mobile and can directly degrade proteins into indistinguishable small "chunks' of amino acids. >>Once the oxygen is gone, and the ATP is all used up, the active transport mechanisms don't work. So the nasty lysozomal enzymes that might tear up the synapses where the memories are stored cannot go anywhere, on top of being limited in their rate of reaction. Free radicals can do some damage, but the pieces of damaged synapses are ALSO going to be inherently limited by the slow speed of diffusion. As long as the pieces are close enough to each other that they can be reassembled like a jigsaw puzzle, we can probably infer the original state of a synapse.>> This clearly does not happen, and the cold ischemic state is a dynamic place, in terms of both protease and lipase activity. Diffusion is slowed, but it is by no means halted, or reduced to biologically insignificant rates. And what's more, phase change in the membranes may open up pores large enough to allow the movement of small, lytic enzymes from the lysosomes... Mike Darwin Content-Type: text/html; charset="UTF-8" [ AUTOMATICALLY SKIPPING HTML ENCODING! ] Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=33156