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
 
 
 
 
 


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