X-Message-Number: 4982
Date: Fri, 13 Oct 1995 14:37:10 -0700 (PDT)
From: Doug Skrecky <>
Subject: more variations on a straight freeze

     Thanks again to Joseph Strout with his analysis of my variations on a
 straight freeze. He is correct that the hamburger technique is going to
 be much better than Mike Darwin's Blender technique. 
     However I would like to focus attention more on the top rated pin
 cushion technique as I feel this might substantially improve the quality
 of tissue preservation in cases of what might otherwise be lost causes. 
 The advantages of the pin cushion technique are: 
     1. It avoids fixation. 
     2. It avoids freezing without cryoprotectant. 
     3. It minimizes mechanical damage. 
     However I agree with Mike Darwin that speed is essential if
 acceptible results are to be obtained. Transport from a remote site using
 just ice water as a preservative is not going to be acceptible in most
 cases, especially where death is due to cardiovascular disease. (see
 other posting on this) A better procedure might be transport in salted or
 perhaps glycerolated ice water to depress the temperature below the
 freezing point. Here the minor ammount of ice crystal damage to the outer
 layers of the brain would be more than offset by enhanced preservation of
 the bulk of the interior of the brain. However if transit times are
 prolonged (>24 hrs) the best we could do would probably be a quick freeze
 using dry ice. With the following I am assuming that transit times are
 short. 

 Note: Permission is again here given for the following to be reprinted in
 any cryonics related publication that wishes to do so: 

                    VARIATIONS ON A STRAIGHT FREEZE
                           By Doug Skrecky

     With the circulatory system being unavailable in many cases for
 cryoprotective perfusion what are the available options? The brain could
 be dipped in cryoprotectant, but this would benefit only a very thin
 layer of tissue on the surface. The major arteries could be injected with
 cryoprotectant, but with the capillaries being blocked by coagulated
 blood no circulation is possible. Something better is needed. 
     Based on my expectations of the quality of the treated tissue I group
 the following 8 techniques in order of increasing merit: 

 1. (Worst) Blender technique: Homogenize brain in a blender with
 cryoprotectant. Pour into a brain shaped mold and freeze. 

 2. Embalming technique: Inject methanol into arteries and veins. Immerse
 brain in methanol for 24 hours at 0 C. (Methanol is the fastest
 penetrating fixative. It penetrates cell membranes 20 times as fast as
 glycerol. ref: The Journal of General Physiology Vol.62 714-736 1973)

 3. Hamburger technique: Pretend that cryoprotectant perfusion was done
 and do the usual slow freeze. The result is hamburger. 

 4. Microwave technique: Microwaves can rapidly penetrate and fix tissue. 
 Pack the brain with ice and place in a microwave with a turntable, which
 has been top rated by Consumer's Reports for uniformity of heating. Cook
 using pulses of radiation until well done. Treat brain with
 cryoprotectants by storing it in a concentrated cryoprotectant solution
 for a week. Store in freezer until needed. (Journal of Neuroscience
 Methods 53:81-85 1994)
 
 5. Freeze Substitution technique: Freeze brain slowly to -10 C to limit
 ice crystal damage. Pickel brain in an anhydrous silica gel desiccant
 packed alcohol based cryoprotectant solution till ice has all melted. 
 Baste in liquid nitrogen. (Note the main difference between this and the
 embalming technique is the temperature used.)

 6. Quick Freeze technique: Freeze brain slowly to -10 C to partly
 dehydrate cells and reduce subsequent intracellular ice crystal
 formation. Then dunk in liquid propane/butane cooled with dry ice. Rapid
 freezing limits extracellular ice crystal growth. Then slowly cool
 further with liquid nitrogen vapour to limit the formation of cracks due
 to increased tissue brittleness. (Biophysical Journal Vol.64 1908-1921
 1993 & Cryobiology Vol.31 506-515 1994)
 
 7. Salami technique: Cut the brain into slices 5 mm thick. 
 Cryoprotectants can rapidly diffuse into thin slices of tissue, thus
 preserving them well when they are frozen. The result is a Dagwood
 sandwich. (eg: see Cryobiology 24:120-134 1987)
 
 8. (Best) Pin Cushion technique: Immerse brain in a cryoprotectant bath. 
 Inject cryoprotectant into the brain via many very fine needles which are
 first slowly inserted into the brain, then slowly removed. Then do a
 quick freeze. 


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