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. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4982