X-Message-Number: 4705
From: 
Date: Wed, 2 Aug 1995 16:43:02 -0700
Subject: Trans Time newsletter   SCI.CRYONICS

THE TRANS TIMES
Life Extension through Cryonic Suspension
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Volume 4 Number 4                                     August 1995


      Determining Electrical Activity in Frozen-thawed Brains

                     by Hal Sternberg, Ph.D.

We have made several recent observations in our laboratory 
concerning low-temperature preservation of mammalian tissues. We 
are collaborating with a UC Berkeley neuro-electrophysiology 
laboratory to help us examine brain activity in animals. The goal 
is to detect neural viability that may not be noticeable by other 
attempts to observe function, such as breathing or reactivity. 

In preliminary studies we have clearly detected neural electro- 
activity in anesthetized hamsters. Upon over-anesthesia and 
death, the electro-activity comes to a halt soon after cardiac or 
respiratory insufficiency. This activity remains undetectable 
unless an excitatory neurotransmitter is injected near the 
placement of the brain probe. This injection will invoke a one- 
time burst of electric activity lasting about 10 seconds.

We also examined neural activity in chilled hamsters. It is 
important to keep in mind that such an animal is in a highly 
subdued state -- the animal does not move or feel pain, and is 
minimally responsive. Widespread neural activity is readily 
detectable in unanesthetized active animals. However, using our 
current equipment it is difficult to detect neural activity in 
very cold hamsters. The cold suppresses neural activity, so the 
cold hamsters are relatively inactive. The colder the animal, the 
less the activity.

Electronic activity is readily observed in animals that are 
cooled to only 20 degrees C, but at much colder temperatures this 
activity is not observable. Based on our very preliminary 
studies, it is my expectation that using standard equipment, one 
is unlikely to detect neural activity in animals that appear dead 
after being frozen.    

I expect that to approximate the extent of neural viability, we 
may need in-vitro brain tissue preparations and methods that 
detect single or grouped neuron firing. I have not yet been able 
to detect brain electrical activity in relatively uncompromised 
hamsters that are simply chilled too long and fail to recover 
when warmed, even after injecting excitatory neurochemicals. I am 
thus doubtful that we will detect a response in post-frozen 
animals. Nonetheless, we will continue our attempts to develop an 
approach to assess neural activity and viability in post-frozen 
animals.    

Unfrozen glycerolized hamsters

We have also made some new observations in reviving hamsters 
after cryoprotection without freezing. We have tested different 
approaches to glycerolizing hamsters. We use up to 15% glycerol 
in the perfusate. While we have found glycerol to be very 
effective in cryopreservation of single independent cells, skin 
and cardiac myocytes, it certainly is not healthy. Glycerol tends 
to destroy tissues. While it is not easy to assess how it 
destroys intact organized tissues, we have an indication from 
certain observations. Upon histologic evaluation it may cause 
some sloughing of endothelial cells from blood vessels. Glycerol 
also causes dehydration of tissues, and some cell nuclei become 
condensed and necrotic. Under gross observation, lung tissue 
exposed to glycerol becomes hepatized and glycerol causes severe 
leakiness of blood vessels. 

Based on observations of glycerolized animals that we 
subsequently revived, I expect that if the tissue extracellular 
space of the brain or lungs is, at any time, saturated with much 
over 3% glycerol, it is lethal. Further, after death the animal's 
circulatory system that delivered the glycerol continually 
becomes increasingly closed off to perfusion. Thus, some tissues 
that receive glycerol cannot be readily accessed to subsequently 
wash out the glycerol.   

Further research

We are currently further evaluating combinations of 
cryoprotectants. While single cell viability seems well preserved 
with glycerol, the integrity of cellular organization, including 
cell-cell interactions and cell matrix interactions, may require 
other cryoprotective measures.

________________________________________________________________

The above is the first two pages of this ten page newsletter. It 
is too tedious to convert the typesetting of the whole newsletter 
to ASCII for posting here.  If you wish to receive a free printed 
copy of this newsletter, just send me your surface mail address.

Art Quaife, President
Trans Time, Inc.


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