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 _________________________________________________________________ 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. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4705