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Cryonics Institute Research Report for 2003

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

For my second research year in the Cryonics Institute (CI) I was able to carry out 67 experiments with rat hippocampal slices performing 200 tasks, 9 experiments with live rat hearts, 18 experiments with perfusion of whole rat heads, and 8 experiments with perfusion of sheep heads.

The purpose of all the experiments was to create a vitrification method for CI. I may not report about the concrete results of the experiments because the institute will probably take a patent using the results. Combinations of all best cryoprotective agents were tested as mixtures for vitrification using live rat brain slices and the functional K/Na ratio assay. Several vitrification mixtures could cryopreserve the brain slices with about 75% survival. The best vitrification mixture could preserve the live brain slices with 85% survival after vitrification to −135º. The CI glycerol method gave only 20% brain slice survival in the best freezing conditions. 85% and 20% is a big difference.

I tried to test the best vitrification mixture for live rat hearts too. A heart can recover according to the principle: all or nothing. There will be coordinate heart beating or only some fibrillation which will stop during incubation of the heart. Unfortunately the rat hearts could not recover after using the best vitrification mixture even without deep cooling. All known cryoprotective agents are toxic for live organs in high, vitrifiable concentrations (55−70%), or they cannot protect the organs from freezing injuries in lower concentrations. Cryobiology cannot still create a cryopreservation method that can protect live organs such as hearts, kidneys, livers, and others with complete recovery for transplantation.

Today cryonics in contrast to modern cryobiology should not wait a completely perfect cryopreservation method because legal dead patients already have defects of human nature that resulted in their death and all after-effects should be cured of future medical technologies. 85% survival of brain tissue is a good result in comparison with the previous cryopreservation CI method and so we should elaborate the better method for whole patient brains and bodies. So, the vitrification method should be tested not only on brain slices but also on whole live animal brains.

When I stared to work with whole rat heads I had an obstacle in a form of the rat blood-brain barrier. The barrier was much less penetrable for cryoprotectants than the human and other mammals such as sheep, rabbits, cats, dogs and so on. My recent experiments with cryoprotectant perfusion of sheep fresh dead heads demonstrated that the sheep blood-brain barrier is more penetrable even for glycerol than the rat one. However, a high degree of brain dehydration was observed side by side with the better cryoprotectant penetration.

Unfortunately the cryonics institute was not allowed to work with live animals except rats without licenses. To get the licenses is too expensive for the Institute. Dead sheep heads are not good for this purpose. I need to use live rabbits in order to employ the sensitive functional K/Na ratio assay for live hippocampal slices. I can perfuse rabbit heads with cryoprotectants, cool them to −130º, keep at this temperature, rewarm, wash out them from cryoprotectants , and prepare hippocampal slices from the washed rabbit brains to evaluate their survival by K/Na ratio assay. I will be looking for a possibility to rent a small room at local universities which have the license to work with live rabbits legally.

My work with live rat brain slices was very useful and the results of the work did not lose their significance for future experiments with cryoprotectant perfusion of whole heads because cerebral cells of mammals are not practically different from a mammal to a mammal. If one can make experimental vitrification conditions for cerebral rabbit cells the same as for rat brain slices, cell survival for these cases could be the same in within experimental errors. It has been verified that cell survival in vitro (for brain slices) and cell survival in vivo (for a whole brain) were similar, for example, for toxic compounds or drugs, if the blood-brain barrier was sufficiantly good penetrable for these substances. My experiments with dehydration of rat brain slices by diffusion and with dehydration of whole rat brain tissue by glycerol or sucrose perfusion of the rat heads showed almost the same cell survival according to the K/Na ratio assay.

I have two sorts of plans for my future research. The first is an ideal plan for obtaining best results.

1. To determine degrees of dehydration and glycerol penetration for the postmortem human blood-brain barrier at 0º in the standard conditions. The human cadavers have to be relatively fresh, namely they should be kept at 0º not longer than 12−24 hours.
2. To determine what type of animal is closest to the human in the respect of their glycerol penetration through the blood-brain barriers. Rabbits, cats, and small dogs should be used. It will be a selection of a proper animal model for subsequent researches.
3. To select the best vitrification mixture of the good vitrification mixtures using the proper animal model and the K/Na ratio assay and electrophysiology.
4. To test the best vitrification mixture using relatively fresh dead human cadavers and trying vitrification of their heads at −130º.
5. To study a possibility to cool the human heads to −196º without cracking.

Fulfilling the plan in the USA would be very difficult and expensive for CI. I think it might be possible in Russia in collaboration with Russian cryonicists and scientists.

My second plan is to work with live rabbits in Michigan area if I can get a room with a license at a university.

1. To find a optimal method of introduction of the best vitrification mixtures into rabbit heads so that to avoid excessive brain dehydration because it is one of strong harmful factors. For this to determine optimal technical parameters of cryoprotectant perfusion: a rate of cryoprotectant administration, temperature and a rate of its decreasing, and a rate of increasing cryoprotectant concentrations in the vitrification mixtures.
2. To verify complete vitrification of the rabbit heads optimally saturated with the best vitrification mixture cooling them to −130º.
3. To find a optimal method of washout the rabbit heads from cryoprotectants and evaluating results with the use of the procedure of the brain slice preparation from the washed brains.
4. Based on the results, to calculate the technical parameters of the vitrification method for sheep and human heads.
5. To verify the parameters for sheep heads using the vitrification method for them practically.