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CI Research Report: 2004

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

For my third research year at the Cryonics Institute I was able to carry out 112 experiments.

In 2003 I elaborated the best vitrification method for rat hippocampal slices. This new method allows preservation on average of 85% cells of the brain slices after slow cooling to −130ºC. However, application of this method to whole brains met a big obstacle in a form of strong dehydration of the brains during cryoprotective perfusion. The brain lost 40 to 60% of baseline volume and it could not be restored by even very long term perfusion. The excessive cerebral dehydration was a harmful factor. The cause of this problem was a very low permeability of the blood-brain barrier for cryoprotective agents. This problem had not been solved in science prior to my work. I did not have such a problem with thin brain slices because they could be saturated with vitrification mixtures by simple diffusion.

I was apparently first in the world to find compounds that could completely open the blood-brain barrier for high cryoprotectant concentrations. So, now I can saturate the whole brain with vitrification mixtures without dehydration. It is a great discovery for science and cryonics technology. I got nearly 100% cell survival after perfusion of the adult rat brains with 40% ethylene glycol with opening the blood-brain barrier and I had only 30% cell survival in the same experimental conditions but without opening the barrier. It is a big difference!

I tried to adapt the new vitrification method for its near future use for CI patients in real conditions of funeral homes. For the brain slices, I had the best results saturating the slices with the best vitrification mixture at −25ºC for the optimal time period 15 minutes. Brain tissue survival strongly depended on exposure time and temperature. The funeral directors cannot use −25ºC but only 0ºC for patient perfusion. The use of 0ºC instead of −25ºC to perfuse whole rat brains with 65% vitrification mixture for the optimal time resulted in average 45% cell survival only. The cell survival will average only 26% if exposure time is 30 minutes instead of 15 minutes at exposure temperature 0ºC. The time period for complete saturation of patients’ brains with vitrification mixture will usually be longer than the optimal one. This will also decrease tissue survival.

I also studied the influence of warm ischemia on viability of rat brain slices. Rats after their death were subjected to 10 minutes, 30 minutes, and 60 minutes exposures at 22ºC and then kept in a refrigerator at 0ºC for 8 hours. Brain slice viability was 95% for 10 minutes of warm ischemia. It was 60% for 30 minutes of warm ischemia and only 40% for 60 minutes of warm exposure. Warm ischemia is a much more harmful factor than cold ischemia. A realistic or typical post-mortem time period for patients may be 60 minutes of warm ischemia and 8 to 12 hours of cold ischemia. My experiments with the real post-mortem time period demonstrated that brain tissue survival was only 7% according to the K/Na ratio assay after perfusion of the rat heads with the best vitrification mixture at 0ºC.

The K/Na ratio assay is a very reliable test of cell and tissue viability. Progress in cryonics research cannot be possible without the use of this evaluation method. But the very sensitive, functional K/Na ratio assay cannot well work in some cases and so we need to use structural, morphological evaluation methods such as light and electron microscopy. However, these morphological evaluation methods are much more expensive, more complicated, less available, more unclear than the K/Na assay. We have already received some light and electron microscopy results from Florida and Canadian laboratories, but we have some problems in this field. This work will be continued to obtain clearer results.

Proper perfusion of real patients with vitrification mixtures as well as with glycerol is the most difficult problem for cryonics because of bad condition of the cardiovascular system for most patients and some other causes. Especially, it is true for a patient’s body. For now, it is impossible to saturate the patient’s body with vitrification mixtures to get its uniform and stable vitrification. It is a reason why Alcor does not offer vitrification of the patient’s body. I think the Cryonics Institute should also not try to vitrify the body at present, but use a different cryoprotectant which will still show improvement over previous methods. Cost of all perfusion solutions for a patient’s head will be about $50 dollars only. Duration of the head perfusion will be about 2 hours.

There will be no standard, typical cases for the patients in order to use a fixed, standard amount of vitrification mixture to get uniform and stable vitrification. My experiments with sheep heads showed that we need to control the adequacy of brain saturation every time. For this, I tried to use burr holes in the skull and measuring refractive indexes of fluid from the holes in order to know complete saturation of the brains with vitrification mixture to get stable vitrification. This work is still in progress. It will be very useful for us to try the vitrification method on a CI patient before an official announcement that the Cryonics Institute offers CI members the vitrification method.

I have plans of some subsequent improvements of the vitrification method. I will design and make a cooling chamber that enables to perform vitrification perfusion of a patient’s head at −25ºC. I will elaborate a procedure of faster cooling a patient’s head through its blood vessels. I will propose to design and build a more perfect and reliable thermocontainers for uniform cooling of patients with very slow cooling rates from −130ºC to liquid nitrogen temperature to avoid cracking the patients’ brains.

I have a lot of experimental materials I would like to publish in scientific journals. But for now I may not do this because we have to make our decision whether we will take patents or not. In any case, I would like to offer some research laboratories and brain tissue banks to demonstrate our achievements in the field of cryopreservation of animal cerebral slices and whole brains. Also, I would like to offer Alcor to compare the CI vitrification method with Alcor’s vitrification method using rats.