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CI Research Report: 2005

by Dr. Yuri Pichgin

For my fourth research year at the Cryonics Institute I was able to carry out 170 experiments. My research work was done in the following directions:

1. An improvement of washout and perfusion solutions.

A buffer for blood washout solution was change in order to have a stable concentrate of washout solution for funeral directors outside Michigan.

Vehicle solution for vitrification mixture one (VM-1) was developed. 70% VM-1 prepared on this vehicle solution did not form any precipitation at 0oC and -20oC for a year.

70% VM-1 instead of 65% one was used to accelerate a saturation of the brain with VM and increase a probability of brain vitrification.

Substance B instead of Substance A was used as a better agent for opening the blood brain barrier (BBB). Concentration of Substance B that can completely open the rat BBB to saturate it with VM-1 without dehydration was found. However, strong edema of non-cerebral tissues of the head was observed. That can worsen a saturation of the brain with VM in some cases for real patients and so for now I did not recommend using those compounds for perfusion of CI patients. I need to perform more experiments on this research theme. This theme is one of priority research themes because some past experiments showed that strong brain dehydration can lessen survival of cerebral tissues by 30%.

The rat cerebral tissue that was completely saturated with 65% VM-1 can be stored at dry ice temperature (-76oC) for at least 48 hours without a decrease of viability after cryopreservation. So, the Cryonics Institute (CI) could transport their patients saturated with 65% VM-1 in dry ice, for example, from UK to USA for 48 hours. However, CI as well as Alcor does not have reliable methods of determination of a complete saturation of the whole human brain with VMs.

The CI vitrification method was applied to the two dogs and to the recent, CI 69th patient.

2. The problem of preservation of patients’ brains during 12-24 hour transportation from regions which are far from CI.

The procedure of perfusion of a patient’s brain with VM solutions is more complicated than the glycerol procedure and so it can be performed at CI only.

2.1 According to Dr. Suda’s experiments, brain tissues can be preserved at -20oC for a long time, probably, for months. However, none of other scientists was able to repeat those experiments for 40 years! Scientists also did not repeat Dr. Suda’s success for other organs those are more resistant to cold storage and freezing than the brain. It is a very important note because organ preservation for transplantation is a multi-millions business and so many scientists tried to repeat Dr. Suda’s success for other organs, I think. My attempts to repeat Dr. Suda’s experiments for the rat brain tissues were not successful, too. My best result was only 20% survival of rat brain tissues. Probably even such a brain tissue survival can produce a bioelectrical activity similar to the one that Dr. Suda recorded.

2.2 Dr. Seki’s experiment. This experiment is much less known for scientists than Dr. Suda experiment because Dr. Seki published only a brief communication of his experiment in the Cryobiology journal in December 1999. He wrote that he can preserve and resuscitate rat isolated hearts for10-26 days! Other scientists can preserve rat hearts at 4oC for 6-8 hours only. There was no publication on this subject by Dr. Seki or by other scientists since that time.

Dr. Seki dehydrated the rat hearts in a certain extent and kept them in a non-aqueous medium. I tried to use some compounds for some dehydration of rat brains to improve cold storage of the brains. However, I had no positive effect.

3. The study of warm and cold ischemia of rat brain tissues.

I give most interesting data of my experiments on this subject.

Brain slice viability was 85% of the control for 1 hour of warm (23oC) ischemia. It was 72% for 2 hours of warm ischemia and only 50% for 3 hours of warm exposure.

Brain slice viability was 80% of the control for 3 hours of cold (4oC) ischemia without warm ischemia. It was 74% for 6 hours of cold ischemia, 59% for 12 hours, 50% for 18 hours, 43% for 24 hours, and 0% for 48 hours of cold exposure. The brain tissues lost 50% of their viability for 3 hours of warm ischemia or for 18 hours of cold ischemia, so warm ischemia is 6 times more harmful for the brain tissues than cold ischemia!

A combination of 10 minute warm ischemia with 6 hours of cold ischemia resulted in 63% survival of the brain tissue. A combination of 1 hour warm ischemia with 23 hours of cold ischemia resulted in 32% survival of the brain tissue.

Another important problem is a quality of VM perfusion for organs with 12 – 24 hour cold ischemia. In this field I found an interesting, positive fact that the CI 69th patient’s head with 12 hour cold ischemia was perfused much better than the rat heads with the same cold ischemia, namely the rat heads had very strong edema during VM perfusion, but the patient’s head had no edema.

4. Testing organ preservation solutions on the rat brain tissues.

The length of time organs for transplant can be preserved outside of the body varies such as: the heart – lung is for 4-5 hours, the heart is for 6-8 hours, the lung is for 12 hours, the liver is 12-24 hours, and the kidney is 48-72 hours. There was no information about the brain because it does not use for transplantation. However, it is a well-known fact that cerebral tissues are most sensitive ones to any harmful factors.

I tested most used organ preservation solutions such as Viaspan (or Wisconsin university organ preservation solution), RPS-2 and its modifications (Renal Preservation Solution), MHP-2 (Mannitol - Hydroxyethyl starch – Perfusion solution; M. Darwin and et al, Alcor), Renasol H (Renal Solution with Hydroxyethyl starch; Dr. Fahy and et al, 21 CM), and New Organ Preservation Solution of Kyoto University (Chen F. and et al, Japan, 2004). None of these solutions showed a positive result for 12 or 24 hour cold (4oC) storage of the rat brains that were perfused with the solutions in comparison with the control, untreated rat brains.

5. Testing some compounds on rat brains to decrease negative effects of long time cold ischemia.

The June 2005 issue of Scientific American reported about "Suspended Animation" of mice by hydrogen sulfide gas (H2S). Some of cryonicists asked the question, Could hydrogen sulfide be an excellent additive to organ preservation solutions to prevent cold ischemia? Although Ben Best as the CI president a relatively long time before this publication made me urgent demands to improve the results of 12 – 24 hour cold storage of the brain tissue. I tested various H2S concentrations (200 mg/L to 2 mg/L) using rat brains that were perfused with these cold H2S solutions and kept at 2-4oC for 24 hours. H2S showed no positive effect in comparison with the control.

I also tested trehalose, N-acetylcystane, and deferoxamine to decrease negative effects of 24, 12 and 6 hour cold ischemia for rat brains. But I had no positive results.

I read many abstracts of scientific articles in which authors wrote that some compounds can, for example, prevent negative effects of 10 minute warm ischemia, but they cannot prevent 20 minute warm ischemia. I studied 3 hour cold storage of rat brains. H2S and deferoxamine demonstrated a very little positive effect for 3 hour cold storage of the rat brains. But we need to improve the results of 12-24 hour cold storage of the brain tissue in order to transport CI patients from far regions to CI.

There is a solution of the problem of warm and cold ischemia, namely CI potential patients can move to areas which are nearest to CI to get a best cryonics service without ischemia.

I was not able to fulfill some of my important past year plans on subsequent improvements of the CI vitrification method because I tried to implement the CI president Ben Best’s urgent demands to carry out experiments in order to improve the results of 12 – 24 – 36 hour cold storage of the brain tissue. First of all I would like to try elaborating VM perfusion of a patient’s head at -25oC and cooling it through blood vessels to -40oC or -60oC as fast as possible because brain tissue survival strongly depended on exposure time and temperature. I would like also to finish the study of the influence of brain dehydration on cerebral tissue survival to solve the question about the use of compounds for opening the blood brain barrier.