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CI Research Report: 2007

by Yuri Pichugin, PhD, Staff Cryobiologist, Cryonics Institute

I was able to carry out 160 experiments for this year. The experiments were devoted to three main research projects.

1. Compounds that may improve results of long term ischemia.

Usual compounds that are used against short term ischemia (ie., anti-oxidants) could not work against long term (12-36 hours) cold storage of the brain. I decided to test some unusual compounds.

There is a method of preservation of blood vessel pieces with low concentrations of formaldehyde. I tested three compounds of the chemical class of aldehydes to verify the possibility of a so-called chemical anabiosis. The aldehydes I tested were formaldehyde, acetaldehyde, and glutaraldehyde. I used very low, non-toxic concentrations of these compounds.

A question of this study was could aldehydes reversibly modify fine cellular structures so that the cell become more resistant to long term cold ischemia? Could aldehydes reversibly "bind", "fix" weak links in molecular cellular structures that are subject to harmful effects of long term cold?

Upper parts of rats were perfused with various non-toxic concentrations of aldehydes at 0ºC and stored for 24 hours at 0ºC. Brain survival was evaluated by the usual hippocampal slice method I performed. No positive effect of the aldehydes was found.

2. Another project was the study of the 12CM ice blockers.

The 21CM employs 1% X-1000 and % Z-1000 ice blockers in its VMs.

I assume that cooling and warming rates for patients is 0.3º/min. I found that the minimal (critical) concentration of CI-VM-1 required to vitrify 20 ml volume without the ice blockers was 55% in cooling to −130ºC (and rewarming) at 0.3º/minute. 52% CI-VM-1 without ice blockers results in ice crystals. It was 52% CI-VM-1 with the ice blockers. 50% CI-VM-1 with the ice blockers results in ice crystals

The results of the toxicity test were as follows:

86.1% viability ± 5.8% for 55% concentration CI-VM-1 without ice blockers

89.6% viability ± 6.2%for 52% concentration CI-VM-1 with ice blockers

Toxicity assay was made using potassium/sodium ratios.

My experiments with the 21CM blockers showed that the blockers can decrease effective concentration of CI-VM-1 required for survival by around 3%. Ice blockers may be of value for organ cryopreservation, but they are not so important for cryonics.

There are many more harmful factors for cryonics patients than insignificant increasing toxicity due to a 3% increase in concentration of vitrification mixtures. I do not even take into account the permeation of the blockers through the blood brain barrier and cell membranes. To get the same effect, we can use 3% more of CI-VM-1 rather than 3% of the ice blockers (which would be much more expensive).

3. An important research project which I performed was a vitrification method for patients who have not experienced long ischemic times.

12-24 hour cold ischemia decreased survival of cerebral tissues to 40% of the control. We should create for CI members an improved CI vitrification method with the best possible cryopreservation of cerebral tissues. For this, first of all, potential CI patients should avoid long ischemic times by arranging for a standby team and by moving to Michigan when in a terminal condition.

A most significant problem of cryopreservation of whole brains is their severe dehydration during VM perfusion. All CryoProtective Agents (CPAs) have much slower penetration rates through the intact brain blood barrier than water has. Water leaves the brain much more rapidly than CPAs enters into the brain during CPA perfusion. As a result, severe dehydration of the brain occurred during perfusion. Severe dehydration is a very harmful factor because it dramatically increases CPA toxicity. CPAs penetrate into thin cerebral slices by simple diffusion and so the slices have no severe dehydration.

As an example of the great benefit that can be gained by using Substance X, 30% ethylene glycol (EG) is not toxic for cerebral tissues under certain conditions. Rat hippocampal slices that were exposed to 30% EG at 0ºC had 100% survival according to the K/Na ratio assay. Rat brains that were perfused with 30% EG at 0ºC without Substance X only had around 40% survival. The brains had about 35% dehydration. When I modified the rat BBB with Substance X, the brains retained their normal volumes during perfusion. As a result, the rat brains that were perfused with 30% EG at 0ºC with proper concentration of Substance X had 100% survival!

The main task of the project was to modify the BBB so that the brain remains its normal volume during VM perfusion. I studied twelve compounds of all the four chemical sub-classes of the class to which Substance X is belong. Four of twelve compounds showed good desirable activity. I describe them as modifiers of the blood brain barrier or BBB modifiers.

I tested the better modifiers on sheep brains. The sheep brains retained brain volume during VM perfusion as well as the rat brains had done. The best one of the good four modifiers was selected for the new CI cryopreservation method for patients who have not experienced long periods of ischemia.

It makes no sense to use BBB modifiers for who have experienced long periods of ischemia because ischemia destroys the BBB so that the brain does not have severe dehydration. I determined limits of cold ischemia duration for the optimal use of the BBB modifiers. Full strength of the best BBB modifier should be used for VM perfusion of rat and sheep brains until 9 hour cold ischemia. We should use only half strength of the modifier between 9 hours of cold ischemia and 18 hours of cold ischemia. We should not use any BBB modifiers for VM perfusion of brains after 18 hour cold ischemia because they can cause tissue edema.

All the known cryopreservation methods result in very poor survival of cerebral cells of whole brains after their cryopreservation. This new CI method significantly increases survival of cerebral cells. The proper use of the method may preserve about 80% of cells after cryopreservation of whole brains. CI submitted a provisional patent application for the method.