TOWARD BETTER VITRIFICATION

Dr. Yuri Pichugin, the Cryonics Institute's Director of Research, has been working on a different approach to vitrification. Elsewhere in this issue he has a brief report on preliminary encouraging results, with photos. I offer a bit of background here for the benefit of new readers especially; the photos are on the page with Dr. Pichugin's report. (He also has a longer report on his work last summer in the Ukraine and Russia.)

Vitrification means formation of a state that is called "vitreous" or "glassy" because, although it seems solid, it lacks the crystalline structure of ordinary solids and, depending on shape, may exhibit very slow flow or creep over long periods of time, something like tar in warm weather. In cryopreserved people, the potential advantage is that ice crystals are not formed, resulting in less damage to tissue.

(Cells do not burst as a result of freezing in almost any circumstances, because not only are animal cell walls generally elastic enough to accomodate a 10% expansion, but most of the ice is formed outside the cells. But there can still be mechanical damage to cell walls from nearby ice crystals and so on, which is avoided by vitrification.)

It must not be thought that freezing is totally destructive or leaves a patient in hopeless condition. Uncontrolled freezing does a great deal of damage, but still leaves many structures and even some functions intact, even in large specimens--that is why frozen tissue banks, including frozen brain banks, are valuable. Past CI methods, using a glycerolbased perfusate, have been proven (e.g. by light and electron microscopy) to leave much less damage than uncontrolled freezing. But vitrification holds potential promise of further advance, much less damage still.

At this point we must address the claims that other organizations already offer vitrification, or will license solutions and

procedures that result in vitrification. In particular, Alcor has now for some time applied a "vitrification" procedure to its neuro (head only) patients. But there are several problems.

First, the solutions and procedures developed by 21st Century Medicine and licensed to Alcor are secret, so we can't verify their degree of effectiveness. 21 CM patents are available, but they are very broad and perhaps outdated, and we don't know which parts of the available patents, if any, represent the current Alcor usage.

Second, to the best of our knowledge, no whole animal brain has ever been subjected to the procedure Alcor uses and then evaluated to show the results.

Third, based on statements by people at 21 CM and at Alcor, their procedures require very fast cooling to around - 130 C to achieve vitrification; then very slow cooling to liquid nitrogen temperature to avoid extensive cracking or shattering; then, if stored at liquid nitrogen temperature (as is currently being done), when the time comes for rewarming that will have to be done very rapidly to avoid devitrification (ice formation) on the way up. For best results their procedure should employ storage at around - 130 C, which would be more expensive and perhaps less reliable.

Again, we are not faulting Alcor for using its "vitrification" procedure on patients without full prior evaluation, because they believe the probable benefits justify it. Nor do we fautl Alcor for its secrecy, which we understand is required by their contract with 21 CM. But we don't want to adopt a new procedure without actually testing and evaluating it with whole animal brains. We hope to reach that stage within the next six months or so with Dr. Pichugin's new approach.

That approach, if it proves out completely in the months ahead, avoids the problems mentioned above. Cooling and warming can be at slow or moderate rates, without any danger of failure to

achieve vitrification on the way down or danger of devitrification on the way up, and storage can be in our regular cryostats in liquid nitrogen. And we will not have the expense of the elaborate procedures believed needed by Alcor to achieve very fast cooling in the first phase.

Also in this issue of The Immortalist Dr. Pichugin has two reports, one following, a long one on his trip last summer to the Ukraine and Russia, and a brief note on recent vitrification experiments with photos.

Dr. Pichugin's Professional Business Trip in the Ukraine and Russia.

I will not mention names of researches in the report because they are still afraid of public links with cryonics.

The experiments were at the Kharkov Institute for Problems of Cryobiology and Cryomedicine, the Ukraine.

My main purposes to perform some experiments at the institute were to use

rabbit hippocampal slices for cryopreservation and bioelectrical activity

(BEA) tests because this was not available for CI in the USA.

Unfortunately the institute's researchers were not ready to record bioelectrical activity of brain slices by a correct method.

I need to use another incubation chamber for rabbit brain slices than one for rat brain slices because rabbit brain slices were 2-2.5 times larger than rat brain slices. I designed and made two portable incubation chambers for the rabbit slices before my trip to the Ukraine. I tested the chambers using rat hippocampal slices in the research laboratory of the Cryonics Institute. The design for the first chamber and for the second chamber was the same.

The rat hippocampal slices were in an interface position in the first chamber and in a submersed position in the second chamber. All the brain slices were untreated. The interface position for the brain slices was much better than the submersed position as well as for the standard Oslo chamber (The Fine Tool Inc. , US) for rat brain slices. The viability of the rat and rabbit brain slices in the interface position in the portable chamber was good.

The slices were prepared according to the standard procedure for rat hippocampal slices. but the portable incubation chambers were used. It was more difficult to work with the rabbit hippocampal slices because they had larger sizes than the rat hippocampal slices and so they can be more damaged by manipulations. The protocol of saturation of the rabbit slices with glycerol was the same as I usually used it for rat brain slices.

The control rabbit hippocampal slices had 2.12 of average K/Na ratio. The rat hippocampal slices usually had 3.0 of average K/Na ratio. This difference may be explained by the larger sizes of rabbit hippocampal slices but less number of live cells in the rabbit hippocampal slices. However a resistance of the rabbit hippocampal cells to toxicity of 40% glycerol (58% survival) was almost the same as for the rat hippocampal cells (62% survival).

The protocol of freezing of the rabbit slices with glycerol was the same as 1 usually used it for rat brain slices and it was described in the past issues of The Immortalist. After the saturatio!; with zl~cerol_ the slices were kept in a freezer (-20°C) for

20 hours and then they were warmed and washed from glycerol. A survival of the rabbit brain slices was only 11% of the control. I carried out similar experiments with adult rat hippocampal slices having the similar results as well. Thus, a resistance of the adult rabbit hippocampal slices to freezing was similar as it was for the adult rat hippocampal slices.

1 performed Dr. Suda's experiments with very young rat hippocampal slices in the Harvard Medical School. The survival of the slices was 42% of K/Na ratio of the control brain slices. The hippocampal slices from very young rats were more resistant to freezing than adult rat or rabbit hippocampal slices

according to K/Na ratio assay.

I described the experiments in details for Dr. Fahy and Dr. Wowk (21 st Century Medicine, Inc.) who decided to subscribe The Immortalist in order to read my scientific reports.

1 worked in the Institute of Higher Nervous Activity and Neurophysiology, the Russian Academy of Sciences, Moscow.

It seems to me all talent scientists have already left Russia and the Ukraine and state research institutes have very old and bad equipment.

There were many difficulties:

1. Dr. X and a Researcher who worked with me did not know their potentials recording system good enough because, most likely, they did not work using it for long time.

2.The Researcher used a vibrating tome to cut brain tissues into thin slices and have no tissue chopper. It is much more difficult to work with vibrating tomes than with choppers. However vibrating tomes can give better brain slices than choppers but when a very skilled experimentalist employed

them. The Researcher was not able to cut rat brain tissues into slices with the same thickness and so this gave varying results for K/Na ratio as well as for BEA. The Researcher usually obtained 2-5 slices from a rat brain only! It was enough for their usual work, but it was not enough for my usual work at all.

3. The recording system and other equipment were very old (about 15-20 years old!) and so they very often were out of order.

Nevertheless my theoretical conversations with Dr. X about the method of measurement of synaptic transmissions and my practical observation of work of the recording system were very useful for me.

Dr. X knows literature in the field of neuroelectrophysiology very well. He tried to convince me that synaptic connections can be damaged by various factors before all other biomicrostructures in brain tissues. To evaluate the state of synaptic connections quantitatively, some researchers record evoked population spikes, but others prefer to record evoked field potentials as a more stable indicator of synaptic transmissions. I have no time to describe details of these methods. I will describe the practical use of the second method that was performed by The Researcher.

The Researcher recorded field potentials in the CAI region of rat hippocampal slices which is the least resistant region and in dentate gyrus which is the most resistant area. We had no time to test the third region, CA3.

The Researcher used the standard recording method, but results were very dependent upon positions of stimulating and recording electrodes. It is necessary to have a good skill in order to perform this work properly. Stimulating current was 500 microAmpere and duration of it was 500 microseconds and 1000 mcSec. The wolfram bipolar concentric electrode (diameter was 200 mcM) was used as a stimulating electrode. The recording electrode was a thin pipette, pulled from borosilicate glass capillary tubing, was filled with ACSF.

If a region of hippocampal has a good state of synaptic connections, increasing of current power via a stimulating electrode must increase an amplitude of a field potential which reflects synaptic transmissions. In other words, an increase of an amplitude of a field potential must be proportional stimulating current power.

As usual, a magnitude of the amplitude for the first field potential from the stimulation by 500 mcA for 500 mcSec can increase two times for the second field potential from the stimulation by 500 mcA for 1000 mcSec. An example. The amplitude for stimulating current 500 mcA for 500 mcSec was 999.3 units. After the stimulation with current 500 mcA for 1000 mcSec, the amplitude became 1435.9 units. The ratio of the first amplitude to the second amplitude is 1.44. 1 will not

give magnitudes of amplitudes but I will give ratios of amplitudes for 1000 mcSec to amplitudes for 500 mcSec.

So, a good healthy hippocampal slice should have the amplitudes ratio 2.00. Let us assume that the ratio of a brain slice after some treatments or influences is 1.44. The ratio 2.00 is accepted for 100% viability (synaptic transmissions) in a region of a hippocampal slice. The ratio of 1.44 to 2.00 is 72% survival (72% of integrity for synaptic transmissions).

A selection of healthy control hippocampal slices for experiments is a very important thing that was not performed by The Researcher because The Researcher was not able to prepare enough number of brain slices for this. It is correct to use healthy slices with the amplitudes ratio 2.00 in experiments only. This was not performed and so talking about another very good and very sensitive test of slice viability as a longterm potentiation (LTP) is not possible.

However the Researcher used 37oC in the BEA recorder cell as other scientists. The first slice had 1.92 K/Na. The second slices had 1.36. Mean was 1.64+/-0.40. BEA of the slices was absent! I think it was artifact of measuring BEA by The Researcher. The Researcher said to me that, maybe, the slices were too thick. They usually used 0.400 mm slices. However, BEA must be at least weak! The slices must have about 55% viability of average control slices (3.00 K/Na ratio).

A thickness of brain slices in all of the next experiments was 0.400 mm.The Researcher was able to prepared three rat hippocampal slices only.

One slice was taken for control. Two slices were used for the standard exposure with 40% glycerol.

The results:

No. Of samples K/Na CAI DG

1. control 0.63 0 1.34

2. 40% . 0.98 0 1.13

3. 40% GI 0.93 1.28 1.13

This is the ratios of the amplitudes in the CAI and in dentate gyrus (DG).

I never used the rat adult hippocampal slices by a thickness 0.400 mm before and so I did not know about K/Na ratios of the control slices. Later in Michigan I performed experiments with rat adult hippocampal slices by a thickness 0.400 mm. The average K/Na ratio of the 0.400 mm control slices was around 2.00. It was lower than the average K/Na ratio of the 0.475 mm control slices (about 3.00) because the live cells were less in the thinner brain slices. The average K/Na ratio of the 0.400 mm control slices in the Moscow experiments was 1.41 only but not about 2.00, maybe, due to the worse slice incubation.

Unfortunately it is not possible to obtain a reliable information from the experiment.

The Researcher was able to prepare three rat hippocampal slices only. They were used as control slices.

No. Of samples K/Na CAI DI.

1. control 1.63 0 1.01

2. control 1.60 1.44 1.38

3. control 1.29 1.15 1.07

Mean for K/Na was 1.51+/-0.19

Exposure of brain slices with 40% glycerol. It is the most important experiment of the entire business trip outside the USA.

The Researcher was able to prepared seven rat hippocampal slices (at last!).

No. Of samples K/Na CAI DG

1. control 1.12 1.07 0.57

2. control 1.23 1.43 1.26

3. control 1.36 0.76 1.34

Mean for K/Na was 1.24+/-0.12

About BEA of the slices before exposure with 40% glycerol. It is impossible to measure K+ and Na+ ions of the brain slices by the K+ - Na+ assay I used so that they were alive for the next step of experiments.

It is results of the amplitudes ratios for the slices before 40% glycerol exposure. It was performed to calculate % of slice survival of the slices after 40% glycerol exposure.

No. Of samples K/Na CAI DG

1. before 40% GI ? 2.85 1.06

2. before 40% GI ? 1.29 1.15

3. before 40% GI ? 1.78 1.27

4. before 40% GI ? 0.76 1.67

Mean is 1.67+/-0.89 for CAI and 1.28+/-0.27 for DG.

A very important note is that the numeration of slices (samples) did not correspond with the numeration for the samples of the same slices used for glycerol exposure because the slices were mixed on the mesh during the process but were not placed on the individual meshes to keep the slice numeration in order. We therefore use a mean of each function to calculate a result.

It is results of the amplitudes ratios for the slices before 40% glycerol exposure.

No. Of samples K/Na CAI DG

1. 40% GI 1.20 0 0

2. 40% GI 1.07 1.00 1.00

3. 40% GI 1.02 1.08 1.15

4. 40% GI 0.86 1.24 1.02

Mean is 1.04+/-0.14 for K/Na, 0.83+/-0.57 for CAI, and 0.79+/-0.53 for DG.

The synaptic transmissions in CAI after 40% glycerol was 50% of the initial synaptic transmissions in CA] before exposure with 40% glycerol.

The synaptic transmissions in DG after 40% glycerol is 62% of the initial synaptic transmissions in DG before exposure with 40% glycerol.

K/Na ratio of the brain slices after 40% glycerol is 84% of the control brain slices before exposure with 40% glycerol. Usually, it was 60 to 70%.

The Researcher checked long-term potentiation of the brain slices but only for after glycerol exposure. LTP was present but it looked worse than The Researcher observed in control slices in usual experiments. It was 30-40% of the control slices on The Researcher's opinion and experience.

We did not know more because The Researcher did not record LTP of the control slices before glycerol exposure of this experiment.

BEA can show viability or survival of neurons. The K/Na assay can show viability or survival of both neurons and glial cells. However it cannot tell one from the other. Ration of neurons to glial cells is 1 to 10 by number and 1 to I by volume. 50% of the total volume of brain cells is a volume of the neurons or the glial cells.

The volumetric ratio but not the numerical ratio influences on magnitude of K/Na ions. If K/Na ratio is more than 50%, it will mean that not only glial cells will be alive but also some amount of neurons will be alive too and vice versa.

My suppose based on the data of the experiment for a correlation between BEA and K/Na ratios of the rat hippocampal slices.

For CAI. If 50% of neurons in the CAI region is preserved, it is 25% of the total K/Na ratio of the hypothetical hippocampal slice because the portion of neurons for K/Na ratio is 50%. If all glial cells in the slice are preserved, it is 50% of the total K/Na ratio of the slice. The total sum of K/Na ratio is 75% of preserved cells. We have 82%! It is not a bad correlation.

For DG. If 62% of neurons in the DG region is preserved, it is 31% of the total K/Na ratio of the hypothetical hippocampal slice because the portion of neurons for K/Na ratio is 50%. If all glial cells in the slice are preserved, it is 50% of the total K/Na ratio of the slice. The total sum of K/Na ratio is 81% of preserved cells. We have 82%! It is a very good correlation! However good statistics were absent and so we cannot be sure of it 100%. I am about 50% sure only. It seems to me this method can work.

The Researcher was able to prepared five ratt hippocampal slices. All the slices were used for 40% glycerol exposure without a control for K/Na assay.

No. Of samples K/Na CAI DG

1. before 40% GI ? 1.17 1.25

2. before 40% GI ? 1.74 1.12

3. before 40% GI ? error 1.12

4. before 40% GI ? 1.59 1.36

5. before 40% GI ? error 1.22

<(Error)) means that magnitudes of the second amplitude were very great so that it was out of the limit of instrument recording. Most likely it happened because The Researcher placed the stimulating and recording electrodes too close each other.

Mean is 1.50+/-0.30 for CAI and 1.21+/-0.10 for DG.

Unfortunately the recording system for measurement of BEA was out of order again while the brain slices were finishing to recover from 40% glycerol exposure in the incubation chamber The Researcher used and so I have K/Na ratio of the slices without BEA.

A very important note is that the numeration of slices (samples) did not correspond with the numeration for the samples of the same slices used for glycerol exposure.

No. Of samples K/Na CAI DG

1. 40% GI 0.87 ? ?

2. 40% GI 0.80 ? ?

3. 40% GI 0.78 ? ?

4. 40% GI 0.77 ? ?

5. 40% GI 1.01 ? ?

Mean is 0.85+/-0.10 for K/Na.

The Researcher was able to prepare five rat hippocampal slices. However The Researcher said to me that all the slices were electrophysiologically dead! Most likely the recording system was out of order again. I took two slices of five to perform K - Na ratio assay later in USA.

No. Of samples K/Na CAI DG

1. control 1.54 ? ?

2. control 1.39 ? ?

Mean is 1.47+/-0.11

Some summarization. I tried to obtain more statistics from all the experiments.

For the control slices. The control slices of the experiment 3 had 1.51 K/Na ratio. It was 1.24 for the experiment 4 and 1.47 for the experiment 6. Mean is 1.41. I excluded the control slice of the experiment 2 because it was one and had too little K/Na ratio. It was a very bad slice.

For the slices after 40% glycerol exposure. The experiment 2, 0.96 K/Na ratio. The experiment 4, 1.04 K/Na ratio. The experiment 2, 0.85 K/Na ratio.

Mean is 0.95.

So, mean for slice survival after 40% glycerol exposure is 67%. Usually, it was 60 to 70% for my previous experiments in the CI lab.

Based on the data, the correlation between BEA and K/Na ratios of the rat hippocampal slices will look so.

For CAI. If 50% of neurons in the CAI region is preserved, it is 25% of the total K/Na ratio of the hypothetical hippocampal slice because the portion of neurons for K/Na ratio is 50%. If all glial cells in the slice are preserved, it is 50% of the total K/Na ratio of the slice. The total sum of K/Na ratio is 75% of preserved cells. We have 67%. It means that 75% - 67% _

8% of the glial cells must be dead additionally.

For DG. If 62% of neurons in the DG region is preserved, it is 31% of the total K/Na ratio of the hypothetical hippocampal slice because the portion of neurons for K/Na ratio is 50%. If

all glial cells in the slice are preserved, it is 50% of the total K/Na ratio of the slice. The total sum of K/Na ratio is 81% of preserved cells. We have 67%. It means that 82% - 67% = 15% of the glial cells must be dead additionally in this area.

Thus BEA was not directly proportional to K/Na ratios because the ratios were dependent upon not ion activity of neurons only but also upon ion activity of glial cells. It was an useful information for me, but I cannot publish the results in research journals because there was no enough statistics.

A VITRIFICATION NOTE FROM DR. PICHUGIN