The procedure of perfusion of a patient’s brain with vitrification mixture (VM) solutions is more complicated than the glycerol procedure and so it can be performed at CI only. There are some of arguments to perform the VM perfusion procedure in Mr. Walsh’s funeral home. Funeral directors cannot carry out Mr. Walsh’s new surgery which is perfect for brain perfusion. The VM perfusion method requires using five - seven 9 liter tanks with VM solutions. Usually funeral homes have no refrigerator to store the tanks. However, the main problem is possible ice crystallization in patients’ brains perfused with VM solutions during 24 – 48 hour transportation in dry ice (-76ºC). According to my experiments, rat cerebral tissues that were completely saturated with 65% VM-1 can be stored at dry ice temperature for at least 48 hours without a decrease of viability after cryopreservation. So, 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 the complete saturation of all the regions of the human brain with final VM concentration. Thus, CI decides to ship unperfused patients in plain ice at 0-4ºC.
What biological alterations occur with human cerebral tissues during long-term cold storage at 0-4ºC? One may ask a question, why can’t the human organism function at low temperature like, for example, the frog organism? Humans are warm-blooded animals (homeotherms) and so the human brain cannot normally function at temperature below 36.6ºC. Brains of cold-blooded animals (poikilotherms: reptiles, insects, arachnids, amphibians and fish) can function at lower temperature up to 0ºC.
With a few exceptions, all mammals and birds are warm-blooded. Some warm-blooded animals, such as bears, groundhogs, gophers and bats hibernate during the cold winter. During hibernation these animals can drop their body temperatures by as much as 35-39°C. However, hibernators can have cold tolerance only in the hibernating stage in distinction from cold-blood animals. Hibernators in the active, non-hibernating stage are cold sensitive as well as non-hibernators.
Ben Best wrote about the state of mice like "suspended animation" that was induced by hydrogen sulfide (H2S) gas in his article Hydrogen Sulfide for Cryonics? (The Immortalist, v. 37, No.7-8, p. 14, 2005). I wrote about the experiments on testing H2S using rat brains below. Here I ask a question, could hydrogen sulfide induce the hibernation state for non-hibernating animals?
A transition from the active state to the hibernating state is controlled by genes which non-hibernators do not have. Brain tissues of hibernators gradually change their microstructure and biochemistry to have the hibernating state. Non-hibernators will not be able to have the hibernating state even if specific hormones are administrated in their bodies because they have no specific genes. So, H2S or any other substances cannot induce the hibernation state for non-hibernating animals.
I present some of scientific articles about a good cold resistance of hibernators’ brain tissues and whole brains.
1. Neuroscience 1990;38(3):591-608 Spontaneous and evoked neuronal activity was investigated extracellularly in slices, taken from the brain of the three groups of animals: hibernating ground squirrels, waking ground squirrels, and guinea-pigs. The difference between active and hibernating ground squirrels was preserved during retesting after deep and prolonged cooling of the slices. The experiments demonstrate stable activity neurons in the hibernating ground squirrels.
2. Brain Res Bull 1994;33(6):719-21. This paper describes preparation of the isolated brain of hibernating ground squirrel maintained by intraarterial perfusion. This technique allows a long-term survival (about 3 days) of the isolated brain of adult animals. The viability was assessed by extracellular investigation of stability of structure-specific electrical activity.
Dr. Pakhotin and et al described an interesting finding in Neuroreport 1997 May 6;8(7):1755-9 Treatment of guinea-pig’s (nonhibernator) cerebral slices with aspirin or indomethacin allowed them to survive overnight deep hypothermia.
I tried to verify this finding using rat hippocampal slices. However, I did not obtain a positive effect of indomethacin and aspirin on the slices at 2-4°C for 12 hours. There were no articles in which the positive effect of those compounds was found by other researchers. See results of my experiments with cold storage of rat brain slices below. It will be very unbelievable if humans have the hibernating state after taking some aspirin.
Let us directly consider the subject of the experimental study of ischemia of cerebral tissues. Ischemia is a restriction in blood supply. In cryonics we usually have a deal with complete ischemia, a lack of any blood supply for tissues. CI may use rats in experiments. I sometimes used fresh dead sheep heads from a local slaughterhouse. I remind that CI does not use live rats in experiments but only very fresh dead rats. The rats were killed by an extra amount of anesthetic (isoflurane) under very deep anesthesia. So, they could not feel any pain in this deanimation procedure. I do not try to reanimate the dead rats as whole organisms. I tried to reanimate, recover only rat cerebral tissues, namely thin hippocampal slices. According to modern neurophysiology, thin cerebral slices can completely be recovered in vitro. The cerebral slice technology can be used to evaluate survival of the whole brain.
The K/Na ratio assay is a most acceptable evaluation method of cerebral tissues for CI because it is a simple, reliable, inexpensive and, at the same time, sensitive functional method. However, this method cannot distinguish viability of neurons from viability of glial cells. It can evaluate general cellular survival. To evaluate survival of neurons, it is necessary to use electrophysiological methods. But electrophysiological equipment is not available for CI.
Any live cells have more potassium ions (K) on the inside of the cells than on the outside of and more sodium ions (Na) on the outside of the cells than on the inside of. This dynamic equilibration is a result of ion pumping and the integrity of cellular membranes. Concentrations of the ions on the inside and outside of the cells become equal when the cells are dead. Live rat hippocampal slices (0.475 mm thickness) have an average K/Na ratio that is equal to 3.0±0.4 according to the standard K/Na ratio assay. The complete dead slices have a 0.1 K/Na ratio. K/Na ratio of a cerebral slice is proportional to quantity of live cells in the slice and so it may be a quantitative indicator of viability, or survival of the cells.
Data on cerebral tissue survival after long time warm or cold ischemia obtained by functional evaluation methods are practically absent in the scientific literature and so the study of cerebral tissue ischemia is actual for cryonics and cryobiology.
1. The study of warm ischemia of rat cerebral tissues.
I present the results of my experiments in comparison with the results of Dr. Leonard and et al experiments (Fig. 1). The title of Dr. Leonard and et al article is The influence of postmortem delay on evoked hippocampal field potentials in the in vitro slice preparation (Exp Neurol 1991 Sep;113(3):373-7). The term "postmortem delay" has the same sense as the term "complete ischemia". Brains from old (9 months) rats were kept in situ at room temperature for delays (warm ischemia) of 5, 30, 60, 90, 120, or 180 min before dissection and slicing of the hippocampal tissue. The experimental conditions for the CI experiments were very close to the Dr. Leonard and et al experiments, but the rats were much younger (two months old).
Fig. 1 The dependence of viability of rat cerebral tissues upon the time periods of warm ischemia. 1 – the CI experiments; 2 – the Dr. Leonard and et al experiments (I present the data for the CA area from the article, the page 375, Fig. 3, "The percentage of slices with a population spike amplitude greater than 1 mV (elicited by approximately one-half maximal stimulus intensity) is plotted as a function of the anoxic delay).
Fig. 1 shows higher survival in the CI experiments than survival in the Dr. Leonard and et al experiments. Maybe, rat age can play a certain role: younger cerebral tissues more resistant to harmful factors than older ones. However, Dr. Leonard and et al and I employed different viability evaluation methods.
The results of my experiments show general cellular survival in rat hippocampal slices according to the K/Na ratio assay. Dr. Leonard and et al results reflect survival of neurons, but the researchers used an unusual method of expression of viability. The authors of the article wrote, "Viability was defined as tissue from which a population spike of at least 1.0 mV could be evoked" (the page 374). Usually electroneurophysiologists define neuronal viability, or survival in a certain area of the slice as a recorded population spike magnitude that was divided by an average control population spike magnitude expressed in %. For example, if recorded population spike in the CA area was 1.0 mV and the average population spike in the CA area of the control slices was 2.0 mV, viability was 50% of the control.
Dr. Leonard and et al expressed viability not as a percentage of survived neuronal cells in the certain area of the rat hippocampal slices but as a percentage of slices with more than 50% neuron viability. This viability evaluation method is a more restricted evaluation one and it can give some underestimated viability in comparison with the usual method. Dr. Leonard and et al used this method because their main task was to determine what number of slices might be used for electrophysiological experiments after different time period of warm ischemia. The researchers concluded "These results indicate that the delay between death and preparation of in vitro hippocampal slices is less important for obtaining physiologically viable slices than previously believed. These data also imply that meaningful electrophysiological information about premortem brain conditions may be inferred from nervous system tissue which is not available immediately after death."
There are different methods of evaluation of cerebral tissue viability. Scientists choose some of the methods in the dependence of their research purposes and of availability of proper experimental equipment. It should be noted that the method recording population spikes evaluates live neurons just in neuronal network. For example, if we inhibit synaptic activity in all live neurons in all the areas of a hippocampal slice the K/Na ratio assay as well as the vital dye evaluation methods demonstrates 100% viability or survival of the slice, but the population spike method shows 0% viability of the slice because the live neurons cannot function as neuronal network.
2. The study of cold ischemia of rat cerebral tissues.
Most CI members live outside Michigan and so they should be shipped to CI for cryopreservation by the CI vitrification method. A time period of a transportation of CI potential patients to CI may be 12-24 hours and longer for patients outside of the USA. So, it is important to study 12-48 hour cold (0-4°C) ischemia of cerebral tissues.
In this series of experiments, I tried to avoid warm ischemia completely in order to study the influence of just cold ischemia on survival of rat cerebral tissues. There were two sorts of the experiments:
1. Rat brains that were not treated with any compounds. To avoid a harmful effect of warm ischemia, the brains were very quickly (for a minute) removed from the skulls under cold physiological solution and cooled in the solution to 2-4°C. The brains were then placed in empty vials and stored at 2-4°C for 3 – 48 hours.
2. Upper parts of rats were quickly perfused with cold (0°C) aCSF through the aorta. The rat heads were cut, placed in plastic bags, and stored at 2-4°C for 3 – 48 hours. aCSF is Artificial Cerebral Spinal Fluid that is usually used for brain slice incubation.
After cold storage, hippocampal slices were prepared from the brains. Cell survival in the slices was evaluated by the K/Na ratio assay. The results of the experiments are in Fig. 2.
Fig. 2 The dependence of survival of rat cerebral tissues upon the time periods of cold ischemia. 1 – survival of hippocampal slices from the untreated rat brains; 2 – survival of hippocampal slices from the rat brains perfused with aCSF at 2-4oC;
Fig. 2 shows that survival of the slices from the untreated rat brains in general slightly higher than survival of the slices from the rat brains perfused with aCSF.
The brain tissues lost 50% of their viability for 3 hours of warm ischemia (Fig.1) or for 18 hours of cold ischemia (Fig. 2), so warm ischemia is 6 times more harmful for the brain tissues than cold ischemia.
I found an interesting article devoted to the study of "Prolonged whole-brain refrigeration with electrical and metabolic recovery" by Dr. White and et al (Nature 1966;209:1320-22). Canine brains were perfused with ice-cold Ringer’s lactate solution and stored at 2°C for various time periods from 2 hours to 15 days. "With brains stored for 2 h and 4 h, cerebral electrical activity was restored at intracerebral temperatures of 22°C or more. Prolonged maintenance perfusion of the rewarmed brain (34°C) for more than 6 h invariably resulted in the disappearance of electroencephalographic activity and development of irreversible oedema. … no recognizable cerebral electrical activity has been recorded from brains stored for 12 h or more." (the page 1322).
So, there was nearly normal electrical activity of the canine brains stored at 2°C for 4 hours. This approximates to 90 – 80% survival of the rat brains evaluated by the slice method and K/Na ratio assay.
It should be noted that even after 15 days of refrigeration, the brain can be perfused though with developing edema. I personally perfused sheep heads with cold (0°C) VM-1 solutions after 1-2 hour warm ischemia plus 22 – 24 hour cold ischemia successfully without edema of head and brain tissues.
3. The study of combination of warm and cold ischemia.
As cryonics practice demonstrated, it was sometimes impossible to avoid warm ischemia before cooling patients and shipping them at ice temperature. I tried to estimate the influence of 0-3 hour warm ischemia of rat brains on their survival after cold storage till 24 hours. For example, there was 2 hour warm storage plus 22 hour cold storage. The results of the experiments present in Fig. 3.
Fig. 3 Combination of 0-3 hour warm ischemia with 24-21 hour cold ischemia for the rat brains.
4. Testing some organ preservation solutions on rat brains and slices.
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 the 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 modification (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 aCSF. Composition of the solutions is in Table 1.
Table 1. Composition of cold storage solutions
aCSF RPS-2 m-v-RPS-2 Viaspan MHP-2 Renasol H
NaCl 124 25 40
NaHCO3 26 10 10 10
KCl 4 28.5 30 125 28.3
KH2PO4 1.4 25
K2HPO4*3 H2O 7.2 7
Mg2SO4*7 H2O 1.25 1.25 5 1.25 1
CaCl2 1.5 1
Glucose 10 180 230 5 10
Mannitol 170
Adenine-HCl 1 0.94 1
Adenosine 5
Allopurinol 1
Chondrioitin
sulfate A 0.1%
chlorpromazine 0.9 mg/l
Glutathione,
reduced 5 3 3 5
Decaglycerol 1.8%
Dexamethasone 16 mg/l 16 mg/l
HEPES 15 8
Tris-HCl 10
HES 50 g/l 50 g/l 50 g/l
Lactobionic acid 100
α-Lactose 45
Raffinose 30
Ribose 0.94
Sodium acetate 2
Tripotassium
citrate 18
aCSF RPS-2 m-v-RPS-2 Viaspan MHP-2 Renasol H
Notes: In general, concentration is expressed in mM/liter. M-v-RPS-2 is a modification of RPS-2 solution that is used by CI as a vehicle, carrier solution for VM-1.
Usually organs are perfused with a cold organ preservation solution for short time period to wash them out from blood. Continuous perfusion of organs with cold organ preservation solutions is more rarely used but it can give slightly better results. I therefore used rat hippocampal slices for 12 hour cold storage to test organ preservation solutions in conditions of continuous perfusion.
There is the typical procedure of treatment of rat brains with organ preservation solutions. Upper parts of rats were quickly perfused with a cold (0°C) organ preservation solution through the aorta. The rat heads were cut, placed in plastic bags, and stored at 2-4°C for 12 or 24 hours.
The results of the experiments with organ preservation solutions are in Table 2.
Table 1 Testing organ preservation solutions. The numerical data are given in % survival.
Time 12 hours 24 hours
Brains Slices Brains
Untreated 49±2 48±4
Solutions
aCSF 59±3 10±2 43±2
RPS-2 55±2 84±3 41±3
m-v-RPS-2 47±2 78±3 25±2
Viaspan 60±3 71±2 40±2
MHP-2 53±7 16±2
Renasol H 52±5 81±3
None of the solutions showed a positive result for 24 hour cold storage of the rat brains that were perfused with the solutions in comparison with the untreated brains. Some of the solutions demonstrated a very little positive effect for 12 hours. Famous Viaspan was not better than a CSF, although it much more expansive than a CSF. M-v-RPS-2 had worse results. However, it is used as CI vehicle solution but not as cold storage one.
The solutions except a CSF and MHP-2 showed higher survival for cerebral slices than for the whole brains. It is difficult for me to explain why these two solutions gave so bad results. In the past, I have performed some similar experiments with rat hippocampal slices using a CSF and had the same bad results. I had two hypotheses to explain why 4 of the 6 solutions in these experiments had higher survival for cerebral slices than for the whole brains.
The first hypothesis was that continuous perfusion could be better than static cold storage. It may be expectable that a rat brain perfused, for example, with cold RPS-2 for 12 hours will have survival close to 84% as for cerebral slices but not 55% (see Table 2). To verify this hypothesis, I performed the experiments with periodical perfusion of the rat brains with cold RPS-2 solution every hour
for 9 hours of their cold storage. The results (45% survival) were worse than the results (55% survival, Table 2) for 12 hour static cold storage of the rat brains with RPS-2. So, the periodical perfusion was not helpful to increase survival of the brain tissues.