Preparing a cryonics patient for cryostorage can involve three distinct stages of alteration of body fluids:
(1) patient cooldown/cardiopulmonary support
(2) blood washout/replacement for patient transport
(3) cryoprotectant perfusion
During patient cooldown/cardiopulmonary support, a cryonics emergency response team or health care personnel may inject a number of medicaments to minimize ischemic injury and facilitate cryopreservation. The first and most important of these medicaments would be heparin, to prevent blood clotting. (For more details on the initial cooldown process, see Emergency Preparedness for a Local Cryonics Group).
Once the patient is cooled, the blood can be washed-out and replaced with a solution intended to keep organs/tissues alive while the patient is being transported to a cryonics facility. At the cryonics facility the organ/tissue preservation solution is replaced with the cryopreservation solution intended to prevent ice formation when the patient is further cooled to temperatures of -140ºC (glass transition temperature) or -196ºC (liquid nitrogen temperature) for long-term storage.
For both organ/tissue preservation & cryoprotection it is necessary to replace the fluid contents of blood vessels & tissue cells with other fluids. The process of injecting & circulating fluids through blood vessels is called perfusion. The passive process by which fluids enter & exit both blood vessels & cells is called diffusion.
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Body fluids can be described as solutes dissolved in a solvent, where
the solvent is water and the solutes are substances like sodium chloride (NaCl, table salt),
glucose or protein. Both water and solute molecules tend to move randomly in fluid with
energy and velocity that is directly proportional to temperature. When there is a
difference in concentration between water or solute molecules in one area of the fluid compartment
compared to the rest of the compartment, random motion of the molecules will eventually
result in a uniform distribution of all types of molecules throughout the compartment.
In thermodynamics this is termed a decrease in potential energy (Gibbs free energy, not
heat energy) due to an increase in entropy at constant temperature -- leading to equilibrium.
The movement of molecules from an area of high concentration to an area of low concentration is called diffusion. The rate of diffusion (J) can be quantified by Fick's law of diffusion:
dc
J = − DA ----
dx
J = rate of diffusion (moles/time)
D = Diffusion coefficient
A = Area across which diffusion occurs
dc/dx = concentration gradient (instantaneous concentration difference divided by instantaneous distance)
Fick's First Law states that the rate of diffusion down a concentration gradient is proportional to the instantaneous magnitude of the concentration gradient (which changes as diffusion proceeds). For movement of molecules from a region of higher concentration to a region of lower concentration dc/dx will be negative, so multiplying by −DA gives a positive value to J. Diffusion coefficient is higher for higher temperature and for smaller molecules.
Diffusion can occur not only within a fluid compartment, but across partitions that separate fluid compartments. The relevant partitions for animals are cell membranes and capillary walls. Cell membranes are lipid bilayers that allow for free diffusion of lipid soluble substances like oxygen, nitrogen, carbon dioxide and alcohol, while blocking movement of ions and polar molecules. But cell membranes also contain protein channels. Protein channels for water allow for very rapid diffusion of water across the membranes. Protein channels for potassium (K+), sodium (Na+) and other ions allow for more restricted diffusion across cell membranes. There is also facilitated diffusion (active transport) of many types of molecules across membranes.
For a normal 70 kilogram (154 pound) adult the total body fluid is about 60% of the body weight. Almost all of this fluid can be described as extracellular or intracellular (excluding only cerebrospinal fluid, synovial fluid and a few other small fluid compartments). Extracellular fluid can be further subdivided into plasma (noncellular part of blood) and interstitial fluid (fluid between cells that is not in blood vessels). Cell membranes separate intracellular fluid from extracellular fluid, whereas capillary walls separate plasma from interstitial fluid. The relative percentages of these fluids can be summarized as:
Intracellular fluid 67%
Extracellular fluid
Interstitial fluid 26%
Plasma
7%
Note that blood volume includes both plasma & blood cells such that adding the intracellular fluid volume of blood cells to plasma volume makes blood 12% of total body fluid.
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Osmosis refers to diffusion of water (solvent) across a membrane that is semi-permeable, ie, permeable to water, but not to all solutes. If solutes are added to one side of the membrane (but not to the other side), water will be less concentrated on the solute side of the membrane. This concentration gradient will cause water to diffuse across the semi-permeable membrane into the side with the solutes unless pressure is applied to prevent the diffusion of water. The amount of pressure required to prevent any diffusion of water across the semi-permeable membrane is called the osmotic pressure.
An important distinction to remember in replacing body fluids is the distinction between two kinds of swelling (edema): cell swelling and tissue swelling. Cell swelling occurs when there is a lower concentration of dissolved molecules (solutes) outside cells than inside cells. Conversely, when a high concentration of solutes surrounds a cell, water rushes out of the cell causing the cell to shrink. To prevent either shrinkage or swelling of a cell there must be an osmotic balance of molecules & ions between the liquids outside the cell & inside the cell. Even if a cell does not burst or collapse due to osmotic imbalance, a sudden change in osmotic balance can severely injure cells.
Like cell membranes, capillary walls function as semi-permeable membranes. The osmotic pressure due to plasma colloids is called oncotic pressure. Tissue swelling occurs when fluids leak out of blood vessels into the interstitial space (the space between cells in tissues). Injury to blood vessels can result in tissue swelling, but tissue swelling can also result from water leaking out of vessels when there is nothing (like albumin) to prevent the leakage.
Both forms of edema (cell & tissue swelling) can impede perfusion considerably, and is frequently a problem in cryonics patients who have suffered ischemic or other forms of blood vessel damage. Maintaining osmotic balance of the fluids outside & inside cells is as important as maintaining oncotic balance, ie, balance of fluids inside & outside of blood vessels.
Sterling's Hypothesis states that at equilibrium there will be a balance between hydrostatic pressure in the capillaries and hydrostatic pressure in the interstitial fluid between capillary walls and tissue cells -- as well as between the oncotic pressure in the capillary and the oncotic pressure in the interstitial fluid. (The hydrostatic pressure is the pressure at any given point of a non-moving fluid.) In most of the body, capillary membranes are permeable to the low molecular weight solutes in the blood, but impermeable to the large proteins. Capillary membranes in the brain are not only impermeable to proteins, but are even impermeable to low moleuclar weight solutes like Na+ and Cl- -- an impermeability referred to as the blood-brain barrier. For the blood-brain barrier, no distinction need be made between osmotic and oncotic pressure because oncotic pressure is mostly due to the osmotic pressure of the small solutes. This fact is relevant for cryonics because infusion of small solutes -- like mannitol or hypertonic saline -- can dehydreate the brain or prevent edema.
Solute concentrations are generally expressed in terms of molarity (moles of solute per liter of solution). But osmotic pressure (like vapor pressure lowering and freezing-point depression) is a colligative property, meaning that the number of particles in solution is more important than the type of particles. One molecule of albumin (molecular weight 70,000) contributes as much to osmotic pressure as one molecule of glucose or one sodium ion. At equilibrium all molecules in a solution have achieved the same average kinetic energy, meaning that molecules with a smaller mass have higher average velocity. Thus, a one molar solution of NaCl will result in twice the osmotic pressure as a one molar solution solution of glucose -- because Na+ and Cl− ions exert osmotic pressure as independent particles.
The osmolality of a solute is the product of the molarity of the solute and the number of dissolved particles produced by the solute. A one molar (1.0 M, one mole per liter) solution of CaCl2 is a three osmolar (3.0 Osm, three osmoles per liter) solution because of the Ca2+ ion plus the two Cl− ions produced when CaCl2 is added to water. For describing solute concentrations in body fluids it is more convenient to use thousandths of osmoles, milli-osmoles (mOsm). Total solute concentration of intracellular fluid, interstitial fluid or plasma is roughly 300 mOsm. About half of the osmolality of intracellular fluid is due to potassium ions, whereas about 80% of the osmolality of interstitial fluid and plasma is due to sodium and chloride ions. Protein contributes to less than 1% of the osmolality of plasma. Cells contain about four times the concentration of proteins as plasma.
An exact calculation of the osmolality of plasma gives 308 mOsm, but the freezing point depression of plasma (−0.54ºC) indicates an osmolality of 286 mOsm. Interaction of ions reduces the effective osmolality.
Whether a cell shrinks or swells in a solution is determined by the tonicity of the solution, not simply the osmolality. A solution is said to be isotonic if cells neither shrink nor swell in that solution. Both 0.9% NaCl and 5% glucose (before glucose is metabolized) are isotonic solutions (roughly 300 mOsm). Hypertonic cause cells to shrink as water rushes out of cells into the solute, whereas hypotonic solutions cause cells to swell as water from the solution rushes into the cells.
Whether a solution is hypertonic or hypotonic depends on the permeability of the cell membrane to the solute, and is not simply a function of osmolality. If the added solute does not cross cell membranes the resulting solution will be hypertonic, causing water to osmotically leave cells. Cryoprotectants that cross cell membranes may cause only transient cell shrinkage (due to the fact that water can leave cells faster than cryoprotectant enters). When extracellular cryoprotectants that do not cross cell membranes are used, the osmolality of the carrier solution should be reduced so that the total extracellular osmolality is not much above 300 mOsm.
Much of the isotonicity of the intracellular and extracellular fluids are maintained by the sodium pump in cell membranes, which exports 3 sodium ions for every 2 potassium ions imported into cells. When ischemia deprives the sodium pump of energy, cells swell from excessive intracellular sodium -- resulting in edema. Inflammation can also cause cell swelling due to increased membrane permeability to sodium and other ions. Interstitial edema can occur when ischemia or inflammation increases capillary permeability leading to leakage of larger plasma solutes into the interstitial space.
[For further details on the sodium pump see MEMBRANE POTENTIAL, K/Na-RATIOS AND VIABILITY]
A critical distinction is made in fluid mechanics between laminar flow and turbulent flow in a pipe. For laminar flow elements of a liquid follow straight streamlines, where the velocity of a streamline is highest in the center of the vessel and slowest close to the walls. Turbulent flow is characterized by eddies & chaotic motion which can substantially increase resistance and reduce flow rate. The Reynolds number is an empirically determined dimensionless quantity which is used to predict whether flow will be laminar or turbulent -- with 2000 being the lower limit for turbulent flow.
Turbulent flow could potentially be a problem in cryonics if it reduced perfusion rate or increased the amount of pressure required to maintain a perfusion rate. It is doubtful that turbulent flow ever plays a role in cryonics perfusion, however. Even for a subject at body temperature (37ºC) Reynolds numbers in excess of 2000 are only seen in the very largest blood vessels: the aorta and the vena cava.
The formula for Reynolds number is:
ρ v D
Re = ------
µ
ρ = fluid density (rho)
v = fluid viscosity
D = vessel diameter
µ = viscosity
The fact that diameter (D) is in the numerator indicates that only high diameter vessels have high Reynolds number. Velocity (v), also in the numerator, is highest in the aorta & arteries. But the use of cryoprotectants and the increase in viscosity (µ) with declining temperature essentially guarantee that turbulent flow will not occur in a cryonics patient.
More serious for cryonics is the
Hagen-Poisseuille Law, which describes the relationship between
flow-rate and driving-pressure:
pressure X (radius)4
Flow Rate = ----------------------
length X viscosity
Typically in cryonics the flow rate will be one or two liters per minute when the pressure is around 80 mmHg. But because flow rate varies inversely with viscosity and varies directly with pressure, pressure must be increased to maintain flow rates when cryoprotectant viscosity increases with lowering temperature. This poses a serious problem because blood vessels become more fragile with lowering temperature. If blood vessels burst the perfusion can fail.
At 20ºC glycerol is about 25% more dense (ρ=rho, in the numerator) than water. But the role of viscosity is far more dramatic, with high viscosity in the denominator reducing Reynolds number considerably. The viscosity of water approximately doubles from 37ºC to 10ºC, but the viscosity of glycerol increases by a factor of ten (roughly 4 Poise to 40 Poise). At 37ºC glycerol is nearly 600 times more viscous than water, but at 10ºC it is about 2,600 times more viscous.
Although turbulence is not a concern in cryonics, the increase in viscosity of cryoprotectant with lowering temperature certainly is. Fortunately, the newer vitrification mixtures are less viscous than glycerol.
The most common strategy in cryonics has been to cool the patient from 37ºC to 10ºC as rapidly as possible and to perfuse with cryoprotectant at 10ºC. Lowering body temperature reduces metabolism considerably, thereby lessening the amount of oxygen & nutrient required to keep tissues alive. Cryoprotectant toxicity drops as temperature declines. But the very dramatic more-than-exponential increase in cryoprotectant viscosity with lowering temperature poses a significant problem for effective perfusion. When open circuit perfusion is used, a higher temperature may be preferable because the opportunity for diffusion time into cells is so limited (about 2 hours -- 1 hour for the head, 1 hour for the body) -- although ischemic damage is difficult to quantify.
With closed circuit perfusion, the perfusion times are longer -- up to 5 hours. If a good carrier solution is used for the cryoprotectant the tissues may receive adequate nutrient. This, along with the oxygen carrying-capacity of water at low temperature, may limit ischemic damage while allowing time for cells to become fully loaded with cryoprotectant. If ischemic damage can be safely prevented in perfusion, the only critical issues for temperature selection are the relative benefits of reduced cryoprotectant toxicity at lower temperatures as against increased chilling injury. The fact that the more-than-exponential increase in viscosity with lowering temperature will increase perfusion time will not be problematic if the risk of ischemia is minimized.
Early in the process of preparing a cryonics patient for cryopreservation it is important to replace the blood. Provided anti-coagulant such as heparin is injected into the patient, blood can remain in a patient whose tissue viability is being maintained by cardiopulmonary support (circulation of oxygenated blood) in the early stages of cooling.
As body temperature approaches 10ºC, however, metabolic rate has slowed greatly and the oxygen-carrying capacity of blood hemoglobin is no longer required. Cool water, in fact, may carry adequate dissolved oxygen at low temperatures. (Water near freezing temperature can hold nearly three times as much dissolved oxygen as water near boiling temperature.) The tendency of blood to agglutinate and clog blood vessels becomes a serious problem at low temperature -- so the blood should be replaced if this does not cause other problems (such as delay and reperfusion injury.)
Typically a cryonics patient deanimates at a considerable distance from a cryonics facility and must be transported before cryoprotectant (antifreeze compound such as glycerol) can be perfused. Blood is washed-out and replaced with an isotonic (ie, osmotically the same as saline) solution, such as Ringer's solution. The patient is then transported to the cryonics facility at water-ice temperature. Freezing must be avoided because ice crystals would damage cells & blood vessels to such an extent as to prevent effective cryoprotectant perfusion. Water-ice temperature will not freeze tissues because tissues are salty (salt lowers the freezing point below 0ºC).
Replacing blood with a saline-like solution for patient transport, however, does not do a good job of maintaining tissue viability or preventing edema. For this reason the organ preservation solution Viaspan®, rather than Ringer's solution, has been used for cryopatient transport. Blood is not simply an isotonic solution carrying blood cells. Blood contains albumin, which attracts water and keeps the water from leaving blood vessels and going into tissues (maintains oncotic balance). Tissues which are swollen by water (edematous) resist cryoprotectant perfusion. One of the most important ingredients in Viaspan® preventing edema is HydroxyEthyl Starch (HES), which attracts water in much the way albumin attracts water -- acting as an oncotic agent by keeping water in the blood vessels. Viaspan® contains potassium lactobionate to help maintain osmotic balance. Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used in organ preservation solutions as a replacement for HES with good results [THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)].
Viaspan® (DuPont Merck Pharmaceuticals) contains other ingredients to maintain tissue viability, such as glucose, glutathione, etc. (the full formula can be found on the Viaspan® website). Viaspan® is FDA approved for preservation of liver, kidney & pancreas, but is used off-label for heart & lung transplants. Viaspan® is being challenged in the marketplace for all these applications by the Hypothermosol (Cryomedical Sciences, BioLife Technologies) line of preservation solutions.
Rather than use these expensive commercial products, Alcor uses a preservation solution developed by Jerry Leaf & Mike Darwin called MHP-2. MHP-2 is so-called because it is a Perfusate (P) which contains mannitol (M) as an extracellular osmotic agent and HEPES (H), a buffer to prevent acidosis which is effective at low temperature. MHP-2 also contains ingredients to maintain tissue viability and hydroxyethyl starch as an oncotic agent to prevent edema. Lactobionate permeates cells less than mannitol and can thus maintain osmotic balance for longer periods of time, but mannitol is much less expensive. Mannitol also has an additional effect in the brain. Because of the unique tightness of brain capillary endothelial cell junctions ("blood brain barrier"), mannitol does not normally penetrate into the extravascular space of the brain. This means that mannitol can act like an oncotic agent for the brain. If the blood brain barrier is intact, mannitol will suck water out of the extravascular space. The brain is the only place that mannitol can do this, and that's why a mannitol is effective for inhibiting edema of the brain -- but only if there is not extensive ischemic damage to the blood brain barrier. (Mannitol has yet another benefit in that it scavenges hydroxyl radical [CHEM.-BIOL. INTERACTIONS 72:229-255 (1989)]).
(For the formula of MHP-2 see Table II of CryoMsg 4474 or Table VII of CryoMsg 2874 -- which also contains the formula for Viaspan in Table V.)
Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used as a replacement for HES in organ preservation solutions with good results [THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)]. The same benefit might not apply to cryonics patients, however, because of the prevalence of endothelial damage due to ischemia. Larger "holes" in the vasculature can mean that a larger molecular weight molecule is required for oncotic support. HES molecular weight is about 500,000, whereas the molecular weight for PEG used in organ replacement solutions is more like 20,000. A PEG with molecular weight of 500,000 would be far too viscous and will form a gel. HES has the benefit of being large enough to always provide oncotic support while being much less viscous than PEG of equivalent molecular weight.
The initial perfusate can also contain other ingredients to assist in reducing damage to the cryonics patient. Anticoagulants can reduce clotting problems, and antibiotics can reduce bacterial damage. Damaging effects of ischemia can be reduced with antioxidants, antiacidifiers, an iron chelator and a calcium channel blocker.
Once the patient is at the cryonics facility the transport solution can be replaced with a cryoprotectant solution. A perfusion temperature of 10ºC gives the best tradeoff of avoiding the high viscosity of lower temperatures and at the same time limiting the ischemic tissue degradation and cryoprotectant toxicity that would be seen at higher temperatures. (Cryonicists usually worry more about ischemic damage than cryoprotectant toxicity due to a belief that ischemic damage has a greater likelihood of being irreversible -- irreparable by future molecular-repair technology.)
Cryoprotectants are used in cryonics to reduce freezing damage by prevention of ice formation (see Vitrification in Cryonics ). Although it seems plausible that our brains may be damaged by freezing in a way that can be repaired, it is possible that freezing damage is really freezing destruction -- destruction beyond all future repair due to scrambling of tissue into molecular debris. If we were certain of future repair, it would make much more sense to immediately thrust cryonics patients into liquid nitrogen upon deanimation rather than to waste time or expense on cryoprotectant perfusion. Cryoprotectants may not only increase the possibility of future repair, they may reduce the estimated time the patient needs to remain in liquid nitrogen -- a vulnerable condition in which a patient could be destroyed due to negligence, accident or malevolence.
Cryoprotectants should be sterilized to prevent the growth of bacteria. Sterilization of cryoprotectants by heating can cause the formation of carbon-carbon double-bonds, which are evident by a yellowing of the cryoprotectant. Only a few such double-bonds can produce the yellow appearance, so the fact of yellowing is not evidence that the cryoprotectant is no longer serviceable. But a preferable method of cryoprotectant sterilization is filtration through a 0.2 micron filter.
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Membrane permeabilities of a variety of nonelectrolytes (including cryoprotectants) have been studied on human blood cells [THE JOURNAL OF GENERAL PHYSIOLOGY; Naccache,P; 62(6):714-736 (1973)]. Critical factors determining membrane permeability are lipid solubility of the substance (which increases permeability) and hydrogen bonding (which decreases permeability). In general, permeability decreases as the molecular size of the substance increases. In contrast to blood cells, human sperm is more than three times more permeable to glycerol than to DMSO [BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)]. For both blood cells and sperm cells permeability to ethylene glycol is very high compared to the other common cryoprotectants.
Water and cryoprotectants both cross cell membranes more slowly at lower temperatures. Cryoprotectants slow the passage of water across cell membranes. Glycerol, DMSO and ethylene glycol all reduce the rate at which water crosses human sperm cell membranes by more than half [BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)].
Aside from the choice of cryoprotectants, a major concern is the way cryoprotectant is administered. For example, glycerol (the standard cryoprotectant used in cryonics for many years) can either be administered full-strength or it can be introduced in gradually increasing concentrations. (Under optimum conditions, glycerol results in 80% vitrification and 20% ice formation. Glycerol has been replaced by better cryoprotectants that can vitrify without any ice formation, but I will sometimes use glycerol as my example cryoprotectant.) A patient should not be perfused with a 100% solution of glycerol or other cryoprotectant. It is prudent to begin perfusion with low concentrations of cryoprotectant because water can diffuse out of cells thousands of times more rapidly than cryoprotectant diffuses into cells. Using gradually increasing concentrations of cryoprotectant (ramping) prevents the osmotic damage this differential could cause.
Cells are much more permeable to water than they are to cryoprotectant. Platelets & granulocytes, for example, are 4,000 times more permeable to water than they are to glycerol [CRYOBIOLOGY; Armitage,WJ; 23(2):116-125 (1986)]. When a cell is exposed to high-strength cryoprotectant, osmosis causes water to rush out of the cells, causing the cells to shrink. Only very gradually does the cryoprotectant cross cell membranes to enter the cell (the "shrink/swell cycle"). For isolated cells, the halftime (time to halve the difference between a given glycerol concentration in a granulocyte and the maximum possible concentration) is 1.3 minutes [EXPERIMENTAL HEMATOLOGY; Dooley,DC; 10(5):423-434 (1982)] -- but tissues & organs would require more time because their cells are less accessible. Even after equilibration, however, the concentration of glycerol inside neutrophilic granulocytes never rises above 78% of the concentration outside the cells.
Rapid shrinking of cells can kill them. When granulocyte cells shrink to 68% of their normal volume, cell survival drops -- and severe cell death occurs below 55% [AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5 Pt 1):C382-389 (1984)]. Increasing glycerol concentration from 0.5 molar (3.6 % v/v) to 1.0 molar (7.3 % v/v) reduces granulocyte survival from 40% to 20% at 0ºC [AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5 Pt 1):C382-389 (1984)]. Cell shrinkage may directly damage the cell (and cell membrane) due to structural resistance from the cell cytoskeleton and high compression of other cell constituents [HUMAN REPRODUCTION; Gao,DY; 10(5):1109-1122 (1995)]. (If damage is maximum at 1.0 molar, the concentrations up to 7.5 molar once used for cryonics protocols may have been of little additional damage.) A rule of thumb is that the osmolality of the perfusate should not be more than twice the osmolality in the cell (which is normally about 300 osmols).
Not only is cell shrinkage damaging, but rapid passage of water across cell membranes physically damages the membranes. The higher the concentration difference between glycerol inside the cell and glycerol outside the cell, the greater the water molecule velocity and the greater the cell membrane damage (up to a maximum) [BIOPHYSICS JOURNAL; Muldrew,K; 66(2 Pt 1):532-541 (1994)]. Even more damage may occur to the blood vessels. Rapid addition of cryoprotectant causes endothelial cells to shrink -- thereby breaking the junctions between the cells [CRYOBIOLOGY; Pollock,GA,; 23(6):500-511 (1986)]. On the other hand, endothelial cell shrinkage by hypertonic perfusate can increase capillary volume, thereby increasing blood flow -- as long as excessive vascular damage does not occur.
For cryonics purposes some vascular damage can actually be an advantage insofar as it increases diffusion -- and vascular repair may be an easy task for future science. In fact, the breakdown of the blood-brain barrier in the 1.8-2.2 molar glycerol range is essential for perfusion of the brain -- as long as damaging tissue edema (swelling) can be avoided. Aquaporin (water channel) expression in the blood-brain barrier could be a safer means of allowing cryoprotectants into the brain [CRYOBIOLOGY; Yamaji,Y; 53(2):258-267 (2006)]. In general, if osmotic damage is similar to types of cryoprotectant toxicity that does not destroy structures containing information essential to personal identity (consciousness, selfhood), then osmotic damage may be far less serious than freezing damage. Unfortunately, it is difficult to draw firm conclusions about what damage is reparable and what structures are essential for personal identity -- and it is wise to not take excessive risk about these matters.
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Osmotic damage by cryoprotectant can be reduced in cryonics protocol by introducing the cryoprotectant in gradually increasing concentrations -- rather than perfusing with cryoprotectant full-strength. Also, allowing more time for cryoprotectant equilibration gives time for cells more distant from blood vessels to receive cryoprotectant. As described earlier, cells placed in a hyperosmotic solution (such as a vitrification solution) will initially shrink rapidly as water leaves the cell, and gradually re-swell as cryoprotectant slowly enters the cell (the "shrink/swell cycle"). As shown in the diagram for mouse oocytes at 10ºC, water leaves the cell in the first 100 seconds, whereas 1.5 Molar ethylene glycol (black squares) or DMSO (DiMethylSulfOxide, white squares) take 1,750 seconds to restore the volume to 85% of the original cell volume [CRYOBIOLOGY; Paynter,SJ; 38:169-176 (1999)].
The best results are achieved with small increases of cryoprotectant initially, with accelerating increase in concentration. An analogous procedure is used in adding cryoprotectant (DMSO) to human embryos being prepared for storage in liquid nitrogen (PBI 10 minutes, 0.25M DMSO 10 minutes, 0.5M DMSO 10 minutes, 1.0M DMSO 10 minutes, 1.5M DMSO 10 minutes) [FERTILITY AND STERILITY; Trounson,A; 46(1):1-12 (1986)].
Closed-circuit perfusion (with perfusion solution following a circuit both inside & outside the patient's body) is contrasted with the open-circuit perfusion used by funeral directors for embalming. In the open-circuit perfusion of embalming, fluid is pumped into a large artery of the corpse and forces-out blood from a large vein -- and this blood is discarded.
A closed-circuit perfusion, as illustrated in the diagram, can be set
up at low cost for gradual introduction of cryoprotectant into cryonics
patients. As shown in the diagram, the perfusion circuit bypasses the
heart. Perfusate enters the patient through a cannula in the femoral (leg) artery
and exits from a cannula in the femoral vein on the same leg. Flowing
upwards from the femoral artery, the perfusate enters the arch of the
aorta (where blood normally exits the heart), but is blocked from entering
the heart. Instead, the perfusate flows through the distribution arteries
of the aorta, notably to the head and brain. Returning in the veins, the
perfusate again bypasses the heart and flows downward to the femoral vein
where it exits. A better alternative to the femoral circuit, however, is
to surgically open the chest to cannulate the heart aorta (for input) and
atrium (for output).
![[ Closed Circuit Perfusion of a Cryonics Patient ]](./perfuse.gif)
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Although it is not shown in the diagram, there will be a pump in the circuit to maintain pressure and fluid movement. A roller pump, rather than an embalmer's pump, should be used. A roller pump achieves pumping action by the use of rollers on the exterior of flexible tubing that forces fluids through the tube without contaminating those fluids. Embalmer's pumps may use pressures much higher than those suitable for cryonics, resulting in blood vessel damage. Embalmer's pumps are also easily contaminated (and hard to clean), unless a filter is used. Contamination doesn't matter much in embalming, but in cryonics contaminants entering the patient through the pump can damage blood vessels, interfering with perfusion. If an embalmer's pump is used for cryonics purposes, ensure that the pressure can be lowered to a suitable level and that it is cleaned and sterilized. The main advantage of roller pumps, however, is the fact that they provide a closed circuit, whereas embalmer's pumps are open-circuit. Roller pumps are generally calibrated in litres per minute, and a flow rate of 1.5 to 2 litres per minute will be necessary to achieve the desired perfusion pressure of approximately 80 mmHg to 120 mmHg (physiological pressures).
Mean Arterial Pressure (MAP) for an normal adult is regarded as being in the range of 50 to 150 mmHg, and Cerebral Perfusion Pressure (CPP) is in the same range [BRITISH JOURNAL OF ANAETHESIA; Steiner,LA; 91(1):26-38 (2006)]. Vascular pressure normally drops to about 40 mmHg in the arterioles, to below 30 mmHg entering the capillaries, and is down to 3 to 6 mmHg (Central Venous Pressure, CVP) when returning to the right atrium of the heart. Perfusing a cryonics patient at about 120 mmHg should open capillaries adequately for good cryoprotectant tissue saturation without damaging fragile blood vessels.
Outside the patient, some of the drainage is discarded, but most is returned to a circulating (stirred) reservoir connected to a concentrated reservoir of cryoprotectant. The circulating reservoir is initially carrier solution which gradually becomes increasingly concentrated with cryoprotectant as the stirring and recirculation proceed. The circulating reservoir can be stirred from the bottom by a magnetic stir bar on a stir table and/or from the top by an eggbeater-type stirring device. The stirring will draw cryoprotectant from the cryoprotectant reservoir, and pumping of the perfusate should also actively draw liquid from the cryoprotectant reservoir. Gradually a higher and higher concentration of cryoprotectant is included in the perfusate and the osmotic shock of full-strength cryoprotectant is avoided.
The carrier solution for the cryoprotectant should perform similar tissue preservation functions as is performed by the transport solution, and should be carefully mixed with the cryoprotectant so as to avoid deviations from isotonicity which could result in dehydration or swelling & bursting of cells. The carrier solution will help keep cells alive during cryoprotectant perfusion.
An excellent carrier solution for cryonics purposes would be RPS-2 (Renal Preservation Solution number 2), which was developed by Dr. Gregory Fahy in 1981 as a result of studies on kidney slices. More recently Dr. Fahy used RPS-2 as the carrier solution in cryopreserving hippocampal slices -- an indication that it is well-suited for brain tissue as well as for kidney. RPS-2 not only helps maintain hippocampal slice viability, it reduces the amount of cryoprotectant needed because it has cryoprotectant (colligative) properties of its own. The formulation of RPS-2 is: K2HPO4, 7.2mM; reduced glutathione, 5mM; adenine HCl, 1mM; dextrose, 180mM; KCl, 28.2mM; NaHCO3, 10mM; plus calcium & magnesium [CRYOBIOLOGY; Fahy,GM; 27(5):492-510 (1990)]. LM5 (Lactose-Mannitol 5) is a carrier solution for use in vitrification solutions that include ice blockers. LM5 does not contain dextrose, which is believed to interfere with ice blockers.
The cryoprotectant reservoir will not in general contain pure cryoprotectant (although in principle it could), but rather a "terminal concentration" solution of cryoprotectant that is equal or slightly above the final target concentration. As perfusion proceeds and drainage to discard proceeds, the level of both reservoirs drops in tandem until both reservoirs are nearly empty, at which point the circuit concentration will have reached the cryoprotectant reservoir concentration. Provided that the two reservoirs are the same size and same vertical elevation, the gradient will be linear over time (if the drainage rate to discard was constant).
For cryoprotectant to perfuse into cells there must be constant exposure to cryoprotectant surrounding the cells -- and there must be pressure to maintain that exposure. In a living animal the heart maintains blood pressure that forces blood through the capillaries and forces nutrients into cells. A dead animal with no blood pressure -- and which is being perfused with cryoprotectant -- also requires pressure for the capillaries to remain open and for cryoprotectant to be maintained at high concentrations around cells.
Alcor found that closed-circuit perfusion must be maintained for 5-7 hours for full equilibration of glycerol, because the diffusion rate of water out of cells is thousands of times the rate at which glycerol enters cells. Of course, it would be possible to pump glycerol into a patient for 5-7 hours with open-circuit perfusion, but only by using thousands of dollars worth of glycerol. The newer vitrification cryoprotectants used by Alcor are vastly more expensive than glycerol. When using expensive cryoprotectants it makes far more sense to recirculate in a closed circuit. Closed-circuit perfusion also has the benefit of allowing for ongoing monitoring of physiological changes occurring in the patient's body during the perfusion process. Open-circuit with an inexpensive cryoprotectant has the advantage of avoiding recirculation of toxins.
Cryoprotectants, particularly glycerol, are viscous -- and cryoprotectants in high concentration are particularly viscous. The introduction of air bubbles into cryoprotectant solutions during pouring and mixing should be avoided because air emboli that enter the cryonics patient can block perfusion. Elimination of air bubbles from viscous cryoprotectant solutions is extremely difficult. Prevention is more effective than cure. Cryonicist Mike Darwin wrote about this problem and possible solutions in a 1994 CryoNet message.
Improper mixing of perfusate containing high levels of cryoprotectant can result in a phenomenon that appears to be high viscosity, but in reality is edema. If, for example, isotonic carrier solution is mixed half-and-half with cryoprotectant solution an open circuit perfusion may have to be halted when no further perfusate will go into the patient. The problem is caused not by viscosity, but by the fact that the isotonic solution became hypotonic due to dilution with cryoprotectant -- causing the cells to swell and forcing perfusion to end. In closed-circuit perfusion, the cryoprotectant concentrate reservoir contains cryoprotectant at about 125% the terminal concentration in a vehicle of isotonic carrier solution so that when reservoir concentrate is mixed with isotonic carrier there is no change in tonicity.
Newer cryoprotectants are less viscous than glycerol, so perfusions can be done in less time. After 15 minutes of perfusion with carrier solution, cryoprotectant concentration linearly increases at a rate of 50 millimolar per minute until full concentration is reached -- in about two hours (a protocol developed on the basis of minimizing osmotic damage when perfusing kidneys). Perfusion is increased for an additional hour or two until the cryoprotectant has fully diffused into cells (as indicated by similarity of afflux and efflux cryoprotectant concentrations).
Only after a few hours of closed-circuit perfusion is the concentration of cryoprotectant exiting the cryonics patient equal to the concentration of cryoprotectant entering the patient. Only an extended period of sustained pressure will keep capillaries open, and otherwise facilitate diffusion of cryoprotectant into cells. And the exiting cryoprotectant concentration will equal the entering cryoprotectant concentration only when the tissues are fully loaded with cryoprotectant. One or more burr holes are made in the skull, and a refractometer is used to verify that terminal cryoprotectant concentration has been reached in the brain.
(A refractometer measures the index of refraction of a liquid, ie, the ratio of the speed of light in the liquid and the speed of light in a vacuum (or air). Light changes speed when it strikes the boundary of two media, thus causing a change in angle if it strikes the new medium at an angle. Because the refractive index is a ratio of two quantities having the same units, it is unitless. Sodium vapor in an electric arc produces an excitation between the 3s and 3p orbitals resulting in yellow-orange light of 589nm -- what Joseph Fraunhofer called the "D line". Insofar as the sodium "D line" was the first convenient source of monochromatic light, it became the standard for refractometry. The refractive index of a liquid is thus a high-precision 5-digit number between 1.3000 and 1.7000 at a specific temperature, measured at the sodium D line wavelength. For example, the refractive index of glycerol at 25ºC -- nD25 -- is 1.4730.)
Closed-circuit perfusion may be necessary for removal of water as well as loading of cryoprotectant if it is true that open-circuit perfusion cannot remove water effectively.
One could imagine that the additional time spent doing closed-circuit (rather than open-circuit) perfusion means increased damage due to above-zero temperature. But most cells are still alive and metabolizing very slowly at 10ºC. Viaspan®, RPS-2 and other organ preservation solutions are designed to keep tissues alive for extended periods at near-zero temperatures -- certainly for the time required for closed-circuit perfusion. Ramping (slowly increasing concentration) of cryoprotectant should be done in such a way that the ion and mannitol or lactobionate concentration remains unchanged in the perfusate. Ramping is not an osmotically neutral process, however, because cryoprotectant is expected to dehydrate tissues.
Ramping at the rate of 2-4 molar per hour increase of glycerol concentration is slow enough to allow full diffusion of glycerol into tissues. Cell shrinkage due to diffusion of water from cells is followed by slow cell swelling due to the gradual diffusion of glycerol into cells. Dehydration of epithelial cells in the blood vessels by glycerol ramping facilitates perfusion by making the vessels wider. If the ramping rate is too slow, the epithelial cells become loaded with glycerol quickly enough that the vessel-widening advantage is lost.
Care must be taken that the concentration of non-penetrating solutes (such as mannitol) in the carrier solution is equal to that in the cells -- normally 300mM (millimolar) concentration. If the concentration of nonpenetrating solutes in the carrier solution is hypotonic -- 150mM, for example -- cells will swell to twice their volume irrespective of the glycerol concentration. This swelling would occur first in the epithelial cells, which could seriously impede the process of perfusion. Faster ramping would lessen this effect by dehydration of the epithelial cells, but the increased rate would be too rapid to allow full diffusion of glycerol into the tissues. It is better to avoid these problems by maintaining a carrier solution concentration of nonpenetrating solutes at 300mM to match that in cells.
Although glycerol has historically been the cryoprotectant used in cryonics, the perfusion process described should be much the same with the newer, more effective, cryoprotectants now being used in cryonics. Cryoprotectant toxicity varies directly with temperature, but cryoprotectant viscosity varies inversely with temperature. There is therefore a trade-off between perfusing at a higher temperature for more rapid cryoprotectant penetration, and suffering the increased toxicity of higher temperature. Unlike osmotic damage, however, toxicity may affect viability through biochemical rather that structural damage and thereby be of less concern for future repair. Ischemic injury is likely to be minimal due to nutrient & oxygen in the carrier solution. by more time at higher temperature, however, is likely to increase structural damage.
Some cryonics protocols are beginning the introduction of cryoprotectant at 10ºC and finishing at 5ºC. A near-term objective is to be able to introduce half the cryoprotectant at 10ºC and the other half at -10ºC. It seems like a good strategy to add the least toxic components of a cryoprotectant cocktail first (at higher temperatures) and the most toxic components at lower temperature -- or simply to increase cryoprotectant concentration as temperature drops. (This technique was used successfully in the Hippocampal Slice Cryopreservation Project.)
| Vertebral Artery | Circle of Willis |
|---|---|
|
|
When perfusing heads, rather than whole body patients, the sternum can be cut (median sternotomy) for direct access to the aorta and superior vena cava. By clamping-off the descending aorta and applying tourniquets to the arms, a closed circuit could be achieved to the head. A closed circuit to the brain could not be established through the carotid arteries and jugular veins because the vertebral arteries also carry blood to the brain. The carotids & vertebrals can anastomose at the Circle of Willis, however, so full perfusion of the brain should be possible even without use of the vertebrals -- by open circuit. But the Circle of Willis is often incomplete -- preventing full brain perfusion when using the carotid arteries alone. Posterior communicating arteries are threadlike or non-existence in a significant number of cases [ANNALS OF OTOLOGY, RHINOLOGY AND LARYNGOLOGY 112:657-664 (2003)]. Atherosclerosis adds to the problem -- nearly 60% of stroke victims have nonfunctional posterior collateral pathways, making circle of Willis deficiencies a risk factor for stroke [CARDIOVASCULAR DISEASES 16:191-198 (2002)].
Attempting to access the vertebrals through the neck is very difficult because they run so close to the spine. But an incision can be made at the collarbone to gain access to the vertebral arteries though the subclavian artery. Perfusing through the vertebrals and the carotids insures that perfusion of the brain is complete even if the Circle of Willis is not compete. If only the brain is to be perfused and not the head and face, then the external carotid artery would be clamped-off, but the external carotid is very difficult to access. Since beginning vitrification in 2005, the Cryonics Institute has used a collerbone access the the carotids and vertebrals. Alcor, however, simply removes the head from the cryonics patient and perfuses directly through the carotids and vertebrals -- and recycles the drippings in a simulation of "closed circuit".
Other tricks may eventually be invoked to improve perfusion, diffusion & vitrification -- and to reduce toxicity. High pressures are currently cumbersome & expensive to use, but future equipment may change that. Ultrasound has been suggested as a means of improving diffusion of cryoprotectant into cells. Sugars, which are cryoprotective, could be included in the perfusate -- along with insulin -- to both increase intracellular vitrification and to boost intracellular resistance to ischemic injury.
There is seemingly no limit to the amount of expense that could be incurred in attempting to reduce the damage done to a patient in cryopreparation. A cost-benefit trade-off must be made between achieving minimal damage an an inexpensive protocol. If the structural basis of personal identity is preserved, protocols to maintain cell viability would be less important since molecular repair technology could eventually reconstruct the person anyway.
Insofar as getting cryoprotectants to the brain is a primary objective of cryonics, understanding the nature of the blood-brain barrier and means of crossing the blood-brain barrier is a matter of primary importance.
The blood-brain barrier (BBB) provides the brain
with a chemical environment that is somewhat more stable than --
and somewhat independent of -- the chemical environment provided
to the rest of the body by the bloodstream. The BBB protects
the brain from variations of ions, amino acids, sugars, hormones,
etc in the bloodstream due to diet, hormone release and other
factors. Many amino acids such as
glycine
and
GABA (Gamma-AminoButyric Acid)
which act as neurotransmitters in the brain are markedly restricted by the BBB
to prevent entry into the brain.
Norepinephrine
in the bloodstream secreted by the adrenal medulla could significantly affect brain
activity.
| Typical Capillary Wall | Typical Capillary versus BBB Capillary |
|---|---|
|
|
Diffusion through capillary walls is the means by which most substances are transferred between the bloodstream and the interstitial fluid (the fluid between cells in body tissues). Capillaries are thin-walled tubes composed of a single layer of highly permeable endothelial cells lying on a thin basement membrane. Lipid soluble molecules such as ethanol, nicotine & diazepam as well as gases like oxygen & carbon dioxide diffuse directly across the capillary membranes. Gaps between adjacent endothelial cells (intercellular clefts) allow for diffusion of water-soluble substances, as do channels through the endothelial cell walls (fenestrae, "little windows"). Plasmalemma vesicles allow for pinocytosis (vesicle transport) of substances across the membrane. Intercellular clefts in the liver are so wide that plasma proteins can pass directly from the blood into liver tissue. Fenestrae are so large in kidneys that substances can be filtered in the glomeruli without having to pass through intercellular clefts.
| Astrocyte |
|---|
|
By contrast, the endothelial cells in brain capillaries are packed so tightly that there are no intercellular clefts. The tight junctions have a high electrical resistance, providing a barrier to ions. Moreover, there are no fenestrae and there is little or no pinocytosis in brain capillary endothelial cells. These tightly packed endothelial cells are the blood-brain barrier (BBB). Because glucose, vitamins and many amino acids can only cross the BBB endothelial cells by active transport, the endothelial cells of the BBB are rich in mitochondria.
Astrocytes are brain glial cells which have "end-foot" processes connected to neurons and BBB endothelial cells, providing biochemical support. The astrocyte processes surround BBB capillaries so completely that they were once thought to be the BBB, although it is now known that they are not. Astrocytes may, however, transmit biochemical signals to the BBB endothelial cells causing them to assume the BBB endothelial cell phenotype.
The BBB actively transports D−glucose rather than its stereoisomer L−glucose. Water crosses the BBB as readily as it crosses most cell membranes. Molecules with a molecular mass less than 400-500 atomic mass units can cross the BBB, but permeation decreases exponentially as molecular size increases for non-lipophilic atoms [MOLECULAR INTERVENTIONS; Pardridge,WM; 3(2):90-105 (2003)]. Substances with more than 8-10 hydrogen bonds show minimal transport across the BBB [NEURORX; Pardridge,WM; 2(1):3-14 (2005)].
Cryoprotectants typically have high hydrogen bonding capability, but because they are small molecules they do (slowly) cross the BBB. In cryonics patients the BBB is often damaged by ischemic-reperfusion injury, which allows for greater passage of cryoprotectant. Ironically, the cryoprotectant DMSO is one of several substances (along with ethanol & detergents) that have been used to assist in drug delivery [NEURORX; Pardridge,WM; 2(1):3-14 (2005)]. The phytochemical capsaicin [BRITISH JOURNAL OF PHARMACOLOGY; Hu,D; 146(4):576-584 (2005)] and the contrast agent Optison [ANESTHESIA & ANALGESIA; Mychaskiw,G; 91(4):798-803 (2000)] have also been used to disrupt the BBB.
The alcohol sugar mannitol is frequently used in cryonics and pharmacology
because of its size and properties. Just as glucose can be
reduced to the
sugar alcohol
sorbitol, fructose can be reduced to the sugar alcohol mannitol. Sorbitol and
mannitol have been used as artificial sweeteners because they are minimally absorbed
from the intestine. Even when sorbitol is absorbed it provides only two-thirds the
calories of glucose.
| Reduction of Glucose to Sorbitol | Fructose | Mannitol |
|---|---|---|
|
|
|
Because mannitol does not cross the BBB it has been used in cryonics to prevent brain edema. Mannitol can cross capillary membranes elsewhere in the body, but not in the brain. Thus, mannitol is an oncotic agent for the brain, but not for the rest of the body. (Mannitol is the M of MHP−2, which has been used as an organ preservation solution by the cryonics organization Alcor.) Raising plasma osmolality from 310 mOsm to 344 mOsm can shrink the brain by 10%, with half of the shrinkage occurring in 12 minutes.
Hyperosmotic (1.5 Molar) mannitol has been used to temporarily open the BBB, allowing for drug delivery. Perfusion of mannitol into the carotid artery shrinks the capillary endothelial cells, thereby opening the tight junctions between the cells for several hours [AMERICAN JOURNAL OF PHYSIOLOGY; Rapoport,SI; 238(5):R421-R431 (1980)]. In an experiment with rats, a 25% mannitol solution infused into the carotid artery for 30 seconds at a rate of 0.25 milliliters/kilogram/second increased BBB transfer of α−aminoisobutyric acid fivefold [JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM; Chi,OZ; 16(2):327-333 (1996)].
Although current cryonics protocol achieves enough saturation of the brain with cryoprotectants, the permeation is incomplete, the brain shrinks, with some of the vitrification achieved through dehydration and intracellular proteins. Finding ways to open the blood-brain barrier to allow greater saturation of vitrifying cryoprotectants without causing edema could be a means of increasing ultimate brain viability, provided cryoprotectant toxicity is less damaging than the effects of dehydration.
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