16. September 2008 · Comments Off on Life in non-aqueous solutions · Categories: Cryonics, Science · Tags: , , , , , ,

Can life exist without water? This is one of the questions that fascinates astrobiologists. The behavior of biomolecules in non-aqueous solutions is also of interest to cryobiologists and cryoenzymologists. Ice formation below zero degrees Celsius can be prevented by high concentrations of cryoprotective agents. But what are the effects of such vitrification agents on proteins?

In 1989 Alexander M. Klibanov published a paper called “Enzymatic Catalysis in Anhydrous Organic Solvents” that reports that enzymes are not only able to function in anhydrous organic solvents, but that some display remarkable properties in such environments like enhanced storage stability, solvent-induced changes of enzyme stereoselectivity, molecular memory, and reactions that are normally inhibited in aqueous solutions.

Upon reading the paper it is clear that when the author speaks of anhydrous solvents it is not implied that enzymes do not require water at all:

“…the key question should be not whether, but how much, water is required for enzymatic activity. Clearly, the enzyme molecule cannot ‘see’ more than a monolayer or so of water around it. Therefore, if this layer of ‘essential’ water is  somehow localized and kept on the surface of the enzyme, then all the bulk water should be replaceable with organic solvents with no adverse effects on the enzyme.”

To assure enzymatic activity in organic solvents two rules must be followed. First, hydrophobic solvents are preferred.  The authors propose that hydrophilic solvents ‘strip’ the essential water from the enzymes, and thereby reduce or eliminate the activity of enzymes. Second, the enzymes to be used in organic solvents need to be lyophilized (freeze dried) from aqueous solutions  with the pH optimal for their activity. This last requirement reflects the phenomenon of “pH memory” in which the enzymes retain the ionization state they had at that pH in the aqueous solution during freeze-drying and in organic solvents.

As surprising as some of these findings may be, the requirement of bound water for enzymes to  function is still consistent with the orthodox view that life requires water. At best, such findings can explain the existence or preservation of life in low water environments.

For cryobiologists, such findings raise interesting questions. In 2004, Fahy, Wowk et al. speculate that one of the mechanisms of cryoprotectant toxicity may involve “reduced hydration of biomolecules.” Understanding how solvents, and the combination of solvents, affect the intracellular milieu and the hydration and stability of biomolecules, should contribute to the design of less toxic vitrification solutions. Such vitrification solutions can be optimized for the human brain to allow for real suspended animation and improved prospects of resuscitation of cryonics patients.

15. September 2008 · Comments Off on Early total body washout experiments in cryonics · Categories: Cryonics · Tags: , , ,

The question of whether cryonics “works” or not is too general and hides the  point that progressive breakthroughs can make the concept more plausible. Human cryopreservation consists of a number of procedures culminating in long term care at cryogenic temperatures. An evidence based approach to cryonics dictates that the limits of procedures that can be reversed by contemporary technologies should be investigated and pushed further. Under ideal conditions viability of the brain of the patient can be maintained until the early stages of cryoprotective perfusion. In other words, cryonics procedures can be reversed with no or minimal brain injury up until the stage where exposure of the brain to higher concentrations of the vitrification agent compromises viability. Although we do not have a good understanding of the extent of ice formation in the brain of  typical patients in which vitrification is attempted, in ideal cases the current limiting factor to reversibility of cryonics procedures is cryoprotectant toxicity.

During its existence as a research program, cryonics researchers have shown great interest in recovering animals from ultra-profound hypothermic temperatures (lower than 5 degrees Celsius). The ability to routinely lower the temperature of mammals to temperatures close to zero degrees Celsius and  recover them without adverse effects to the brain does make the initial stages of cryonics reversible. Although this procedure does not necessarily require that the blood of an animal is removed at the lowest temperatures, theoretical considerations and experimental evidence dictate that  the use of an universal organ preservation solution will improve outcome. In the 1980s, the Alcor Life Extension Foundation engaged in a series of experiments that recovered dogs perfused with a mannitol-based organ preservation solution called MHP after 5-hours of bloodless perfusion at  5 degrees Celsius without adverse neurological outcome after rewarming.

Less known than those record setting experiments are earlier explorations in cryonics into whole body asanguineous hypothermia. The following document by cryonics researcher and Alcor patient Jerry Leaf documents a Trans Time experiment during the early days of total body washout experiments in cryonics. This account was published in the November/December 1977 issue of Long Life Magazine and the scan contains an introduction by Art Quaife and an afterword by Saul Kent.

Jerry Leaf – A Pilot Study in Hypothermia (1977) PDF

14. September 2008 · Comments Off on D(+)-Lactose and other sugars in organ preservation and cryonics · Categories: Cryonics, Science · Tags: , , , , , ,

A PDF file of this document is available with images and structural visualization of various sugars.

D(+) lactose monohydrate is the principal sugar in mammalian milks. The monohydrate part is easiest to explain; it simply means that the lactose molecule has one water molecule attached to it. This is important because some chemicals can have a lot of water molecules attached to them. For instance, you can have magnesium chloride with two attached water molecules (dihydrate) or six attached water molecules (pentahydrate). This becomes very important when you are weighing out a chemical and you need the chemical to be present in the correct amount. You’ll understand how important this is if you consider that someone proposes to sell you a kilo of some very valuable chemical (say 100 times more valuable than gold per milligram). There is going to be a considerable difference in the amount (by weight and usually by volume) of the actual active chemical you get per milligram or gram (weight) depending upon how hydrated it is (how many water molecules it has attached. The molecular weight (molecular mass) of magnesium chloride is 203.30 and the molecular weight (MW) of water is 18.01. Now, if you have 6 water molecules for each magnesium chloride molecule you have a total mass of water of  (18.01 x 6) = 108.06. That means if you have the pentahydrate salt of magnesium chloride you must add the weight of the 6 water molecules to the MW of magnesium chloride: 203.30 + 108.06 = 311.36. So, if someone is selling you a gram of magnesium chloride pentahydrate at the same price you can buy magnesium chloride anhydrous (no water) you are getting cheated because you are paying the same price for a gram of product that is 1/3rd water!

In biology and chemistry the same principle applies because if you need a certain amount of a chemical for critical reasons, say to maintain normal cell function or inhibit cell swelling in hypothermia, then you must account for any water molecules that may be attached to the chemical. In the case of magnesium chloride pentahydrate versus anhydrous magnesium chloride you are going to have to weigh out about 1/3rd more of the powder of the pentahydrate salt in order to get the same amount of magnesium chloride present in one gram of the anhydrous salt.

Why have pentahydrate, monohydrates, dihydrates and so on of chemicals? The answer is that some chemicals are almost impossible to handle in room air without rapidly absorbing water. Some chemicals will absorb just so much water and no more and thus are very stable under conditions of normal use, so they are supplied in this form. Some chemicals, especially organic chemicals, are virtually impossible to economically produce without one or more attached water molecules. Magnesium chloride is a really good example because it is intensely hygroscopic; it will literally pull water out of the air right before your eyes. So, not only is anhydrous magnesium chloride more expensive than the pentahydrate, it is virtually impossible to handle. If you try to weigh it out it will literally be grabbing water from the ambient air so fast that you can’t tare it on the scale. In seconds you will see tiny droplets of water on the weighing boat or paper where the magnesium chloride has literally pulled so much water out of the air it is fully dissolved in a tiny droplet of water! Calcium chloride is just about as bad, so, you’ll notice that we don’t even bother trying to weigh these chemicals out as dry powders, but rather buy them as pharmaceutical products for injection because they are already dissolved in solution in very precise concentrations. Thus, it is much simpler to draw up the correct volume of these salts dissolved in solution to add the desired amount of these two chemicals to perfusate. It is possible to weigh them, but you have to be quick and it helps if you live the desert where the humidity is very low.

Now we come to the D(+) part which is much harder to explain. In the early part of the 19th Century the French physicist Dominique F.J. Arago noticed that when he passed polarized light through quartz crystals the light could be rotated either to the left or right depending upon the individual crystal. Shortly thereafter the brilliant physicist and mathematician Jean Baptise Biot (the Biot number, a dimensionless number used in unsteady-state (or transient) heat transfer calculations, is named after him) also observed this same effect in liquids and gases of organic substances such as turpentine and some other petroleum products. About 10 years later the English astronomer Sir Joun F.W. Herschel discovered that different crystal forms of quartz rotated the linear polarization in different directions. Simple polarimeters have been used since this time to measure the concentrations of monosaccharide sugars, such as glucose, in solution. In fact, one name for glucose, -dextrose-, is so named because it causes linearly polarized light to rotate to the right or “dexter” side. Similarly, levulose, more commonly known as fructose (fruit sugar) causes the plane of polarization to rotate to the left. Fructose is even more strongly levorotatory than glucose is dextrorotatory. Invert sugar which is formed by adding fructose to a solution of glucose, gets its name from the fact that subsequent structural conversion causes the direction of rotation to “invert” from right to left.

The reason for this behaviour of these seemingly identical substances was not understood until the mid-19th Century when Pasteur was working on the problem of why wine was souring as opposed to fermenting into an alcohol solution. The culprit was yeast that metabolized the fructose in the grape juice to tartaric acid. A solution of tartaric acid derived from living things (the wine lees yeasts) rotated the plane of polarization of light passing through it, whereas chemically synthesized tartaric acid prepared by non-organic means in the laboratory did not have this effect. This was puzzling because both the synthetic and the biologically derived tartaric acid undergo the same chemical reactions and are identical in their elemental (atomic) composition. Pasteur noticed that the crystals came in two asymmetric forms that were mirror images of one another. He meticulously sorted the crystals by hand and then dissolved each of the two forms of crystals in water; solutions of one form rotated polarized light clockwise, while the other form rotated light counter-clockwise. An equal mix of the two had no polarizing effect on light. Pasteur deduced the molecule tartaric acid molecule was asymmetric and could exist in two different forms that resemble one another; as would left- and right-hand gloves, and that the organic form of the compound consisted purely of the one type.

This phenomenon is referred to as isomerism and occurs when two molecules have the same molecular formula (atomic composition) yet have different structures and therefore different chemical and physical properties. There are many different kinds of isomers. The two major divisions of isomers are the geometric and the structural. Structural isomers are isomers that have the same number of atoms but different arrangement of atoms. One structural isomer of glucose is fructose. Geometric isomers are identical in arrangement of covalent bonds but are different in the order that the groups are arranged.

A major category is stereoisomers which are two isomers that have the number of atoms in the same order. A stereoisomer of glucose is galactose. In the Fischer projection all of the atoms are the same except for one rotated group. There are two categories of stereoisomers, enantiomers and diastereomers. Enantiomers are two isomers that are mirror images of each other when looked at in 3D while diastereomers are not. Galactose is just one of many diastereomers of glucose. To find out the possible number of stereoisomer forms a monosaccharide can have, you can use the formula 2x where x is the number of chiral carbons the molecule has. Molecular chirality occurs when a sugar has a carbon with four different groups attached to it. Any carbon with a double bond on it is never chiral nor are the end carbons. Because glucose has four chiral carbons there are 24 different stereoisomers; which means that there are sixteen different stereoisomers for glucose.

Two of the main divisions of glucose’s many forms are l-glucose and d-glucose. These two are enantiomers which are determined by whether the two molecules are symmetrical at the last chiral carbon. When the hydroxyl group is on the last chiral carbon on the right it is considered d-glucose  and when it is on the left it is classified as l-glucose. The “d” means that the glucose rotates polarized light to the right (dextrorotatory) and the “l” stands for  levorotary (rotates polarized light to the left). These refer to how a plane of light rotates as it passes through a solution of it. First light is passed through a polarizing filter, then a polarimeter containing a solution made with the molecule. When a d-solution is in the polarimeter it will cause the light to turn to the right or at positive angle, while an l-solution will cause the light to turn to the left or a negative angle. Both d-glucose and l-glucose exist naturally but d-glucose, also called dextrose, is the most abundant sugar on the planet.

The practical biological and chemical implications of these isomeric structural differences is profound. D-glucose (dextrose) is the principal sugar used by the body to generate energy. By contrast, l-glucose cannot be significantly metabolized and an animal or human would starve to death if this was the only carbohydrate available in its diet and no other sources of energy (fats or proteins) were available. L-glucose looks, tastes and has the same mouth feel as d-glucose and there has been considerable interest in producing it in large quantities as an artificial sweetener.  Unfortunately, the synthetic pathway to produce l-glucose, and more importantly, the separation of the d- and l-glucose isomers after synthesis is currently prohibitively expensive.

What does all this have to with cryonics and organ preservation? Under normal metabolic conditions the cells of the body produce chemical energy in the form of ATP and about 1/3rd of this energy is used to pump ions into and out of the cells. This is necessary because the most common salts (ions) are very small and can easily pass through the cell membranes. Two straightforward examples are very much on-point. Cells need high concentrations of the potassium ion inside them to be able to function properly including carrying out some vital chemical reactions and doing things like contracting in the case of muscles or transmitting signals in the case of nerve cells. Conversely, cells must not have too much sodium in them or they  become swollen (edematous) and while this can ultimately rupture or lyse the cell, long before this happens cell swelling disrupts the meshwork of supports that maintain the cell’s shape and probably serve as scaffolding for various enzymes to be anchored on and to facilitate efficient chemical processing (metabolism). Unfortunately, sodium has a net negative charge and the protein inside cells has a net positive charge. Thus, sodium will flow into cells and carry water with it resulting in cellular edema. This process is prevented by active pumping of sodium out of the cell. Similarly, calcium is extremely toxic to cellular mitochondria in high concentrations and calcium is also used as a critical signalling molecule inside cells. Thus, the calcium concentration outside cells is typically 10,000 times higher than that present inside cells. Again, this difference in concentration is maintained largely by active pumping which requires energy expenditure and on-going metabolism.

So, sodium gets pumped out and potassium gets pumped in and this process is linked and carried out by the same molecular machine; the sodium-potassium pump. Of course, all of this presumes that there is both available energy in the form of ATP and that the cellular pumping machinery can use that energy. There are a number of things that can interrupt ion pumping. There can be a lack of energy due to starvation, hypoxia or ischemia, and there can exist situations where the energy is available but cannot be used. Some chemicals poison enzymes critical to ion pumping; a classic example is tetrodotoxin which comes from blowfish and which poisons sodium pumping. The other condition where adequate energy (ATP) can exist but cannot be used is deep hypothermia. Non-hibernating animals have enzymes that shut down or become inactive when the temperature is reduced well below that of normal body temperature. In humans (and most non-hibernating mammals) the  enzyme responsible for pumping sodium out of cells and potassium into them, sodium-potassium-ATPase, is largely inhibited at 10oC and is virtually shut down at few degrees above 0 oC.

Cell swelling in brain cells occurs with incredible rapidity after interruption of blood flow in ischemia (cardiac arrest). While cell swelling is not the only, or even primary, cause of injury in cerebral ischemia, it is a major player in causing injury in cold ischemia; conditions which obviously obtain in organ preservation and ultra-profound hypothermia in cryonics patients. The way this edema is prevented in organ preservation is to replace almost all of the small cell membrane permeable ions with big molecules that cannot pass through the cell membrane and which are osmotically active; in others words can hold water outside of the cell.  The first solution to do this with any success was Collin’s Solution invented by Geoff Collins. It used comparatively large phosphate salts to keep water outside of the cells and prevent cellular edema. However, phosphates do leak across the cell membrane and they are incompatible with DMSO and also precipitate as crystals when solutions are cooled to low temperatures or frozen.

Thus, the organ preservationists turned to sugars and sugar alcohols as molecules to serve as an osmotic agent and prevent cell swelling. Sugars are comparatively large molecules and some are very large. They do not typically pass through cell membranes rapidly, if at all. Some of the first sugars tried were glucose and sucrose and the sugar-alcohol mannitol. Neither glucose nor sucrose worked well. Glucose leaks across cell membranes fairly rapidly and has facilitated transport in the brain. Sucrose makes quite viscous solutions in the necessary concentrations (~180 mM) and for unknown reasons is toxic to the kidney tubule cells. Mannitol was much more successful in the laboratory but never made it into clinical organ preservation solutions.

In the 1980s, a biochemist named Jim Southard and a transplant surgeon named Folkert Belzer began to systematically study molecules to inhibit cold ischemic swelling, as well as other molecules to help conserve ATP, inhibit free radical damage, and otherwise address the derangements that occur under deep hypothermic conditions. They identified two sugars as particularly effective in inhibiting cold ischemic cellular edema, raffinose and lactobionate. They combined these two sugars along with other ingredients to create the first and still most successful “universal” organ preservation solution, UW-Solution, or as it is commercially marketed, Viaspan.

Unfortunately, ViaSpan does not work for the brain. We tried it extensively in the early 1990s and got serious cerebral edema followed by convulsions and death in dogs that had been perfused with ViaSpan for as little as two hours! By contrast, we could recover dogs perfused with MHP (a mannitol based perfusate) after 5-hours of bloodless perfusion with the solution at  5oC with no neurological or other problems; most of the dogs were placed with cryonics members and lived out the rest of their lives normally.

Recently, 21st Century Medicine has been systematically investigating hypothermic organ preservation and they have made a number of stunning breakthroughs. One thing which was long overdue to be done was to systematically screen various molecules for their cell swelling inhibiting effects. They found that one sugar in particular was highly effective, D(+)-lactose. Only the d-isomer worked well.

Why some sugars work and others do not, or actually cause harm, is a mystery. The molecular weight is certainly a factor, but different sugars with nearly identical molecular weights may perform totally differently. Also, the isomer of the sugar appears critical in some cases, as is the situation with lactose.  21st Century Medicine has patented an organ preservation based on D(+)-lactose and is in Phase II clinical trials for this solution, which they call TranSend. They are currently getting 72-hour simple flush and store on ice preservation of kidneys (rabbit and dog), pancreases and livers (dogs) and are getting similar results in the human clinical trials. They have achieved 48-hour heart preservation using a derivative of this solution which combines periods of trickle-flow cold perfusion with brief intervals of modest warming to ~15 oC.

09. September 2008 · Comments Off on The hostile wife phenomenon in cryonics · Categories: Cryonics · Tags: , ,

On August 23, Chana and Aschwin de Wolf drafted a blog entry on the phenomenon of partners who are hostile to cryonics. We sent our draft for review to a number of high profile cryonicists and received a message from Mike Darwin telling us that he still had an unpublished article on this topic. Because Darwin’s experience and observations resembled our own, and we believed there was a lot of value to his analysis, we decided to integrate our observations and his article to produce an even more comprehensive piece on the topic. The resulting draft  was distributed again to other cryonics activists and produced some additional text that was added as an appendix to the original article.

Although we do not have any illusions that the problem of hostile partners, and hostile female partners in particular, will be disappearing anytime soon, we believe it is important that this matter of life and death is documented. We also hope that this article will be of help to cryonicists, or people who want to make cryonics arrangements, who have hostile family members.

Michael Darwin, Chana de Wolf, Aschwin de Wolf – Is That What Love is? The Hostile Wife Phenomenon in Cryonics.

The article is also available as a PDF file with images and appendixes

04. September 2008 · Comments Off on Cryoenzymology and cryoprotectant toxicity · Categories: Cryonics, Science · Tags: , , , ,

The major limiting obstacle to reversible cryopreservation of complex organs is cryoprotectant toxicity. Elimination of ice formation through vitrification requires high concentrations of cryoprotective agents. These high concentrations of cryoprotectants can be toxic to tissues. Over the years, major advances by the cryobiology research company 21st Centrury Medicine have been made to reduce the toxicity of vitrification agents, culminating in the least toxic vitrification agent to date, M22.

In 2004, Fahy et al. published a landmark paper that proposed a model to predict general cryoprotectant toxicity. Although the authors speculate about the mechanisms of cryoprotectant toxicity in the discussion section of the paper, the emphasis of their investigations is to formulate less toxic vitrification solutions. Whereas general cryoprotectant toxicity is proposed to reflect cryoprotectant-induced perturbation of intracellular water, the mechanisms underlying specific cryoprotectant toxicity involve the effects of individual cryoprotective agents on macromolecules (for example, metabolic conversion of glycerol to a toxic compound).

A number of viability measures are available to investigate the toxicity of cryoprotective agents. One such measure is the potassium/sodium ratio. In complex organs such as the brain, other viability measures  are possible such as measuring electrical activity after vitrification and rewarming. These viability measures can be used to improve vitrification agents but they do not throw much light on the actual mechanisms of cryoprotectant toxicity. More “sophisticated” viability assays such as measurements of post-vitrification gene expression are available to help elucidating those mechanisms. Another technique that may hold promise for investigating cryoprotectant toxicity is cryoenzymology.

Cryoenzymology is the study of of enzymes at subzero temperatures in fluid solvents. The study of enzymes at low subzero temperatures overcomes two problems in studying enzyme reactions in steady state conditions: 1) the rapidity of the reactions and 2) the low concentrations of intermediates present. By starting enzyme-catalyzed transactions at low subzero temperatures the progressive transformation of intermediates into a subsequent one can be studied as the temperature is gradually increased.  This method can produce detailed structural and kinetic information of substrate-enzyme reactions which are not available at room temperature.

Because cryoenzymology requires a fluid aqueous environment at low subzero temperatures, organic cosolvents are used to prevent ice formation. Because the organic solvents used in cryoenzymology serve a similar function as cryoprotectants in vitrification, it is not surprising that we often find the use of the same solvents such as DMSO and ethylene glycol. An ideal solvent for cryoenzymology should inhibit ice formation without adverse effects on the structure or kinetics of the molecules that need to be studied. Researchers in cryoenzymology have also found that the presence of high concentration of organic solvents decreases the temperature at which proteins denaturate. Similarly, in cryobiology, there is a need  to expose biological tissues to low subzero temperatures without causing cryoprotectant-induced protein denaturation.

Although an ideal organic solvent for cryoenzymology is not necessarily an ideal cryoprotectant, observations of the interaction of organic solvents and proteins at subzero temperatures can throw light on  phenomena such as solvent-induced versus temperature-induced protein denaturation, chilling injury, cold shock, and solvent-water-protein interactions. The field of cryoenzymology also had to address a lot of challenges encountered in cryobiological research such as selection of proper buffers for use with organic solvents at cryogenic temperatures and the effect of solvent on solution viscosity.

Cryoenzymology is also of interest to other areas in biology such as the study of life under extreme conditions. The study of extremophiles is flourishing because of its relevance to astrobiology, the study of life (or the potential for it) in the universe.

Review papers on cryoenzymology:

Fink AL: Cryoenzymology: the use of sub-zero temperatures and fluid solutions in the study of enzyme mechanisms (1976)

Fink AL, Geeves MA: Cryoenzymology: the study of enzyme catalysis at subzero temperatures (1979)

Douzou P: Cryoenzymology (1983)

Travers F, Barman T: Cryoenzymology: how to practice kinetic and structural studies (1995)

31. August 2008 · Comments Off on Structure-function analysis of neuroprotectants · Categories: Cryonics, Neuroscience, Science · Tags: , , , ,

In “The chemistry of neuroprotection”, the author argues convincingly that there could be great benefit from a systematic and rigorously scientific study of the physical chemistry of putative neuroprotectants vis-à-vis their pharmacological effect. However, the first example used of the earliest thinking in this direction (which comes, not surprisingly via V. A. Negovskii, the father of resuscitation (1) medicine) is instructive as to some of the potential barriers standing in the way of this approach.

“It is not surprising that all the agents which are effective in shock carry a negative charge. This applies both to heparin, which possesses a very strong negative charge, and to hypertonic glucose solution. The same may be said about a substance now in wide use – dextran – which has small, negatively charged molecules, and also about the glucocorticoids 21, 17, and 11, which also have a negative charge.” – Professor Laborit in: Acute problems in resuscitation and hypothermia; proceedings of a symposium on the application of deep hypothermia in terminal states, September 15-19, 1964. Edited by V. A. Negovskii.

In the intervening decades since Laborit wrote the words quoted above, supraphysiologic (high) steroids have not only failed to demonstrate benefit in cerebral resuscitation and shock, they have been found to be actively harmful in every well designed RCT undertaken to test their utility (a). This also extends to their lack of utility in trauma, spinal cord injury and sepsis. Similarly, the utility of heparin in treating the encephalopathy of the post-resuscitation syndrome, or improving survival after cardiac arrest has recently been called into question. Glucose, hypertonic or otherwise, was long ago demonstrated to markedly increase neurological injury if given immediately after reperfusion following cardiac arrest, and elevated blood levels of glucose, both pre- and post cardiac arrest have a strong negative correlation with both survival and neurological outcome.

Determining the seriously harmful effects of steroid administration in critical illness took decades. Despite the compelling evidence for their injurious effects, administration of large, supraphysiologic doses of steroids is still a practice both used and defended by some clinicians (albeit not ones who rely on evidence based criteria) and the use of glucose in shock, trauma and cardiac arrest took a nearly comparable period of time to discredit. These two examples are noteworthy because they comprised mainstays of therapy for most kinds of neuroinjury for decades, and they had compelling theoretical appeal, as well as many positive small clinical and animal research studies. Indeed, the debate continues to this day with controversy centred mostly on the use of low or “physiological replacement” doses of steroids in critical illness. As the eminent pulmonologist and intensivist Neil Macintyre observed in 2005, “Patients die, but steroids never do.”  This raises the twin problems of bad research (i.e., junk science) and statistically under powered or otherwise flawed studies. Combined, it has been estimated that these two types of defective studies comprise the bulk of published peer-reviewed scientific work.

High dose corticosteroid therapy for neuroinjury offers another complication in determining the therapeutic efficacy of any drug that merits consideration as a neuroprotectant (new or old). While there is no doubt that high-dose corticosteroids are ineffective and deleterious in the clinical setting, there is also little doubt that these agents are neuroprotective in the laboratory setting under certain conditions and for discrete subpopulations of neurons. The reasons for the failure of translational research in the case of corticosteroids are complex, but are mostly attributable to crucial differences between the laboratory and the real world of clinical medicine. In the case of corticosteroids these differences are most significantly:

a.    Delay from time of insult to time of treatment; in the laboratory the timing of interventions is uniform and is typically much shorter than is the case in the clinic where delays in both presentation and treatment are both long and highly variable.
b.    Heterogeneity of injury in humans compared to animals; animal models of neuroinjury are highly standardized (location, extent, mechanics) whereas human patients present with diverse injuries inflicted in many complex and often poorly understood ways.
c.    Species differences; not only are there large genetic differences between humans and rodents in general, there are dramatic differences in the native ability of rodents to both resist and overcome infection in comparison to humans.
d.    Demographics and comorbidities: laboratory animals are comparatively very uniform genetically, are typically young and healthy and of the same age, do not have comorbid conditions such as hypertension, diabetes, atherosclerosis, obesity or the diminished physiological capacity and repair and regenerative capacity increasingly present in humans over the age of 25.
e.    Rodents aren’t people and do not interact with investigators in ways that facilitate straightforward determination of an adverse affect such loss of short term memory, or other cognitive deficits. It is now understood that the corticosteroids are toxic to the neurons of the hippocampus in both rodents and men. However, injury from this adverse effect is not only more evident in men than in mice (or rats for that matter), it is only men who are capable of complaining about it.

It is notable that all of these effects, with the possible exception of increased resistance to steroid-induced immunosuppression-mediated infection, obtain in the case of other translational models of drug development. The conclusion that corticosteroids are very likely neuroprotective in humans (in terms of the direct pharmacological effect on selected subpopulations of neurons in injured central nervous tissues under ideal conditions) is highly likely. However, the confounding realities of the clinic and the genetic differences between men and rodents (the animals almost exclusively used in this type of research) mask this effect. This poses yet another serious challenge to investigators seeking to establish common moieties in prospective neuroprotective molecules.

Clinical trials of putative neuroprotective substances have been overwhelmingly negative. This has been the outcome despite often stellar results achieved in animal models; often in diverse species in studies conducted by multiple investigators in different institutions and sometimes in different countries; none of whom have any obvious relationship, let alone one that might raise the specter of conflict of interest. In the last 6 years alone, over 1000 experimental papers and over 400 clinical articles have appeared on this subject. What this suggests is that the same deficiencies seen in studies reported upon in rest of the peer-reviewed biomedical literature also apply to studies of pharmacological intervention in neuroprotection. An inevitable conclusion is that until the signal to noise ratio improves, attempts to draw general conclusions about  the shared, essential properties of neuroprotective molecules will be difficult at best, and unreliable or misleading at worst.

Perhaps a good place to start this kind of analysis is in an area where the molecular structure of the agent(s) is extraordinarily simple and the animal and clinical data are both robust and show good to fair agreement. Hypertonic sodium chloride solutions have demonstrated efficacy in providing both systemic (splanchnic) and cerebral protection in a broad class insults including hemorrhagic/hypovolemic shock, closed head injury and less robustly in stroke and global cerebral ischemia. Interestingly, other cation salts of chloride given at comparably high tonicity do not have this effect. Furthermore, animal as well as small human clinical studies have demonstrated isochloremic hypertonic solutions to be as effective as hypertonic sodium chloride at restoring microcirculatory flow and reversing metabolic acidosis in haemorrhagic shock without the potentially troublesome side-effect of raising the mean arterial pressure to levels where re-bleeding may occur in trauma or subarachnoid haemorrhage.  A relative lack of effectiveness of the chloride salt of magnesium compared to the sulfate salt of this ion has also been noted. Understanding the mechanics of these paradoxes would seem to be a worthwhile and comparatively straightforward place to begin such structure-activity relationship analyses.


Cerebroprotective drugs not infrequently possess a multiplicity of pharmacological effects that are known to be neuroprotective but that may be accomplished by very different and even indirect means in terms of their structure-function relationship. Some cerebroprotective molecules, such as the female hormone 17β-estradiol and the mixed estrogen antagonist-agonist tamoxifen share common physiochemical properties such as free radical scavenging, N-methyl-d-aspartate (NMDA) receptor inhibition, and modulation of volume regulated anion channels (VRAC); which play a role in ischemia-induced release of excitatory amino acids. There is considerable evidence that some of 17β-estradiol’s neuroprotective effect is via signal transduction as well as its neurotrophic effects, even at doses below those necessary for its direct effects on reactive oxygen species production and its NMDA receptor inhibiting effects. While the structure of the molecules shares some important features, they are also structurally very different and the signal transduction and neurohormonal effects are almost certainly very different. Thus, these molecules also present a fascinating opportunity to probe structure-function relationships in neuropharmacology.


Finally, an admission, or perhaps a confession is order in ending this discussion. This author has been responsible for the application of at least one putative neuroprotective drug to cryopatients which ultimately proved ineffective in human clinical trials when administered during and after cardiopulmonary resuscitation (CPR). This drug, nimodipine, performed well in animal trials, but failed to show benefit in human trials, possibly as a result of its hypotension-inducing effect. Adequate mean arterial pressure (MAP) following resuscitation from cardiac arrest is essential to survival and a post arrest bout of hypertension has been demonstrated to provide substantial cerebral rescue in animal models of global cerebral ischemia. Reduction of MAP in cryopatients is a serious concern because achieving adequate perfusion pressure is problematic under the best of conditions. It is also worth noting that cryopatients have been given a variety of other ineffective neuroprotective drugs over the past 30 years, including the opiate agonist naloxone, the corticosteroid methylprednisolone and the iron chelating drug desferroxamine.

While these drugs, with the possible exception of nimodipine, are not likely to have been injurious (except perhaps to the pocketbook), their use raises important questions about when and how promising animal research should be translated to the setting of clinical cryonics. Unique among all other populations of human and animal patients, cryopatients have the opportunity to be treated with neuroprotective drugs that show great promise, absent the long delays of regulatory vetting, and independent of the economic pressure experienced by pharmaceutical companies to not only market drugs that are effective, but to market ones that are also profitable. The question thus becomes what criteria do we use in applying these drugs absent the extensive pre- and post marketing evaluation that obtains with approved ethical drugs? In essence the question we must ask and answer is “can we do better, much better in fact, than our colleagues in conventional critical care medicine?

Michael G.  Darwin, Independent Critical Care Consultant

Click here for this entry with references in PDF format.


(1) Resuscitation medicine is properly termed reanimatology, and is so-called in the non-English speaking world

(a) The one condition in which there is unequivocal benefit to supraphysiologic administration of steroids is meningococcal meningitis with substantial evidence also supporting a similar degree of efficacy in Typhoid  and Pneumocystis carinii pneumonia.

25. August 2008 · Comments Off on The chemistry of neuroprotection · Categories: Neuroscience · Tags: , , , , ,

In a review of the 1998 21st Century Medicine seminars, Cryonics Institute president Ben Best writes:

“The presentations impressed upon me how much witchcraft and how little science has gone into the study of cryoprotectant agents (CPAs). This might be understandable in light of the fact that most cryobiologists are, in fact, biologists. I suspect that a great deal could be accomplished by a thorough study of the physics of the chemistry of CPAs.”

Such an observation could equally apply to the study of neuroprotectants in cerebral ischemia. There has been a growing literature investigating the potential of numerous molecules for the treatment of stroke and cardiac arrest. Although some approaches have been more successful than others, systematic reviews of the chemical and physical characteristics of effective drugs are lacking and discussion of the topic is  often confined to isolated remarks.

A number of examples:

“It is not surprising that all the agents which are effective in shock carry a negative charge. This applies both to heparin, which possesses a very strong negative charge, and to hypertonic glucose solution. The same may be said about a substance now in wide use – dextran – which has small, negatively charged molecules, and also about the glucocorticoids 21, 17, and 11, which also have a negative charge.” – Professor Laborit in: Acute problems in resuscitation and hypothermia; proceedings of a symposium on the application of deep hypothermia in terminal states, September 15-19, 1964. Edited by V. A. Negovskii.

“We report that estrogen and estrogen derivatives within the hydroxyl group in the C3 position on the A ring of the steroid molecule can also act as powerful neuroprotectants in an estrogen-receptor-independent short term manner because of to their antioxidative capacity.” Christian Behl et al. Neuroprotection against Oxidative Stress by Estrogens: Structure-Activity Relationship. Molecular Pharmacology 51:535-541 (1997).

“Minocycline’s direct radical scavenging property is consistent with its chemical structure, which includes a multiply substituted phenol ring similar to alpha-tocopherol (Vitamin E)” – Kraus RL et al. Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity. Journal of Neurochemistry. 2005 Aug;94(3):819-27.

“It is notable, however, that NAD+ and minocycline share a carboxamide and aromatic ring structure. A common structural feature of competitive PARP inhibitors is a carboxamide group attached to an aromatic ring or the carbamoyl group built in a polyaromatic heterocyclic skeleton. This structure is also present in each of the tetracycline derivatives with demonstrated PARP-1 inhibitory activity. Alano CC et al. Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations. Proceedings of the National Academy of Sciences of the United States of America. 2006 Jun 20;103(25):9685-90.

Systematic study of structure-activity relationship of neuroprotectants would not only contribute towards the development of a general theory of neuroprotection in cerebral ischemia, it would also contribute to the design of multi-functional neuroprotectants. Although it is now increasingly accepted that combination therapy offers more potential for successful treatment of stroke and cardiac arrest than mono-agents, parallel or sequential administration of multiple drugs present non-trivial challenges in research design and clinical application. Such problems may be better addressed by designing molecules with different mechanisms of action in the same structure, an approach that is currently recognized and investigated by forward-looking biomedical researchers.

Although the field of cerebral resuscitation has known some notable researchers  like Vladimir Aleksandrovich Negovskii and Peter Safar, who devoted their lives to a thorough study of the mechanisms of cerebral ischemia and its treatment, the field as a whole shows a never ending stream of trial and error publications to investigate yet another drug (before moving on to other areas in neuroscience and medicine). Although there is an increased interest in meta-analysis of all these experiments, meta-analysis that places its findings in a broader biochemical and pharmacological context is rare.

The emphasis on theory and research design can be taken too far. As Nassim Nicholas Taleb recently argued, the role of design in biotechnology is overestimated at the expense of chance observations and unexpected directions. But in the area of cerebral resuscitation the risk of too much theory and systematization is low at this point. As evidenced by the successful development of vitrification agents with low toxicity in cryobiology, a committed long-term and systematical effort to find solutions to human medical needs can pay off.

21. August 2008 · Comments Off on Edvard Munch's Death in the Sick Chamber · Categories: Arts & Living, Death · Tags: , , , ,

Edvard Munch’s painting “Death in the Sick Chamber” (1895) portrays death as expressed through the survivors. A striking aspect of this work is that all the people in the room do not console one another and are physically and emotionally isolated.

In “Modern Art and Death”, Carla Gottlieb writes:

….the faces are contorted, not in mourning for a beloved lost member, but in fear of the unknown which has just swallowed the deceased, fear for themselves who are eventually to meet the same fate. In this agony, each person is alone; each survivor turns away not only from the dead but also from the other participants in the scene. Faced with death, the family bonds fall apart, revealing their superficial character. Thus Munch experienced death as dissolving family ties…

The accompanying black and white lithograph evokes an even bleaker atmosphere, as can be seen in the sunken eyes and grim mouth of the woman facing the viewer.

Munch shows the destabilizing and alienating effects of death. Although the people in the room seem to be at a loss how to proceed in life, there is closure. In cryonics such closure is not available. Cryonics also destabilizes the fabric between people because some may survive and others may not. The idea of cryonics can also produce guilt about loved ones who died and never got the chance. These factors, and not just technical feasibility alone, may explain why cryonics is so unpopular.

20. August 2008 · Comments Off on Dietary supplements induce neurogenesis after stroke · Categories: Health, Neuroscience · Tags: , , , ,

A recent study in Rejuvenation Research reports that a combination of dietary supplements confer neuroprotection in stroke. Over a 2 week period rats received either a proprietary formulation of blueberry, green tea, Vitamin D3, and carnosine  called NT-020 or vehicle (i.e., the same solution minus the compounds of interest) before stroke was induced through middle cerebral artery occlusion (MCAo). Two weeks after the insult the rats were subjected to behavioral tests and histological examination. Rats treated with the dietary supplements scored better on behavioral tests, had less histological damage, and showed evidence of neurogenesis.

This study is interesting for a number of reasons. Foremost, it highlights the possibility that dietary choices can positively affect outcome after ischemic insults. These findings complement research that found that caloric restriction improves behavioral and histological outcome after stroke.  The findings also reinforce that some of the most effective neuroprotective agents to date are ordinary nutrients, vitamins, and hormones. In this study the investigators combine a number of these agents to greater effect. Although the authors do not present specific data on bioavailability in the brain for these compounds, they argue that a multi-agent approach relaxes the dosage requirements for individual agents.

The paper reviews assays that demonstrate improved neurogenesis in the rats that received NT-020 such as endogenous birth of new neurons, neuronal phenotype expression of newly formed cells, and alterations in neurogenic factors. Pharmacological modulation of neurogenesis after ischemia is a young research field and the results reported in this paper provide additional evidence for the (only recently accepted) phenomenon of adult neurogenesis. Unresolved questions at this point include how neurogenesis differs among species and whether post-ischemic neurogenesis can improve long term outcome in humans.

The  design of the current study does not allow a rigorous answer to the question of whether neurogenesis contributed to or accompanied improved outcome. The possibility that other mechanisms (such as  increased free radical scavenging) were primarily responsible for the observed improvements cannot be ruled out based on this study.

Link: Dietary Supplementation Exerts Neuroprotective Effects in Ischemic Stroke Model

18. August 2008 · Comments Off on Revitalize aging feet · Categories: Health · Tags: , , , ,

My mother, being a decidedly well put-together woman, impressed upon me the importance of self-care from an early age. She was obsessed with skin maintenance and especially careful to instruct me in hand and foot care. I was given my first bottle of moisturizer at the age of fourteen (“I heard your skin starts losing its elasticity at that age”) and vividly recall sharing in an invigorating bi-weekly foot soak and pedicure. Later on, after developing severely fallen arches (aka “flat feet”) and enduring the pain associated with that condition, foot care became an especially important part of my self-care routine and I have since become somewhat of a foot care proselytizer.

As such, I was delighted to see an article entitled “Revitalize Aging Feet: The Importance of Proactive Foot Care” in the latest issue of Life Extension Magazine. This article, by Dr. Gary Goldfaden, begins with a spiel that I also frequently employ, alerting readers to the fact that the feet are the most overworked and undercared for part of the human body. For these reasons, our feet are particularly susceptible to injury, fatigue, infection, and skin aging –more so as we age and they lose their protective fat cushioning and have been exposed to a lifetime of ultraviolet radiation.

But, as with most things related to the body, an ounce of prevention is worth a pound of cure. Preventative foot care can not only make your feet look better, it can also lessen pain and muscle fatigue, which ultimately makes your entire body feel better.

As Dr. Goldfaden points out, many commercial foot creams consist primarily of water, which only serves to “plump up” the skin, thus smoothing out wrinkles, for as long as the water remains. Additionally, many of these products also contain oils that can actually increase free-radical oxidation and accelerate skin aging! Fortunately, there are some natural products that can significantly improve the look and condition of the feet.

Essential oils such as eucalyptus and menthol are a great place to start. Eucalyptis oil contains a compound called 1,8-cineole, which helps facilitate the production of skin lipids (ceramides), an important factor in retaining moisture in the skin. Eucalyptus oil also serves to protect feet from microorganisms that cause odor and infection, and acts as a natural analgesic for soothing achy joints and muscles. Menthol is also an effective pain reliever, and has the added benefit of providing a cooling sensation which is very refreshing for tired feet. Both eucalyptus oil and menthol also have beneficial effects on foot circulation, increasing blood flow to the feet and promoting the delivery of oxygen and nutrients to the deepest layers of the skin.

Also discussed is tea extract, which is rich in anti-oxidants which can protect feet from oxidative stress and inflammation. Other properties of antioxidant tea blends, such as their vitamin C activity, are also believed to contribute to improved skin tone and structure by strengthening connective tissues. Squalene, found in olive oil, is a natural emollient that hydrates and nourishes tissue while also providing anti-oxidant effects and inhibiting the proliferation of microorganisms. Coconut oil has an abundance of medium chain triglycerides that are “almost identical to the medium chain fatty acids found in human sebum” and is also a proven antibacterial, antiviral, and antifungal agent. Last, but not least, shea butter is touted for its abundance of vitamin E, a powerful antioxidant that is known for its ability to diminish wrinkles and smooth skin tone. I have personally found that buying vitamin E oil from a pharmacy is also very cost effective and works wonders to keep my feet looking and feeling soft and supple.

While the LEF article is timely and full of good advice, I was somewhat disappointed that it did not discuss other aspects of preventative and therapeutic foot care such as wearing appropriately supportive shoes, inserts and orthotics, visiting a specialist in case of foot disorders such as flat feet or neuromas, and the benefits of massage and reflexology. Expect to see a follow-up at this blog covering these topics in the near future.