How many neurons need to survive for cryonics to work?

On this page a calculation is attempted to determine how many neurons need to survive for cryonics to work. The flaw in this approach should be obvious when the author writes :

According to The Stroke Association, a stroke is a brain injury with effects which may include difficulty thinking, learning, concentrating, remembering, making decisions, reasoning and planning. Rehabilitation consists of relearning skills, not having your brain recover naturally.

So a reasonable position is that the cryonic chilling process should cause less damage to the brain than a stroke

The debilitating effects of a stroke are the result of the (delayed) neuronal death that follows an ischemic insult to the brain. In cryonics, biochemical or freezing damage to cells does not necessarily produce irreversible cell death because damaged cells are stabilized by cold temperatures. As such, morphological preservation of brain cells can co-exist with loss of viability. Therefore, securing viability of brain cells is a sufficient but not a necessary condition for resuscitation of cryonics patients.  Future cell repair technologies are assumed to infer the original viable state of the cells from their morphological properties.

This does not mean that conventional stroke research does not have any relevance for evaluating the technical feasibility of cryonics. Extensive delays between the pronouncement of legal death and the start of cryonics procedures could alter the structural properties of cells to such a degree that meaningful resuscitation is even problematic with advanced nanomedical cell repair technologies. This is one of the reasons why Alcor complements the cryopreservation process with stabilization procedures to secure viability of the brain after pronouncement of legal death.

The science of personal survival

There are various competing strategies how to achieve meaningful life extension or rejuvenation, including , but not limited to, genetic manipulation, periodical elimination of damage, caloric restriction,  molecular nanotechnology and mind uploading. A useful review of these strategies has been published in the book The Scientific Conquest of Death: Essays on Infinite Lifespans (2004) by the Immortality Institute. Most people will recognize that these strategies are not mutually exclusive. Some of them can be practiced right now (e.g., caloric restriction) and others ( e.g., periodical elimination of damage) could serve as a bridge to more comprehensive interventions such as a comprehensive genetic overhaul of human biology. As has often been recognized on this website, cryonics holds a special place among life extension strategies because it enables one to benefit from progress in the biomedical sciences that may not occur during one’s lifetime. We would like to think we can escape death by jumping from one successful biomedical innovation to another and that, of course, all the good things will happen in our lifetime, but reality often interferes with such optimism.

One thing that might greatly accelerate the pace of progress in the field of longevity science is the development of an integrated framework that studies the logical and empirical relationships among all these strategies. For example, a recent blog entry on the technical challenges surrounding chemopreservation of the brain triggered a meaningful private exchange about issues concerning the perfusion of ischemic tissue, empirical criteria for information-theoretic death, and the options for histological validation of cryonics technologies.  Such overlapping areas of investigation are plentiful and it would be helpful to explicate them.

Too much focus on “the big picture” can interfere with the identification of original ideas and rapid progress. Too little attention to the adverse effects of compartmentalization risks the waste of resources, which is not a trivial concern in the still poorly funded life extension community.

Reducing compartmentalization can have other sobering effects as well. For example, it is not unusual to see a group of researchers advocating a new approach to their field that is routine in other areas of investigation. For example, the idea that anti-aging research could benefit from less emphasis on illuminating the exact molecular mechanisms of aging and simply treat the observable manifestations of aging is no news to researchers in the field of cerebral ischemia. The pathophysiology of stroke is so complex that greater progress could be achieved by identifying clear targets for pharmacological intervention. But after decades of research it has become abundantly clear that such a paradigm change is no guarantee for more rapid progress. Despite this goal-oriented approach not one single neuroprotective agent has survived clinical trials.  This does not mean that such pragmatic approaches should be abandoned. It does mean, however, that research ideas should be evaluated on their empirical success and not just on their logical merits.

There are obvious examples where the claims in one field seem to make the claims in another field redundant. The most obvious example is the case of molecular nanotechnology. The projected timescales that are envisioned for this technology are not much different from the timescales that are envisioned by some anti-aging researchers to develop meaningful rejuvenation. In that case one could argue that (exclusive) preference should be given to those research programs that allow for the most comprehensive manipulation of biology. For example, a mature nanotechnology would be able to rejuvenate people, resuscitate cryonics patients, and alter the human endoskeleton to make us far less prone to fatal accidents. Such an argument would be a logical extension of the argument against devoting too much time to find treatments for specific age-related diseases instead of tackling aging itself.  Similar reasoning can be employed against anti-aging research. If accelerated change will bring the prospect of general molecular control within reach in the next few decades it makes little sense to spend vast amounts of time agonizing over specific anti-aging interventions. Why not just launch a “Manhattan Project” to pursue the much more comprehensive vision of molecular nanotechnology?

From a logical point of view, this is a persuasive argument. The limitations of such a perspective should now be obvious too.  We do not have certainty about the future of technological progress, let alone its specifics. As a matter of fact, in such matters it is not even evident how we should think about statistical or inductive probabilities.  To some people, the progress in one field is indicative of the progress we are going to observe in other fields, including fields in which there has been little progress to date. The problem with such naive inductivism is that it can just as well be used to  make the opposite case if a different reference class is chosen.

The logical empiricist philosopher Rudolf Carnap once wrote:

The acceptance or rejection of abstract linguistic forms, just as the acceptance or rejection of any other linguistic forms in any branch of science, will finally be decided by their efficiency as instruments, the ratio of the results achieved to the amount and complexity of the efforts required. To decree dogmatic prohibitions of certain linguistic forms instead of testing them by their success or failure in practical use, is worse than futile; it is positively harmful because it may obstruct scientific progress.

A related argument can be made about the science of personal survival. We should be cautious about privileging any line of research on  “logical” grounds. The fate of competing visions should be decided through empirical investigation.  This position should not be interpreted as saying that there is no place for logic in choosing research programs.  Logic has a central place in research design and interpretation of experimental observation but it cannot be solely relied upon a guide for decision making. Empirical observation disciplines thinking and ample room should be left for the unexpected. As Nassim Nicholas Taleb has pointed out:

There is a lot more randomness in biotechnology and any form of medical discovery. The role of design is overestimated. Every time we plan on trying to find a drug we don’t because it closes our mind. How are we discovering drugs? From the side-effects of other drugs.

Many experimental researchers have had the experience of engaging in research to find a solution to one problem but to discover the solution to another problem instead. Researchers who have recognized and embraced this phenomenon by becoming less fond of their own ideas and more open to run with such unexpected discoveries have reaped great benefits.

Gender differences in stroke treatment and prevention

Over the years, experimental science has developed a standard protocol for the testing of medical hypotheses using animal models which calls for the use of males only. Why? Because no laboratory scientist wants to deal with those pesky female hormones. Female hormone fluctuations are viewed as just another variable to be controlled (generally by excluding females altogether) — all the better for making interpretation of results simple and straightforward.

But, as common sense might dictate, it turns out that results from male-only animal models often give a less-than-accurate view of the whole picture when this research is translated and applied to treatment of disease in humans. Why? Because, as most people without a doctorate in physiology can tell you, physiological gender differences exist. Is it any surprise, then, that disease treatment and prevention should also be prescribed with these physiological differences in mind?

And so the buzz for the past few years in the medical community is the astonishing fact that stroke treatment and prevention are not the same in men and women. In labs that have recently begun to investigate these differences, drugs that were found to protect male brains against stroke in animal models did nothing to protect female brains. The major message behind all this press: doctors cannot continue to apply one-size-fits-all prescriptions for stroke prevention and treatment.

The real fact is that it is even more complicated than a “simple” physiological difference. Traditionally, cardiovascular disease has been viewed as a “man’s disease” (men have about a 19 percent greater chance of stroke than women). Accordingly, studies have found that women are less likely to receive prescriptions for blood pressure medications or be advised to take aspirin, both of which have been shown to reduce stroke risk. Strangely, women are less often treated after having a stroke, even though they appear to respond better to acute stroke treatment (such as tissue plasminogen activator) than men. So while men do indeed have more strokes, women are still more likely to die from stroke.

Women are also at increased risk if they take birth control pills, use hormone replacement therapy, have a thick waist and high triglycerides, or are migraine sufferers. And, contrary to anecdotal evidence, women appear to be less likely to go to the hospital at the first sign of stroke symptoms.

Oregon Health and Science University is at the forefront of research into gender differences in medicine, having developed the first research institute of its kind, the OHSU Research Center for Gender-Based Medicine. Given that Oregon recently ranked 46th out of 50 states for incidence of stroke deaths among women (as reported by Making the Grade on Women’s Health: A National and State-by-State Report Card, 2007), there is obviously a need for gender-based medical research to save the lives of women at increased risk of cardiovascular and other disease.

The chemistry of neuroprotection

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.

Dietary supplements induce neurogenesis after stroke

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

Critical cooling rate to prevent ischemic brain injury

Induction of hypothermia can reduce injury to the brain when it is deprived of oxygen. How fast do we need to cool a patient during cardiac arrest or stroke to prevent irreversible injury to the brain?

It is an established fact that induction of hypothermia prior, during, or after circulatory arrest can reduce brain injury. As a general rule, the lower the temperature is dropped, the longer the brain can tolerate circulatory arrest. The neuroprotective effects of hypothermia are often expressed using the Q10 rule which says that for every 10 degrees Celsius drop in temperature metabolic rate decreases by 50%. Or to put it differently, the Q10 rule states that ischemic damage susceptibility is decreased by a factor of 2 for every 10 degrees Celsius temperature drop.  Q10 may vary between species and in different organs and cells. For example, different temperature sensitivities were observed for release of the neurotransmitters glutamate, aspartate, glycine, and GABA during cerebral ischemia by Nakashima et al. Because even very modest reductions of brain temperature can have profound neuroprotective effects, the Q10 rule may not tell the complete story.

Other things being equal, it would be very useful to have a measure of brain injury when hypothermia is induced prior and/or during cardiac arrest. At least two authors have made an attempt to produce such a measure of ischemic exposure. In Cryonics Magazine (2nd Quarter, 1996), Michael Perry started initial work on this in an article called “Toward a Measure of Ischemic Exposure” (PDF).  Perry’s Measure of Ischemic Exposure (MIX) calculates how long the patient has been at a given temperature, with a higher weighting used for higher temperatures. A related measure has been proposed be Steve Harris called the E-HIT. E-HIT stands for Equivalent Homeothermic Ischemic Time. In his (unpublished) manuscript, Harris uses the E-HIT formula to calculate the equivalent normothermic ischemic time for different cryonics case scenarios and real cases. Clearly, the availability of such a measure (and its routine calculation in case reports) would constitute a major contribution to cryonics as evidence based medicine. It could aid in deciding if viability of the brain was maintained during cryonics procedures by estimating the equivalent warm ischemic time.

What makes such a measure complicated during cardiopulmonary resuscitation (CPR), or cardiopulmonary support (CPS) in cryonics stabilization procedures is that hypothermia may only constitute one intervention to mitigate brain injury. In an ideal cryonics case, pronouncement of legal death is followed by rapid restoration of oxygenated blood flow to the brain by (mechanical) cardiopulmonary support, administration of neuroprotective drugs and induction of hypothermia. Such a combination of interventions might avoid any injury to the brain, reducing the equivalent warm ischemic time to zero. A more realistic scenario is that such a combination of interventions may reduce the extent of ischemic injury compared to cooling only. Another complicating factor is that oxygenation in combination with low perfusion pressures might produce more injury than “anoxic cardiopulmonary support” (chest compressions without ventilation). It is clear that calculating a measure of equivalent ischemic time for real cryonics cases can become very complicated.

It would be interesting to know the cooling rate that would be necessary to stay ahead of brain injury, using contemporary medical criteria, during circulatory arrest. For this purpose we use some very simplifying assumptions:

1.The patient is not ischemic prior to pronouncement of legal death.

2. Cooling is initiated immediately after pronouncement of legal death.

3. There is no cardiopulmonary support or administration of neuroprotective agents.

4. Brain injury starts at 5 minutes of warm ischemia.

5. Q10 is 2.0: for every 10 degrees Celsius we decrease the temperature , metabolism is dropped 50% , which doubles the time a patient can tolerate ischemia.

6. No other forms of injury occur other than ischemic injury.

7. Ischemic injury is completely eliminated at the glass transition temperature of the vitrification agent M22 (-123.3°C).

8. A constant cooling rate is assumed.

Using these assumptions, Alcor’s Mike Perry calculates that a cooling rate of 2.89 degrees Celsius per minute is necessary to stay ahead of the equivalent of 5 minutes of warm ischemia.

Let Ehit = total ischemic time limit in hours, 1/12 corresponding to 5 min
Q10 = factor of decrease in metabolism per 10 degrees
Tdrop = desired temperature drop, from 37 degrees (body temp) down to -123.3= 160.3 degrees Celsius
ch=desired cooling rate in deg/hour
cm=desired cooling rate in deg/min = ch/60


ch = 10*(1-exp(-Tdrop*ln(Q10)/10))/(Ehit*ln(Q10))

For Q10=2, Tdrop = 160.3, cm = 2.89 deg/min

If some of the assumptions are slightly changed we find the following for Q10=2.2

For Q10=2.2, Tdrop = 160.3, cm = 2.54 deg/min

If we assume negligible ischemic insult below 0 Celsius and only worry about cooling down to that temperature, so Tdrop is only 37 rather than 160.3, it doesn’t change these amounts drastically:

For Q10=2, Tdrop = 37, cm = 2.66 deg/min
For Q10=2.2, Tdrop = 37, cm = 2.40 deg/min

Clearly, such high cooling rates cannot be achieved during either conventional cardiopulmonary resuscitation or cardiopulmonary support in cryonics. The cooling rates we can hope for during the initial stages of cryonics procedures may exceed 1.0 degrees Celsius per minute at best. It is therefore not realistic to assume that cooling alone may be able to limit brain injury to a degree that allows resuscitation without adverse neurological effects using contemporary medical criteria. This should strengthen the case for the use of other interventions such as administration of neuroprotective agents and oxygenation of the patient. Although the latter intervention may produce adverse effects on the brain itself, the calculations above indicate that anoxic cardiopulmonary support is not compatible with maintaining viability of the brain as the objective of cryonics stabilization procedures. The case for rapid stabilization of cryonics patients remains strong.

Cerebral ischemia and impairment of circulation

Cryopreservation of the brain depends on the removal of blood from the brain’s vasculature and its replacement with cryoprotective solutions in order to prevent ice crystal formation (freezing) during cooling (i.e., facilitate vitrification). Ultimately, the success of a good cryoprotectant is limited by perfusability of the brain, or the ability of cryoprotective solutions to penetrate all areas and cells of the brain via the cerebral vessels. Long periods of global cerebral ischemia detrimentally affect reperfusion of the cerebral vessels, thereby significantly degrading perfusability of the brain. Many possible causes for post-ischemic impaired perfusion have been hypothesized in the past, including swelling of the endothelial cells that make up the inside lining of blood vessels. However, a landmark 1972 paper by Fischer & Ames provided evidence implicating changes in the blood itself as the probable reason for post-ischemic reductions in perfusability of the brain.

Ames, et al. had already demonstrated impaired reperfusion in rabbits after cerebral circulatory arrest followed by infusion of a suspension of carbon black and examination of coronal brain sections. Brains undergoing less than five minutes of arrest perfused well and were evenly stained by carbon black ink. Ischemia in excess of five minutes resulted in patchy white areas where perfusion was impaired and blood vessels did not fill with ink. In an extension of this work, Fischer and Ames investigated the effects of perfusion pressure, anticoagulation, and hemodilution on post-ischemic perfusability.

In their experiment, the researchers induced cerebral circulatory arrest in rabbits and then perfused the head and neck with carbon black immediately following various ischemic periods. The perfusion solution was introduced to the cerebral circulation from a reservoir placed above the rabbit and through tubing that was inserted into and secured in the ascending aorta. Perfusion pressure may thus be modulated by varying the height of the reservoir above the animal. In some animals, acute hemodilution was achieved by rapid infusion (50 ml/kg) of saline into the femoral vein for moderate hemodilution or by a combination of saline administration and blood removal for extreme hemodilution prior to ischemia and carbon black infusion. A series of animals were also anticoagulated by giving heparin (500 units/kg) intravenously 15 minutes prior to ischemia. Brains were then removed and diffusion fixed in 10% formalin, allowing coronal sections of the brain to be examined macroscopically.

Brains from 5 groups of animals were studied:

Group 1: 4.5 minutes’ ischemia, reservoir at 28 cm above the heart (two animals), 40 cm (two animals), and 110 cm (two animals).

Group 2: 15 minutes’ ischemia, reservoir at 70 cm (six animals), 110 cm (eight animals), and 170 cm (seven animals).

Group 3: 30 minutes’ ischemia, reservoir at 170 cm (six animals).

Group 4: Hemodilution, 15 minutes’ ischemia, reservoir at 70 cm, hematocrit (HCT) 21 to 32 (five animals); HCT 4 to 13 (six animals).

Group 5: Heparin, 15 minutes’ ischemia, reservoir at 70 cm (six animals).

As previously observed, “animals with 4.5 minutes of ischemia failed to demonstrate impaired cerebral reperfusion even with perfusion pressures as low as 28 cm of water, all brains being completely and evenly black.” However, significant impairment of perfusion was observed after 15 minutes of ischemia. The effect was greatest in the thalamus and brain stem, while cerebral cortex remained adequately perfused after 15 minutes of ischemia at all levels of perfusion pressure. White (non-perfused) areas were significantly greater after 30 minutes of ischemia than after 15 minutes at comparable pressures and in all areas examined (including cortex). Hemodilution greatly improved reperfusion after 15 minutes of ischemia, but no difference between anticoagulated animals and controls was observed.

The authors speculate that blood viscosity, rather than clotting or cellular swelling, was the most probable cause of impaired reperfusion following ischemia. They further noted that “improved postischemic cerebral circulation has also been noted to follow the infusion of hyperosmolar agents,” which they speculated was also due to reducing blood viscosity. Red cell aggregation during blood stasis was assumed to be a major contributing factor to the increase in viscosity, and differences in vascular resistance throughout the brain manifesting as differences in circulatory impairment were thought to underlie the observation of impairment of discrete, macroscopic regions of the brain — particularly subcortical regions.

The results of Fisher et al. support rapid restoration of perfusion pressure (mechanical cardiopulmonary support) after cardiac arrest in cryonics patients, and hemodilution with agents like Dextran-40. It is harder to reconcile with the emphasis in cryonics to administer fibrinolytics and anticoagulants to eliminate and prevent blood clotting. The results of Fisher et al. are also at odds with those of Böttiger et al., who found that activation of blood coagulation after cardiac arrest is not balanced adequately by activation of endogenous fibrinolysis. Perhaps these findings can be reconciled if we allow for the possibility that cardiac arrest and cerebral ischemia induce formation of (micro)thrombi, but that these are not clinically significant, or at least do not affect reperfusion as greatly as other blood abnormalities such as red cell aggregation. And perhaps the formation of thrombi in small cerebral vessels can adversely affect cryoprotectant perfusion without being visible by gross examination. Formation of large clots may still be a problem after longer periods of circulatory arrest. Tisherman et al. have observed large vessel blood clots in rats and dogs after normothermic cardiac arrest of more than 20 minutes. Finally, cryonics patients may present with existing blood clotting problems as a result of (septic) shock.

Although the emphasis on antithrombotic therapy to maintain circulatory patency in cryonics patients seems to be warranted, more emphasis on other factors that affect cerebral (micro) circulation in cryonics patients seems desirable. As the work of Fisher et al. indicates, hypertension and hemodilution during cardiopulmonary support may be just as, if not more, important. The relationship between in vivo stasis of blood circulation and coagulation remains elusive and could benefit from more research.

Wide therapeutic window for melatonin in stroke

Neuroprotective agents for stroke continue to fail in clinical trials. One important reason is that the therapeutic window for many of those agents is too narrow to confer benefits to acute stroke victims. It would be desirable to have a potent neuroprotectant agent that has a wide therapeutic window, few side effects, and can be easily obtained at low costs. Melatonin is a compound that seems to confer strong neuroprotective benefits in cerebral ischemia and is available in the United States as a dietary supplement.

Melatonin is a potent antioxidant whose small molecular size and high lipophylicity makes for superior blood-brain barrier (BBB) permeability and penetration of the cell nucleus. Melatonin inhibits the production of inducible nitric oxide (iNOS), which exacerbates post-ischemic cell-death, and is a potent scavenger of peroxynitrite. The neuroprotective effects of melatonin have been observed in animal models of ischemic stroke when delivered after stroke, or as prophylaxis up to 9 weeks pre-stroke.

Additionally, melatonin exhibits very few side effects in humans. A previous (2005) meta-analysis of fourteen melatonin experiments documented an estimated improvement in outcome of 42.8%, though no studies used aged, hypertensive, or diabetic (well-known risk factors for stroke) animals, nor was melatonin administration investigated beyond 2 hours post-ischemia. However, In their recent article, Kilic et. al demonstrate that melatonin continues to provide significant neuroprotection even when administered 24 hours post-ischemia.

The researchers induced mild focal cerebral ischemia in a mouse model of middle cerebral artery (MCA) occlusion (30 minutes of MCA occlusion followed by reperfusion). They were then either left untreated, treated with vehicle only (ethanol in drinking water), or treated with melatonin (0.025 mg/mL) dissolved in ethanol in drinking water. Treatment was administered 24 hours after reperfusion via intraperitoneal bolus injection of 4mg/kg body weight (b.w.) melatonin, or via injection of the diluent in animals treated with vehicle. Continued treatment was administered through the animals’ drinking water at approximately 4 mg/kg b.w./day for 29 consecutive days. Two other groups of sham-operated animals were also either treated or untreated to serve as controls.

Prior to ischemic induction a baseline of grip strength, motor coordination, and spontaneous locomotor activity was measured in all animals. They were assessed again 7 and 30 days post-ischemia. Immunohistochemistry was then performed to characterize cell proliferation and neural progenitor cells.

The authors note that in untreated animals, 30 minutes of MCA occlusion resulted in “disseminated neuronal injury in the striatum, but not in the overlying cortex,” and that the percentage of surviving striatal neurons was significantly higher in ischemic animals treated with melatonin. This indicates a strong “structural rescue” effect of melatonin even when administered with a 24 hour delay. Melatonin also stimulated cell proliferation in the ischemic animals’ brains, evidenced by increased numbers of BrdU and DCX positive cells in animals treated with melatonin as compared to controls.

Melatonin exhibits a beneficial psychomotor effect in post-ischemic animals. Both grip strength and motor coordination measures were significantly higher in melatonin treated animals — in fact, motor coordination was improved almost to nonischemic animals’ level by 7 days, an effect which persisted at 30 days. In contrast, animals treated only with vehicle exhibited severe deficiencies in grip strength and motor coordination at 7 and 30 days post-ischemia.

Post-ischemic anxiety and hyperactivity were also attenuated in melatonin treated animals as measured by open-field observations of spontaneous locomotor activity. Because post-ischemic anxiety in animals is hypothesized to be analagous to post-ischemic depression in humans, these beneficial psychomotor effects could be especially advantageous in ameliorating the psychopathological symptoms associated with stroke.

These results indicate a wide therapeutic window for melatonin administration to reduce brain injury after stroke. Melatonin has now been shown to demonstrate potent neuroprotective effects when given before, immediately after, and up to 24 hours after stroke. Melatonin is also a core component in an anti-oxidant “cocktail,” developed by Michael Darwin et al., called VitalOxy, to mitigate reperfusion injury after cardiac arrest in cryonics patients. But its greater potential lies in prophylactic use. For cryonics patients, premedication of melatonin in the days or weeks leading up to pronouncement of medico-legal death can be a real, practical, and easy way to ensure appropriate “pre-mortem” protection against brain injury after legal pronouncement of death.

In their review article, Reiter et al. (2005) point out that melatonin not only limits cellular destruction of the brain due to ischemia, but also of other tissues, such as the heart. Melatonin’s ability to significantly improve outcome in stroke warrants serious consideration of melatonin as a neuroprotectant in everyday medical settings. Because melatonin is an endogenous molecule (i.e., it is produced by the body naturally), it is unpatentable so pharmaceutical companies do not stand to make much of a profit from the widespread use of melatonin, which severely limits the promotion of melatonin for use in stroke treatment.

Melatonin is widely available and inexpensive, making it an attractive option for both stroke patients and candidates for human cryopreservation. However, an advisable human dosage for cerebroprotection is difficult to determine — although the side effects of melatonin are minimal, it is well known that melatonin causes lethargy and induces sleep in most humans at much lower dosages than those described in the experimental literature. Unfortunately, the relevant research articles leave us clueless as to the general physiological effects of high doses of melatonin: the animals’ sleeping patterns or activity levels are not mentioned.

Improved protection of the brain after stroke may be achieved when melatonin is combined with other dietary supplements that have a wide therapeutic window and complement the action of melatonin by mitigating other elements in the ischemic cascade such as supporting energy generation in the brain, inhibition of PARP, and inhibition of apoptosis.

Preventing vegetative patients through cryonics

The blog Practical Ethics reports on pioneering research from a group of scientists in Cambridge who are using fMRI scans to study the brains of people who have been diagnosed as being in a vegetative condition. A Persistent Vegetative State (PVS) is a condition that is characterized by a state of wakefulness without detectable awareness. The researchers found that some patients who have been diagnosed as being in a vegetative state were able to respond to certain stimuli, indicating the possibility of awareness. Although it is encouraging that new medical technologies can assist in preventing misdiagnoses of patients with severe brain injury, the fact remains that few of these patients will ever recover with their former personality and memories intact.

Patients who have been diagnosed with conditions such as Persistent Vegetative State (such as Terri Schiavo) or the Minimally Conscious State (MCS) often suffered serious damage to the brain as a result of severe stroke or cardiac arrest. Although there are rare cases of remarkable recoveries, most patients with such diagnoses have ceased to exist as persons because the parts of their brains that encoded their personalities have ceased to exist.

It is now a well established fact that brain cells do not immediately die after severe hypoxic insults such as stroke or cardiac arrest. Actual necrosis (or apoptosis) takes many hours, or sometimes even days (as a result of a phenomenon called “delayed neuronal death.”). Unfortunately, ischemic insults to the brain exceeding 5 minutes are often sufficient to set parts of the brain on an irreversible path to destruction of the person, even if resuscitation of the patient is possible. Currently, there is no single approved neuroprotective agent that can salvage these brain cells from destruction. Although hyperacute combination therapy may offer hope for people suffering severe hypoxic insults, most of such patients currently would be better served by placing them in a state of biostatis through cryonics before the complete ischemic cascade can run its course.

Although cryonics is often dismissed as speculative, it can be argued that long term preservation of the neuroanatomy of such patients through vitrification offers better hope of recovery as the same person (or any person at all) than immediate resuscitation after the insult. But for acceptance of cryonics as a treatment for patients at great risk of (delayed) severe brain damage to become acceptable, the general public will need more exposure to the technical feasibility of cryonics and perspectives on death that offer a more prominent place to the concept of personhood.

Neuroprotection for ischemic stroke

The journal Neuropharmacology recently published a new review of the current state of the art in neuroprotection for ischemic stroke. A strict definition of a neuroprotectant excludes agents that have as their goal circulatory patency or the reversal of vascular occlusion, such as thrombolytics and anticoagulants. As a consequence, the only medication that is approved for (ischemic) stroke patients, tPA, is not a neuroprotectant. Despite the explosion of interest and research in the field (as documented in Ginsberg’s review), no single neuroprotective agent has successfully survived human clinical trials. The author discusses a number of reasons why encouraging results fail to translate into human success and stresses the fact that most agents in clinical trials are administered too late to confer positive benefits, and even states that “there is practically no evidence that neuroprotection for acute ischemic stroke is possible with any agent beyond ~6h.” It is no surprise, then, that the author does not report many promising neuroprotective strategies except for therapeutic hypothermia, high-dose human albumin therapy, and hyperacute magnesium therapy.

What does this mean for cryonics? As discussed in this review about medications in human cryopreservation stabilization, neuroprotection in cryonics has never been approached as a quest to find one single “magic bullet” to protect the brain after cardiac arrest. Cryonics stabilization medications protocol consists of a number of agents that intervene at different points in the ischemic cascade, reverse and inhibit blood clotting, and improve circulation. If rapid stabilization is possible, the time-window for treatment in cryonics is usually excellent in comparison to (focal) ischemic stroke where treatment within 1-2 hours is considered “hyperacute.” But cardiac arrest after an (often) long terminal and agonal period is not equivalent to (focal) ischemic stroke, and evidence that the medications that are given to cryonics patients are of great benefit is confined to a series of (non-published) experiments on (young) healthy animals in cryonics-associated laboratories.

When the author discusses future directions to find successful neuroprotective agents, he highlights the challenge of finding funding for neuroprotective trials that include metabolic treatment and combinations of (non-proprietary) drugs. In light of the predictable failure of mono-agents that the author reports, the discussion of the potential of combination treatment is remarkably brief and confined to the point that potential neuroprotectants need to be validated in combination with thrombolytic treatment. There is now an accumulating number of research papers on combination treatment in animal models that would warrant a more systematic analysis than the obligatory acknowledgement that combination therapy might produce better neuroprotection. Perhaps the most novel part of this new review of neuroprotective agents is the discussion of the author’s own research into high-dose human albumin therapy and the brief mention of a new paper (2007) that discusses the prospects of neuroprotective strategies that “are based on the principle that drugs should be activated by the pathological state that they are intended to inhibit.”