Tag: Cerebral Ischemia

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.

No-reflow as a post-mortem artifact

It is common medical knowledge that after 5 minutes of cardiac arrest the prospects of successful resuscitation without neurological impairment become progressively bleak. But there is less consensus on the mechanisms of such injury. One strong candidate is what is called the “no-reflow” phenomenon. No-reflow refers to the impairment of perfusion of the brain after circulation is restored. A number of researchers (e.g., Ames, Fisher) have demonstrated the existence of the no-reflow phenomenon in cerebral ischemia by perfusing the brain with carbon black after various periods of ischemia. Areas that are perfusable thus turn black, while non-perfusable areas of no-reflow do not.

In a remarkable 1992 paper by De Le Torre et al., the researchers report that the no-reflow phenomenon is only observed in the  presence of agonal or post-mortem cardiopulmonary failure.  Perfusion impairment was not observed in brains of rats that maintained stable cardiopulmonary function during, and received intravenous carbon black after, up to 30 minutes of cerebral ischemia. Clearly, these results are hard to reconcile with traditional explanations of no-reflow (e.g., blood rheology changes, edema-induced changes in the vessel lumen) and the authors offer little guidance in the discussion section of the paper on how to explain the finding that co-existence of stable cardiopulmonary function and global cerebral ischemia does not produce post-ischemic vessel filling defects after restoring blood flow to the brain.  Unless blood flow to the brain was not completely eliminated in the animals in which stable cardiopulmonary function was maintained it is difficult to imagine a mechanism of no-reflow that makes sense.

It may be that reperfusion of blood that has been circulating continuously throughout the body during the period of  cerebral ischemia accounts for improved perfusability of ischemic brains as compared to those reperfused after whole-body ischemia (i.e., cardiac arrest). Maintaining cardiopulmonary function during cerebral ischemia could improve subsequent perfusion of ischemic tissues by preventing red cell aggregation in the blood and/or inflammatory responses (e.g., neutrophil activation) initiated by reduction in, or lack of, blood flow. If this would be the case, the adverse effects of ischemia in the brain would be offset by the maintenance of physiological blood flow in the rest of the body.

The findings in this paper do not offer encouragement for the practice of human cryopreservation because cryonics patients invariably experience cardiopulmonary failure prior to stabilization intervention. Classic cryonics interventions such as administration of streptokinase and heparin do not seem to be very effective in reversing cerebral perfusion impairment,  which raises some important  questions about the phenomenon of post-mortem blood coagulation. The only effective intervention in animal research remains rapid hemodilution and high perfusion pressures, which is not easy to implement in typical cryonics casework. But why this intervention works in the light of the findings above remains something of  a mystery. Other interventions to prevent no-reflow after cardiac arrest in cryonics patients are currently being investigated and detailed reports will be forthcoming in the future.

Microvasculature perfusion failure in cryonics

Under ideal circumstances cryonics patients are stabilized immediately after pronouncement of legal death by restoring  blood flow to the brain, lowering temperature, and administering medications. In most cryonics cases, however, there is a delay between pronouncement of legal death and start of cryonics procedures. In some cases there are no stabilization interventions at all. Provided that these periods of warm and cold ischemia are not too long, such patients can still be perfused with a vitrification agent. But how thorough cryoprotectant perfusion (and thus vitrification) in these cases can be remains an unresolved issue.

Since the late 1960s a number of studies have been published that document that cerebral blood flow cannot be completely restored after prolonged periods of cerebral ischemia. Brains that have been perfused with black  ink after increasing periods of ischemia have shown progressive development of no-reflow areas in the brain (as evidenced by the absence of ink). In 2002 Liu et al. used a technique that allows direct visualization of trapped erythrocytes by treating fixed brain tissue with sodium borohydride (NaBH4), which renders trapped erythrocytes fluorescent. In a rat model of focal ischemia the authors found that a significant fraction of the capillary bed (10% to 15%) in the penumbra (the area surrounding the ischemic core) is blocked by trapped erythrocytes, even after 2 hours of reperfusion.

The authors discuss a number of clinically relevant issues. They propose that the lower density of trapped erythrocytes in the ischemic core of the brain reflects hypoxia-induced lysis (which releases cytoxic hemoglobin). They further speculate that the older ink methods may have underestimated the degree of no-reflow because areas that are not accessible to red blood cells may still be accessible to other molecules. This presents an opportunity to deliver oxygen to the brain by using small oxygen carrying molecules such as perfluorocarbons.   The authors did not investigate variations in perfusion pressure or the efficacy of volume expanders to restore no-flow areas to circulation.

A focal ischemia model is not a good model for cryonics and one can only speculate what the effects of various periods of complete ischemia would be on cerebral blood flow and erythrocyte trapping. Older studies on ischemia and perfusion impairment, however, indicate that periods of 30 minutes of complete ischemia can produce substantial areas of no-flow in the brain. Unless these areas are opened to circulation during either stabilization or cryoprotectant perfusion, straight freezing of  pockets of the brain is a real possibility. It remains to be investigated if areas that are obstructed by trapped red blood cells are accessible to cryoprotectant agents and  how much of  these areas can be opened by a combination of hemodilution and non-penetrating perfusate components (through dehydration). Although cryopreservation of  ischemic brains is the norm in cryonics, our knowledge about the effects of ischemia on vitrification of the brain remains limited.

Promoting cerebral blood flow in cryonics patients

It has been shown that perfusability of the brain is significantly compromised after long-term (>5 min) ischemic events (the “no reflow” phenomenon). Improving cerebral blood flow after circulatory arrest is one of the fundamental objectives of human cryopreservation stabilization protocol.  To that end, cryonics organizations administer the resuscitation fluid Dextran-40 and the drug Streptokinase to dilute the blood (and inhibit  red cell aggregation / cold aggulination) and  break up blood clots, thereby improving macro and microvascular circulation. Research by Fischer and Ames, who investigated the effects of perfusion pressure, hemodilution, and anticoagulation (i.e., the use of heparin) on post-ischemic brain perfusion, indicated that hemodilution is the most effective component of the post-ischemic perfusion protocol for enhancing brain perfusability. However, a later study by Lin, et al. (1978) reported significant improvement of cerebral function and blood flow with combined dextran and Streptokinase administration after cardiac arrest in dogs.

In their study, the researchers measured regional cerebral blood flow and cardiac output as well as EEG (i.e., brain wave activity) during five hours of post-resuscitation physiological maintenance following 12-16 minutes of cardiac arrest. Animals were divided into three groups as follows:

Group I:   no treatment

Group II: 1 g/kg dextran 40 in 10% saline following arrest and 10 mg/kg/minute during the five hour maintenance period

Group III: combined therapy of dextran-40 and Streptokinase — same dose of dextran as Group II and 5,000 u/kg rapid infusion and 25 u/kg/minute during the five hour maintenance period

The duration of flat EEG was significantly shorter in Group III animals (20 to 45 minutes with a mean of 28.8 +/- 2.8) than in Groups I (20 to 120 minutes with a mean of 59.5 =/- 10.8) or II (20 to 62 minutes with a mean of 46.9 +/- 4.8) and showed a faster recovery pattern than in Group I (significant difference was reached at three hours). Group II also showed a faster EEG recovery than Group I, reaching significance at five hours.

Cerebral blood flow, particularly in the hippocampus and grey matter (the areas most detrimentally affected by ischemia) in Group III was significantly improved as compared to Group I as early as three hours post-arrest, and was greater than that in Group II (significantly better only in the hippocampus). There was no difference in cardiac output found between the treated and untreated groups. All groups suffered a decrease in cardiac output of nearly 50% of baseline level (measured at 3 and 5 hours post-arrest).

Hematocrit — the proportion of blood volume occupied by red blood cells — was measured in each group and was found to be significantly increased during the post-arrest period in Group I, decreased to 25% of the baseline measurement in Group III (at both 3 and 5 hours post-arrest), and unchanged in Group II.

The authors speculate that “the improvement in cerebral circulation at the microvascular level after infusion of low molecular weight dextran was thought to be 1) related to the rapid increase in plasma volume with resultant lowering of hematocrit and reduction in blood viscosity, 2) a direct effect on the RBC [red blood cell] which increases its negativity and reduces the tendency to cellular aggregation.” They also note that though some doubt had been cast by the Fischer and Ames paper on the hypothesis of vascular endothelial cell swelling as a cause of no reflow, they did observe a higher proportion of smaller diameter capillaries in ischemic brains as compared to controls, and that “if capillary narrowing does play a role in microvascular deterioration, then hemodilution and prevention of cellular aggregates such as occurs with dextran would be beneficial in minimizing poor flow in narrow capillaries.”

Taken together, these findings indicate that combined dextran-40 and Streptokinase therapy improve brain perfusion after cardiac arrest — at least for arrest periods of up to 16 minutes.– supporting the choice for these agents in cryonics. One limitation of this study, however, is that the experiments did not include a group which received only Streptokinase. Including a Streptokinase group would have given more  precise data about the individual effects of the two agents in improving post-ischemic cerebral blood flow. Recent clinical trials with clot busting agents in cardiac arrest have failed and some contemporary authors question the phenomenon of post-arrest blood clotting. Perhaps streptokinase is useful in the treatment of circulatory arrest but its efficacy is dependent upon other blood flow improving interventions such as hemodilution. The case for post-ischemic hemodilution (and interventions to reduce RBC aggregation) is strong but the case for antithrombotic therapy in cryonics (and resusctation medicine) remains to be made.

Structure-function analysis of neuroprotectants

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.

17β-Estradiol

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.

Tamoxifen

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.

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(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.

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.

Incomplete ischemia during cardiopulmonary support

One concern about prolonged cardiopulmonary support in cryonics is that its decreasing effectiveness may not be able to meet cerebral oxygen demand, and may even become detrimental. Some investigators have  observed that severely reduced flow (cerebral blood flow less than 10% of control) to the brain may actually be more harmful than no flow at all.  Explanations of why incomplete (“trickle flow”) ischemia may be worse than complete ischemia include aggregation of slow moving blood cells,  glucose-induced excessive lactate production, and oxygen-induced free radical damage to membranes.

In contrast, a study by Steen et al. concluded that some blood flow is better than no flow at all. The authors found that dogs could sustain only 8 to 9 minutes of complete ischemia but 10 to 12 minutes of incomplete ischemia (cerebral blood flow less than 10% of control) without neurological impairment. These results are at odds with the findings of Hossmann et al. who found better electrophysiological recovery in cats and monkeys after complete ischemia, and studies by Nordstrom et al. who observed increased metabolic recovery in rats after complete ischemia.

The authors speculate that these differences may reflect the different durations of (in)complete ischemia. Hossmann et al. studied 60 minutes of ischemia and Nordstrom studied 30 minutes of ischemia. The authors note that the durations they studied (8-14 minutes) are more clinically relevant because neurological recovery with contemporary technologies is not possible after 30 or 60 minutes of cerebral ischemia. Although these findings provide support for restoration of any kind of cerebral circulation after cardiac arrest, it does not offer much guidance in evaluating the practice of prolonged cardiopulmonary support in cryonics.

The authors also draw awareness to the difficulty of correlating electrophysiological and metabolic recovery to neurological recovery. They quote a study by Salford et al. who observed some return of metabolism even though histological abnormalities had already been developed. Such studies warrant caution about using return of electrophysiological activity as an indicator of cerebral viability because it is not likely that such viability can be sustained over the long term, let alone predict functional recovery of the brain.  It is doubtful that viability in the latter, stricter, sense can be maintained during stabilization of most, if any, cryonics patients. At best, the studies that demonstrate recovery of electrophysiological and metabolic activity after prolonged cerebral ischemia offer hope that such periods of circulatory arrest do not produce acute information-theoretic death.

No metabolic or histological evidence was found to support the implication of no-reflow, lactate accumulation, and free radical damage in incomplete ischemia.  Again, the authors speculate that no-reflow may be more pronounced during longer periods of incomplete ischemia, an observation that seems to be indirectly supported by Fisher et al. who observed progressive impairment of perfusion for longer periods of ischemia.

Cryonics patients often experience shock, blood coagulation abnormalities, and decreased cerebral perfusion prior to pronouncement of legal death and cardiopulmonary support.  An additional complicating factor in cryonics is that cardiopulmonary support is often supplemented by induction of hypothermia and administration of vasopressors and neuroprotective agents. Although the paper by Steen et al. addresses a lot of issues that are important to evaluate cryonics procedures, it is clear that for real empirical guidance regarding the wisdom of prolonged cardiopulmonary support specific cryonics research models are required.

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

Then

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.

Polyethylene glycol and cryonics

The blog Al Fin reports on polyethylene glycol (PEG) as an acute treatment for traumatic brain and spinal cord injury. PEG is hypothesized to confer cytoprotection by sealing damaged cell membranes. As such, PEG would also seem a promising candidate for the treatment of acute neural insults in which progressive cell permeability / damage plays a part such as stroke and cardiac arrest. Unfortunately, the inability of high molecular weight polymers to cross an intact blood brain barrier (BBB) may limit the use of PEG as a treatment for cerebral ischemia. One study that investigated PEG in a model of middle cerebral artery occlusion (MCAo) did not find any benefits for PEG. In cryonics, membrane sealing polymers like PEG may still be useful because they can cross the compromised BBB and prevent cell lysis when transport of the patient is delayed. Its membrane sealing properties may also be useful for extending the time cryonics patients can be perfused without causing edema.

As a high molecular weight polymer, PEG has also been investigated as a component for cold organ preservation solutions. A number of studies have found that PEG can be substituted for  hydroxyethyl starch (HES) as the oncotic agent in University of Wisconsin solution (commercial name: Viaspan). Replacing HES with PEG in UW solution also decreases red blood cell aggregation and viscoscity.

PEG is also briefly discussed by Mike Perry as an embedding medium in his article about low-cost alternatives to cryonics.