November 26 , 2007
by Aschwin de Wolf
Abstract: A comprehensive review of contemporary human cryopreservation combinational pharmacotherapy is provided. Individual drugs are reviewed and, in some instances, substitutes are suggested. The article concludes by proposing general principles for human cryopreservation combinational pharmacotherapy, and specific future research directions are suggested.
* General Anesthesia
* Blood Coagulation
* Vasoactive Medications
* Free Radicals
* Nitric Oxide and PARP
* Hemodilution and Osmotic Therapy
* Coenzyme Q10
* Na+ /H+ Exchange Inhibition
* Immunosuppressive Drugs
* Gastrointestinal Ischemia
* Depressed Metabolism
* Combining Medications
Introduction: Neuroprotection in Human Cryopreservation Versus Clinical Trials
There is a striking contrast between the large number of medications that are typically administrated during a cryonics case to mitigate the effects of ischemia and the fact that, despite many years of extensive research, no single neuroprotective agent has proved beneficial in human stroke trials. From this two different conclusions may be drawn: either pharmacological therapy in cryonics lacks evidence and may be mostly futile, or the use of neuroprotective medications in cryonics reflects a different perspective that is not being recognized in conventional medicine. Perhaps it is a combination of both. A review of the reasons why neuroprotective agents may typically fail in human clinical trials, despite encouraging results in animal studies, may throw some light on this issue.
Most of the evidence for the efficacy of neuroprotective agents has been obtained in animal models and rodent models in particular. As a result we now have better knowledge of how to protect animals from cardiac arrest and stroke than we have about humans. Despite significant similarities between the human and animal genome, subtle differences may explain why certain interventions work in one species but are less efficient in others. Another limitation of animal models is that the animals used are generally young and healthy without the typical comorbidities that are seen in stroke and cardiac arrest patients, let alone reflect the prolonged terminal pathophysiology of cryonics patients. It also is increasingly recognized that a lot of research on neuroprotective agents suffers from poor research design. Some encouraging findings about neuroprotective agents in (small) animal models later turned out to be due to hypothermia, or a combination of hypothermia and the neuroprotective agent.
Concerns have also been expressed about optimal dosage and endpoints in clinical trials. For many medications the relationship between dosage and effectiveness is represented by an inverted U curve and one explanation for the results obtained in human clinical trials has been that the dosages have been too low, too high (less often), or ineffective because of limited ability to cross the blood brain barrier and/or cell membranes and the mitochondrial and nuclear membranes in particular. In animal models the efficacy of neuroprotective interventions is often measured in terms of infarct size, short term recovery, and “superficial” outcomes such as motor performance. In contrast, human clinical trials often use long term neurological outcome as an endpoint. As a result, encouraging short term results do not necessarily translate to better long term outcome, possibly because of delayed activation of apoptosis and mitochondrial failure.
But the two most important reasons for disappointing results of neuroprotective agents in human clinical trials may be delayed administration of the agent and a lack of interest in validating multimodal approaches that target the diverse mechanisms of injury. The cascade of ischemic injury triggered by cardiac arrest is complex and multi-factorial in nature and opportunities for intervention range from prevention of cell membrane depolarization to inhibiting apoptosis mediated delayed cell death. Although there are few studies quantifying the temporal aspects of the ischemic cascade, many clinical stroke trials have allowed treatment of patients for as long as 12 hours after the ischemic insult using agents for which benefits were found only for pre-treatment or immediate administration in laboratory animal models of stroke. Although this extended time window may sometimes be justified in the case of focal ischemia, where the region outside of the ischemic core, the penumbra, is still salvageable, in cardiac arrest, where there has been a complete and prolonged interruption of blood flow, the temporal aspects of the ischemic cascade impose specific limits on the nature of interventions that are available at different times after the ischemic insult.
The lack of rational combination therapy is a serious limitation to the development of effective therapies for ischemia-reperfusion injury. Because different and independent biochemical pathways lead to cell death, blocking or reversing only one is insufficient. This feature of ischemic cell death likely also explains why some neuroprotective strategies seem to have short term beneficial results (versus controls) but show no difference in outcome over the long term; one “rapid” form of cell death is eliminated but other, independent, modes of cell death have not been eliminated, or in the case of some therapies (e.g. , PARP inhibition), may even have been activated by the treatment. Although combination therapy presents a formidable research challenge, the failure of mono-agents would seem to mandate a paradigm change, provided the tension between the rigorous requirements of biomedical research and the real world needs of medicine are overcome.
Scientists necessarily seek to limit as much as possible the number of variables in an experiment and the addition of multiple drugs or other therapeutic interventions (such as hypothermia), and their complex interaction with each other, greatly complicates interpretation of experimental results. At a minimum, combination therapies greatly increase the complexity, number and cost of experiments required to understand not only the outcome of such interventions, but also of the relative effects, of each individual component in the combination or “cocktail. ” The insistence on the explication of mechanism and relative contribution of therapeutic agents in a cocktail is largely an artifact of the very recent ability to do this. In the past, single and multiple drug (or other) therapies were embraced solely on the basis of their ability to deliver the desired results .
However, during the mid and late 90s Michael Darwin and Steve Harris investigated the efficacy of acute post-insult administration of a large number of neuroprotective agents and succeeded in resuscitating dogs from up to 17 minutes of normothermic cardiac arrest. These results seem to validate the view that rapid multifactorial treatment of global cerebral ischemia using a combination of medications is a highly promising therapy for recovering animals (dogs) with a similar sensitivity to cerebral ischemic injury as humans from prolonged periods of cardiac arrest with normal neurobehavioral function. Because concerns about the proper window of opportunity for protection and the limited efficacy of monoagents were addressed in these experiments, the failure of neuroprotective agents in human clinical trials and their use in cryonics can be reconciled. However, concerns about interspecies differences, optimal (human) dosages, and most importantly, the lack of validation of these protocols in models that represent the typical pathologies of cardiac arrest and stroke victims, let alone cryonics patients, are still valid.
General anesthetics are administered to cryonics patients for two reasons; to eliminate the slim possibility of return of consciousness and to reduce brain metabolism. Currently the agent of choice is propofol. Although this medication may have been considered a sub-optimal solution compared to the use of barbiturates, there is growing evidence in favor of propofol as a potent neuroprotective agent. The neuroprotective properties that are attributed to propofol include preservation of glutamate uptake during ischemia, antioxidant and free radical scavenging properties (due to its structural resemblance to Vitamin E), and its effectiveness as a mitochondrial permeability transition antagonist .
Unfortunately, a major disadvantage of propofol is that it decreases blood pressure. The exact mechanism for this is not clear but may include modulation of nitric oxide, inhibition of the sympathetic nervous system, and/or the medication’s intralipid solvent . As a result, the first medication that is administered to stabilize cryonics patients undermines the important objective of restoring adequate cerebral perfusion. In the clinical setting of giving propofol for sedation or anesthesia, the decrease in blood pressure is compensated for by increased cardiac output, principally by increasing heart rate. During stabilization of the cryonics patient, however, circulation is restored by manual or mechanical closed chest compressions, a method that is comparatively ineffective in generating adequate vital organ blood flow.
Does the hypotension from propofol administration outweigh or cancel its positive effects in inhibiting cerebral ischemia-reperfusion injury? It is hard to answer this question without quantitative data on pre- and post-infusion of propofol in cryonics patients during cardiopulmonary support. One complicating factor is that propofol is just one of a number of medications in cryonics with hemodynamic effects such as vasopressin, epinephrine, s-methyl-isothiourea (SMT), and 4-hydroxy-tempo (TEMPOL).
Propofol induced hypotension might be prevented by careful titration of the agent during stabilization but unless real time measurements of blood pressure and depth of anesthesia are available during stabilization this would be a highly theoretical approach. Another alternative would be to reduce the required dose of propofol by administration of magnesium sulphate as an adjuvant . Considering the fact that magnesium also has neuroprotective properties, there is good reason to investigate this agent in more detail.
A superior alternative would be to identify either a non-controlled agent that inhibits metabolism without negative hemodynamic effects or an agent that induces such profound reductions in metabolic demand that optimizing cerebral perfusion becomes a less pressing issue. Because propofol has a number of separate neuroprotective properties that other intravenous anesthetic agents, such as etomidate, do not have, it might be more beneficial to look into the possibility of moving from general anesthetics to hibernation or “suspended animation” inducing molecules in order to secure electrocerebral silence and reduce metabolic demand.
Antithrombotic therapy is considered a very important element of stabilization in cryonics. Breaking up and preventing blood clots eliminates obstructions to cerebral blood flow during stabilization. This in turn facilitates better distribution of neuroprotective medications and improved blood substitution, which ultimately contributes to better cryoprotectant perfusion of the brain and body.
Although blood coagulation after cardiac arrest is generally taken for granted in cryonics, the mechanisms of cardiac arrest induced coagulation are complex and multifactorial. Some mechanisms that have been proposed include the pre-mortem disease of the patient (e.g. , sepsis, trauma), endogenously released catecholamines, inflammation, and free radical mediated endothelial injury.
The importance of ischemia induced coagulation is a subject of debate. A study on the impairment of cerebral perfusion following ischemia found that hypertension and hemodilution were more effective in restoring complete cerebral circulation than administration of heparin  . Alternatively, more specific studies on ischemia induced blood clotting found a significant decrease in anthithrombin and a marked increase in coagulation after prolonged cardiac arrest [6, 7]. The finding that blood clotting seems to increase with the duration of cardiac arrest provides evidence for blood clotting as an indirect downstream effect of ischemia and reperfusion injury. Adding more complexity is the observation that clots autolyse again after a prolonged post-mortem period. Perhaps post-mortem blood clotting can be best represented as an inverted U curve although specific data on this phenomenon are hard to locate.
Currently the fibrinolytic agent of choice is streptokinase. Could this choice be improved on by switching to Tissue Plasminogen Activator (t-PA)? There are a number of advantages to the use of t-PA. First, unlike streptokinase, t-PA only activates plasminogen that is bound to existing clots and does not affect the general coagulability of the blood. As a result, the use of t-PA does not predispose the patient to unnecessary bleeding. Second, although thrombolysis decreases with temperature for all thrombolytics, t-PA is still active at 10 degrees Celsius whereas streptokinase’s clot busting activity is negligible at this temperature . Third, past clinical administration of streptokinase can produce antibodies to streptokinase that will render the drug largely ineffective and/or produce an allergic reaction .
Do these advantages outweigh the much higher price of t-PA? t-PA’s more selective action can be considered a positive or negative for cryonics. Although excessive bleeding may present a problem in some cryonics cases, complete inhibition of the coagulation cascade is a more desirable goal. t-PA’s ability to lyse clots at lower temperatures is a distinct advantage but this advantage may only be relevant for patients that are treated with thrombolytics when they are already at (ultra)profound hypothermic temperatures. t-PA does seem to have a clear immunological advantage over streptokinase, but only for a very small number of patients. In light of the substantial costs of t-PA, streptokinase still has much in its favor as the fibrinolytic agent of choice. For patients who are at risk for an adverse immune response to streptokinase, t-PA may be considered on a case by case basis.
Recent clinical trials of thrombolytic drugs given at the start of CPR have failed to show the expected benefit and a period of hypertension coupled with hemodilution imposed immediately upon restoration of circulation may be more effective than anticoagulation at restoring adequate cerebral circulation. The problem with this approach, both in cases of sudden cardiac arrest and stabilization of cryonics patients, is the (current) impossibility of successfully inducing hypertension using closed chest CPR .
While the role of microvascular clotting in ischemic injury may be debated, there is no argument that subsequent large-scale clot formation in the circulatory system is undesirable because of its obstructive effects and should be avoided. This macroscopic, systemic clotting can be effectively prevented by prompt post-arrest administration of heparin. Prompt post-arrest administration of heparin is considered such an important part of cryonics protocol that even cryonics organizations that do not offer standby and stabilization encourage hospitals and funeral directors to administer this drug after pronouncement of legal death.
One disadvantage of heparin is that it does not directly inhibit the coagulation enzyme thrombin. Because thrombin does not only convert fibrinogen to fibrin but also stimulates platelet aggregation and inflammation, heparin is less effective compared to direct thrombin inhibitors such as the hirudin derivatives. Direct thrombin inhibitors have also been shown to be superior to heparin in combination with fibrinolytic agents . Like t-PA, direct thrombin inhibitors such as hirudin are produced by recombinant biotechnology and may be beyond the reach of cryonics organizations. It also needs to be pointed out that heparin may have neuroprotective properties that are independent from its actions as an anticoagulant.
The antithrombotic objective of cryonics stabilization is further enhanced by the anti-platelet agent aspirin. Aspirin is a relatively weak inhibitor of platelet aggregation and only prevents platelet aggregation through the thromboxane A-2 pathway. As a result, platelet aggregation can still occur through other pathways such as the adenosine diphosphate (ADP) pathway. Although some cryonics organizations have switched to injectable aspirin as an alternative, regular aspirin is not very water soluble and needs to be dissolved in a solubilizing medium such as tromethamine (THAM) prior to administration. This effectively makes the medication a slow drip, moves it further down the protocol, and eliminates flexibility in dosage if this combination is prepared prior to a cryonics case.
COX-2 inhibition has been found to be neuroprotective in CA1 hippocampal neurons in a rat model of global ischemia but aspirin is a non-selective inhibitor of COX and requires high doses for inhibition of COX-2 . In light of the fact that aspirin is neither a very potent anti-platelet agent nor a COX-2 inhibitor agent at low dosages, glycoprotein IIb/IIIa inhibitors, which inhibit platelet aggregation at multiple pathways, and more selective COX-2 inhibitors, may warrant further investigation as candidates for cryonics stabilization protocol.
Citrate is a potent anti-coagulant that is routinely used for blood storage in blood transfusions. Citrate prevents blood clotting by chelating calcium, which is an essential step in the clotting cascade. Because calcium also plays an important pathological role in ischemia and reperfusion injury, citrate may also be beneficial as a neuroprotective agent. In cryonics sodium citrate has traditionally been included in the protocol as optional for cases where long transport times can be expected. Since the early 2000s citrate, in the form of citrate dextrose, has been added as a standard medication and as a solvent for NiKy (niacinamide and kynurenine).
The use of citrate in a stroke or cardiac arrest model has never been validated. A number of issues still need to be investigated. Since calcium is essential for normal functioning of an organism, can administration of citrate be reconciled with the objective of stabilization to secure cerebral viability? Is calcium necessary for the metabolism of other drugs (such as epinephrine), production of (endothelial) nitric oxide, and stability of cell membranes? Considering the adverse effects of hyperglycemia on ischemic injury, should an alternative form of citrate, such as sodium citrate, be used in cryonics? The stoichiometry of the amount of citrate used in cryonics needs to be quantified to determine how much citrate is enough for anticoagulation without disrupting these important functions.
As has become clear from this brief review of antithrombotic pharmacology in cryonics, not much is currently known about ischemia induced and “postmortem” clotting. Encouraging results with hemodilution and hypertension in cardiac arrest and stroke indicate that ischemia induced blood abnormalities may not be necessarily restricted to the blood coagulation cascade. Agents that are designed to mitigate blood rheology (such as drag reducing polymers) should be investigated as well.
A major limitation of closed chest CPR is that it cannot generate enough cerebral flow to maintain cerebral viability. In conventional medicine CPR is only a bridge to facilitate restoration of spontaneous cardiac activity, whereas in cryonics chest compressions need to be maintained until extracorporeal bypass can be initiated or metabolic demand is sufficiently depressed to permit temporary circulatory arrest without brain injury. Because initial cooling and administration of propofol does not drop metabolic demand enough to tolerate the degree of cardiac output achieved by manual chest compressions and ventilations, the efficiency of cardiopulmonary support can be enhanced by using mechanical cardiopulmonary support in conjunction with airway adjuncts like the impedance valve and vasopressors like epinephrine and vasopressin.
In cryonics, the two vasopressors epinephrine and vasopressin are used in combination. A clear disadvantage of epinephrine is that it is pH sensitive and has a relatively short half life compared to vasopressin, necessitating intermittent administration of this agent if optimal results are desired. Another disadvantage of epinephrine is partial reversion of the effects of general anesthetics like propofol .
Combination treatment with epinephrine and vasopressin has not been investigated in much detail in a model of cardiopulmonary resuscitation. Some research points to a beneficial synergy effect of combining epinephrine and vasopressin. Other research reports decreased cerebral perfusion when both agents were combined .
Vasopressin and epinephrine are included in cryonics protocol because of their vasoactive properties but other medications have vasoactive properties as well. A notorious example is the profound drop in blood pressure that propofol produces. The spin trap agent 4-hydroxy-tempo (TEMPOL) also decreases blood pressure. Conversely, the selective inducible nitric oxide (I-NOS) inhibitor S-methyl-isothiourea (SMT) increases blood pressure. As a result, stabilization pharmaceuticals may produce unintended and undesirable alternations in blood pressure as a side effect. Cryonics stabilization pharmacology may benefit from investigating this phenomenon in a realistic model and hemodynamic monitoring during actual cryonics cases can be used to develop a medications protocol in which the neuroprotective benefits of medications can be reaped without adverse hemodynamic consequences. One method to investigate this during a case would be to trend end tidal CO2 (ETCO2) readings against the administration times of individual medications and fluids.
Currently cryonics stabilization protocol combines technologies that aim at “resuscitation” and depressing metabolic demand. Could vasopressor support be dispensed with if induction of hypothermia and substantial metabolic depression and/or hypoxic tolerance could be achieved rapidly after legal pronouncement of death? There is no reason to believe that this would eliminate the need for vasopressors in cryonics. Vasopressors not only assist in securing adequate cerebral blood flow, they also contribute to more rapid and complete distribution of medications to the brain. Large molecules such as streptokinase require good turbulent flow and shear to effectively lyse clots. Finally, higher perfusion pressures may also be beneficial in reversing red cell sludging and no-flow areas in the brain.
In light of these observations, the question can be asked why a vasopressor like vasopressin is not even higher up the list in cryonics protocol. In cryonics cases where not all medications can be pushed in the hospice or hospital, or immediate transport is mandated, the traditional response has been to push “the first three meds. ” Considering the beneficial effects of vasopressors on cerebral perfusion, induction of hypothermia and efficacy of the other medications, it might be more beneficial to push “the first four meds” before transporting the patient: propofol, streptokinase, heparin and vasopressin.
Depolarization and calcium overload of brain cells triggers the release of massive amounts of the neurotransmitter glutamate into the extracellular space. Absent energy dependent glutamate re-uptake, excitatory receptors are activated leading to a further increase in intracellular calcium. This again releases more glutamate into the extracellular space, triggering a positive feedback loop of excitotoxity induced calcium overload. Intracellular calcium overload also activates calcium dependent enzymes that break down membrane lipids and proteins, activate apoptosis and synthesize nitric oxide. It is not surprising that strategies aimed at inhibiting glutamate release, increasing glutamate uptake or blocking receptor medicated intracellular calcium overload have been intensely investigated as a neuroprotective strategy in cerebral ischemia. Although encouraging results have been obtained with glutamate receptor blockers in animal models, so far these agents have failed in clinical trials and in some cases were halted because of psychotomimetic side effects (like the NMDA receptor antagonist MK-801).
An excitotoxicity inhibitor that is assumed to be essential in the normothermic cerebral resuscitation experiments at Critical Care Research (CCR) is L-kynurenine. L-kynurenine is a blood brain barrier permeable precursor of kynurenic acid, an endogenous antagonist of NMDA receptors, and at higher doses, AMPA receptors. Although administration of exogenous L-kynurenine has been shown to prevent the generation of epileptiform bursting activity in brain slices there may be trade-offs in administration of this molecule as a neuroprotective agent. A study of the kynurenine pathway shows that this molecule not only produces kynurenic acid, which is cerebroprotective, but also produces the NMDA receptor agonist quinolinic acid and its intermediaries 3-hydroxykynurenine and 3-hydroxyanthranilic acid, both of which can cause neuronal damage, presumably through non-NMDA mediated free radical generation. In light of this concern, it might be beneficial to investigate administration of kynurenine hydroxylase inhibitors (such as nicotinylalanine) which change the balance of kynurenic acid and quinolinic acid in favor of kynurenic acid, or better, combined administration of L-kynurenine with a potent inhibitor of the kynurenine hydroxylase enzyme to further increase the total amount of kynurenic acid produced . Encouraging results have been obtained when L-kynurenine was administered together with nicotinylalanine and probenecid (which slows the loss of acidity induced release of kynurenic acid from the brain) . On the other hand, a recent rat study found that that the metabolites of the kynurenine pathway, kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and anthranilic acid adversely affect mitochondrial bioenergetics, raising concerns about increasing endogenous kynurenic acid in general, and administration of L-kynurenine in particular .
One of the damaging effects of ischemia/reperfusion is the generation of reactive oxygen species (ROS) like superoxide, hydrogen peroxide, and the hydroxyl radical; and reactive nitrogen species (RNS) like nitric oxide and peroxynitrite. The brain is particularly susceptible to free radical generation because of its high oxygen consumption, high content of polyunsaturated acids and non-heme iron, and a relatively inefficient enzymatic antioxidative defense system . Although many agents with free radical scavenging and spin trapping properties have shown potential in animal models and pre-clinical experiments, no single agent has survived a successful phase 3 clinical trial. The highly promising PBN derivative spin trap agent NXY-059 ultimately failed in a second phase III trial in 2006. Due to NXY-059’s relatively poor blood brain barrier permeability, its primary mechanism of neuroprotection is assumed to be the attenuation of inflammation mediated free radical injury .
Considering the diverse and complementary nature of endogenous antioxidant defense mechanisms, and the limited efficacy of mono-agent antioxidant strategies in treating cerebral ischemia, an alternative direction would be to combine several agents with free radical scavenging or spin trapping properties. For example, a spin trapping agent like PBN can be combined with the pineal hormone and free radical scavenger melatonin. Although melatonin has shown promise in a wide variety of animal models and is considered a serious candidate as a neuroprotective drug, its nonpatentable nature does not provide a strong incentive for drug development.
An important mainstay in human cryopreservation stabilization to mitigate ischemia-reperfusion injury is the antioxidant “cocktail” VitalOxy. VitalOxy consists of melatonin, PBN, Vitamin E (alpha-tocopherol), and the anti-inflammatory agent carprofen. Vital-Oxy is typically administered in combination with other agents with (indirect) antioxidant properties like propofol, TEMPOL, s-methyl-isothiourea (SMT), and mannitol. This raises the question of the relative efficacy and redundancy of these different antioxidants.
Considering the magnitude of potent antioxidant and free radical agents in cryonics protocol more benefit might be obtained from investigating the current medications in more detail than from identifying and adding new molecules. For example, antioxidant and free radical agents can be screened on hemodynamic effects. Is administration of both PBN and TEMPOL redundant in cryonics protocol? Can reperfusion injury be minimized by delaying ventilations until antioxidants and free radical scavengers have been circulated? If evidence is found in favor of VitalOxy as a treatment for cardiac arrest induced ischemia/reperfusion, the individual components of VitalOxy can be compared to the complete cocktail to get a better understanding of relative efficacy of each individual component. Additional variants that can be explored are the substitution of gamma-tocopherol or the alfa-tocotrienol form of Vitamin E for alpha-tocopherol or newer spin trip agents as a substitute for PBN.
Nitric Oxide and PARP
Contemporary cryonics stabilization protocol includes the inducible nitric oxide synthase inhibitor S-methylthiourea (SMT). Cerebral ischemia has a marked effect on production of nitric oxide, a short lived signaling molecule, in the brain. During ischemia three isoforms of nitric oxide are expressed: endothelial nitric oxide (eNOS), neuronal nitric oxide (nNOS) and an immunologically induced form (iNOS). Research has shown that the beneficial effects of nitric oxide depend on the stage of ischemia and the specific form of nitric oxide that is expressed . eNOS appears to enhance blood flow by modulating vascular tone and inhibiting ischemia induced platelet aggregation and leukocyte adhesion. nNOS and iNOS appear to have detrimental effects in cerebral ischemia by causing free radical damage and limiting ATP formation. iNOS is particularly harmful because it produces large busts of nitric oxide, which combines with the superoxide radical to create the damaging lipid permeable peroxynitrite radical.
Considering the findings that inhibition of iNOS is beneficial in models of cerebral ischemia and cardiac arrest, the potent selective iNOS inhibitor S-methylthiourea (SMT) seems to be a rational component of human cryopreservation protocol. More important than the search for more potent and selective iNOS inhibitors would be to investigate the efficacy of combined iNOS and nNOS inhibition versus selective iNOS inhibition. Selective nNOS inhibitors like 7-nitroindazole have been demonstrated to reduce ischemic brain injury. Alternatively nNOS inhibition turned out to be detrimental in a cardiopulmonary resuscitation model . Perhaps nNOS expression is beneficial for cardiac function but detrimental for neuronal survival. Differences in nitric oxide expression between various species raise questions about the efficacy NOS inhibition in humans. A related question is the time course of NO expression during cerebral ischemia. If iNOS is expressed relatively late during reperfusion, would that make iNOS inhibition irrelevant in cryonics?
Peroxynitrite induced DNA strand damage activates the DNA repair enzyme poly (ADP ribose) polymerase (PARP). Excessive PARP activation during ischemia produces futile cycles of NAD+ and subsequent ATP depletion and production of inflammatory mediators leading to cell dysfunction and necrosis. Pharmacological inhibition of PARP has been found to be beneficial in a number of medical conditions including cerebral ischemia. PARP activation is a shared downstream event in a number of different pathways in ischemic injury, potentially offering a greater degree of protection and providing a longer window of opportunity as a neuroprotective strategy. One limitation of PARP inhibition might be that it is not very effective in inhibiting apoptotic forms of neuronal death . As such, PARP inhibition may only confer selective protection against ischemic cell death.
The current PARP inhibitor of choice in cryonics is niacinamide (nicotinamide), a B3 vitamin and NAD+ precursor and relatively weak PARP inhibitor. Niacinamide restores ATP levels in the brain and, at large dosages, inhibits PARP. Because of its multi-factorial protective properties it may be harder to separate its efficacy as a PARP inhibitor versus its other beneficial properties such as supporting the citric acid cycle and mitochondrial function . Substantial decreases in brain injury were obtained by intranasal administration of NAD+ in a model of transient focal ischemia . Intranasal administration of niacinamide was not found to be neuroprotective in this study, however. Perhaps the dose of niacinamide (10 mg/kg) used in this study was too low (doses higher than 125 mg/kg have been found to be neuroprotective) to confer any benefits. This raises some concerns about the effectiveness of niacinamide as a PARP inhibitor in cryonics protocol because the molecule is currently being administered at a fixed dose of 500 mg. An alternative PARP inhibitor that has been investigated during the normothermic cerebral resuscitation experiments at CCR is 3-aminobenzimide (3-AB), a structural analogue of niacinamide. 3-AB is a more potent PARP inhibitor and hydroxyl radical scavenger but was suspected to produce necrosis of the tracheal mucosa in a number of experiments .
The usefulness of PARP inhibition in cryonics needs further investigation. PARP inhibition may be beneficial for a number of reasons. It may reduce rapid energy depletion during stabilization. In the case of niacinamide administration, it may also provide a substrate for the citric acid cycle in energy deprived cells. The large window of opportunity for PARP inhibition provides an opportunity for neuroprotection that can no longer be secured further upstream in the ischemic cascade. The downside of this is that many harmful events may have already run their course (membrane depolarization, intracellular calcium overload, excitotoxicity, generation of reactive oxygen and nitrogen species etc). But this concern is not really relevant in cryonics because PARP inhibition is used in conjunction with other agents that intervene at other, more upstream, parts of the ischemic cascade.
PARP inhibition can change the mode of cell death from necrosis to apoptosis. This seems to be a relative advantage in cryonics because apoptosis-induced cell death requires more time to complete as a result of gene transcription and protein synthesis. Inhibition of both necrosis and apoptosis might be preferable, however. This may present a problem in conventional medicine because it could prevent the body from removing injured and non-viable cells, but in cryonics preservation of ultrastructure should be considered more important for future resuscitation of the person.
Perhaps a more potent PARP inhibitor could improve outcome for cryonics patients but only research can determine whether the more potent PARP inhibitors will outweigh the multi-factorial benefits of an agent like niacinamide. It should also be remembered that very potent PARP inhibitors like INO-1001 are patented by biotechnology companies which may either limit availability or drive up the costs of cryonics medications protocol. PARP inhibitors can also be investigated as components in hypothermic organ preservation solutions.
Cardiac arrest and reperfusion induced activation of leukocytes and inflammatory mediators contribute to increased coagulation, microvascular dysfunction, free radical formation, and ultimately, cell death. Although the inflammatory mechanisms induced by ischemia are complex and multi-factorial in nature, modulation of inflammation is perceived to improve outcome. Cryonics medication protocol includes the non-steroidal anti-inflammatory drugs (NSAIDs) aspirin, carprofen, and ketorolac. Although it is not clear how much redundancy there is in such an approach, aspirin seems to compare favorably to the other NSAIDs because it irreversibly acetylates the cyclooxygenase enzyme and has additional anti-platelet properties. The actual benefits of administering multiple NSAIDs needs further investigation, especially in light of the fact that research on ketorolac and carprofen as neuroprotectants is non-existent.
Perhaps the cryonics protocol can be improved by substituting selective COX-2 inhibitors for non-selective NSAIDs in conjunction with specific glycoprotein IIb/IIIa inhibitors plus selective cytokine- and endothelial adhesion molecule inhibitors, but the most immediate medical and practical benefits may be secured by substituting medications and fluids with potent immunosuppressive properties for existing ones such as the antibiotic minocycline for gentamicin and hypertonic saline for dextran 40 and/or mannitol.
Patients that present for human cryopreservation are at high risk for bacterial infections, or, in the case that a patient is pronounced legally dead after septic complications, bacteremia is already present. Cardiac arrest aggravates this situation because absent adequate circulation and oxygen tension the human body is defenseless against foreign invaders [26, 27]. The use of invasive medical procedures during stabilization, such as intravenous catheter placement and surgery, present additional risk factors. Although hypothermia has multiple neuroprotective properties, downregulation of leukocytes and inflammatory mediators increase the likelihood of bacterial overgrowth. Because some cryonics patients experience extended periods of normothermic and hypothermic circulatory arrest, cryonics stabilization and transport medications typically include one or more antibiotics such as the aminoglycoside gentamicin. One limitation of gentamicin is that it is less effective against anaerobic bacterial infections.
Although the benefits of antibiotic therapy in cryonics have never been systematically validated with cryoprotective perfusion as an endpoint, some classes of antibiotics have additional anti-inflammatory and neuroprotective properties that could enable improved cerebral resuscitation and a reduction of the number of medications that are currently given. For example, the tetracycline derivative minocycline is an antibiotic with potent neuroprotective properties.
Minocycline is a broad spectrum bacteriostatic antibiotic with good tissue and brain penetration that possesses a broad variety of neuroprotective properties including inhibition of -metalloproteinases, -iNOS, -mitochondrial cytochrome c release and -apoptosis, which can be explained by its antioxidant and free radical scavenging activities . Minocycline’s free radical scavenging potency is comparable to alfa-tocopherol which may be explained by their structural similarity . Because minocycline has been found to have neuroprotective properties in a lower molar range than required for its antioxidant and free radical scavenging effects, other mechanisms such as PARP inhibition may play a part in this. In a 2006 paper minocycline was found to be a PARP inhibitor in the nanomolar range . Similar to its antioxidant and free radical scavenging properties, this may be related to its structural similarity (a carboxamide and aromatic ring structure) to competitive PARP inhibitors.
Because of its multi-factorial mechanisms, minocycline seems to be a promising candidate for cryonics stabilization protocol. Observations about the chemical structure of minocycline raise a broader issue about the biochemistry of neuroprotection that warrants continued research. Other classes of antibiotics that have neuroprotective and anti-inflammatory properties include the macrolides such as erythromycin  .
Within minutes of the beginning of cerebral ischemia extracellular and intracellular pH drops and the patient becomes acidic. This process is aggravated in patients who are hyperglycemic during an ischemic insult. The drop in pH may be detrimental for a number of reasons. A lowered pH could increase sodium concentrations in the cell by activation of the Na+/H+ exchanger, release iron and increase free radical formation through the Fenton reaction, suppress neurotrophin synthesis, and decrease the ability to lower intracellular calcium accumulation . Acidosis may also impair cerebral vascular autoregulation. An acidic environment may further reduce or eliminate the effectiveness of drugs that are pH sensitive such as heparin and epinephrine.
Sodium bicarbonate administration during CPR has come under increased scrutiny because it can cause a paradoxical rise in intracellular pH as a result of diffusion of the carbon dioxide constituent into the cells. In cryonics the buffer agent tromethamine (THAM) is used instead. Administration of THAM in a feline model of focal ischemia restored extracellular pH (and possibly intracellular pH) and reduced infarct volume by 40% . The use of an alternate buffer cocktail, tribonat, did not improve discharge from the hospital after cardiac arrest in a randomized double blinded trial . Perhaps the role of pH management in cerebral ischemia is modest and can only confer benefits in conjunction with other neuroprotective strategies. Currently THAM is administered in cryonics as a large volume drip. Considering the immediate drop in pH after cardiac arrest, giving a portion of this fluid (with or without aspirin) as a bolus needs to be considered again in cryonics protocol. Monitoring and treatment of hyperglycemia should be a related objective.
Hemodilution and Osmotic Therapy
Cryonics protocol includes the synthetic colloid dextran-40 and the osmotic agent mannitol. Dextran-40 is not just used as a hypertonic volume expander to restore blood pressure in hypovolemic patients, it can also improve cerebral blood flow during reperfusion by decreasing viscosity and ischemia induced leukocyte-endothelial adhesiveness. Dextran-40 also has a mitigating effect on blood coagulation and hypothermia induced cold agglutination and rouleaux formation. Cerebral edema and intracranial pressure can be mitigated by administration of mannitol, a sugar alcohol which has the additional advantage of being an effective hydroxyl scavenger. Mannitol is not only the osmotic agent of choice in stabilization, it is also a core component in the hypothermic organ preservation solution MHP-2, which is used during remote blood substitution.
There are extensive debates in the medical literature about the relative merits of various plasma substitutes and oncotic agents in resuscitation and cerebral ischemia but there has not been conclusive evidence to strongly support one agent over the other. The exceptional rheological effects of dextran-40 and the antioxidant properties of mannitol seem to support the choice for these agents in cryonics. Concerns have been raised about the risk of anaphylactic shock with dextran-40. This risk can be reduced by prior administration of dextran-1, but its limited availability has been a problem for cryonics organizations. Crystallization of mannitol in solution has been an even more frustrating problem for cryonics organizations. Although warming pads to reverse this situation are typically included in stabilization standby kits, not much success has been reported in using them in time-sensitive situations. Other solutions include diluting the solution or using heated storage chambers, which will respectively require in-house preparation of mannitol or challenging logistics. One clinical concern about mannitol is that repeated administration or high dose administration may have adverse effects by opening the blood brain barrier and reversing the osmotic gradient, aggravating cerebral edema as a result.
An interesting substitute for mannitol could be hypertonic saline. Because sodium chloride has a higher reflection coefficient than mannitol (1.00 vs. 0.90) it can exert a more effective osmotic gradient across the blood brain barrier . Hypertonic saline also improves regional cerebral blood flow and has anti-inflammatory effects on neutrophils, free radical generation, and cytokine release . Because of its rapid equilibration between intracellular and extracellular compartments, hypertonic saline is sometimes combined with a dextran or hydroxyethyl starch to create a more potent and longer lasting osmotic agent. Although the high sodium concentration may present a risk for cryonics patients with compromised blood brain barriers, use of a dextran or hydroxyethyl starch containing hypertonic solution may combine, or improve upon, the beneficial effects of dextran-40 and mannitol administration while reducing concerns about volume overload and the logistical problems associated with mannitol. Administration of hypertonic saline may be contraindicated in dehydrated hypernatremic cryonics patients. A related concern is that administration of isotonic solutions could cause serious cerebral edema in patients with high sodium contents in the brain. Unlike normal patients, cryonics patients do not eliminate excess ions from the body .
One agent that is currently not a part of human cryopreservation protocol is Coenzyme Q10 (CoQ10). CoQ10 has gained popularity as a supplement to prevent and treat cardiovascular diseases. Recent interest in the medical application of CoQ10 has included such diverse medical conditions as cancer, AIDS, and stroke.
CoQ10 is currently being researched as a neuroprotective agent in stroke and cardiac arrest because of its presumed effect in supporting cellular metabolism, as a free radical scavenger, and as a cell membrane stabilizer in brain ischemia-reperfusion injury. CoQ10 is a lipid soluble coenzyme and can cross the blood brain barrier.
There are a number of papers on the use of CoQ10 in cerebral ischemia and cardiac arrest. Zhen et al. found cerebroprotective effects of CoQ10 in a canine model of deep hypothermic circulatory arrest . Damian et al. reported improved neurological outcome and long-term survival for out-of-hospital cardiac arrest when CoQ10 was combined with mild hypothermia . Li et al. , on the other hand, did not find evidence for CoQ10 as a neuroprotective agent in a rat model of focal and global ischemia .
More evidence of the benefits of CoQ10 as a monoagent in cerebral ischemia, or CoQ10 as an additional medication in cryonics combination therapy, is needed to support using this agent in cryonics. An additional modality that could be investigated is to compare the two different isomers of CoQ10: ubiquinol and ubiquinone. Ubiquinol has superior bioavailability, and does not require initial reduction by the body to function as a free radical scavenger (electron acceptor).
Magnesium seems to be an ideal candidate for cerebral resuscitation. It is a safe, inexpensive, natural cation with multi-factorial neuroprotective properties. Magnesium’s neuroprotective mechanisms include inhibition of glutamate receptor mediated excitotoxicity and intracellular calcium overload, positive effects on energy metabolism, blood flow, and membrane integrity. For this reason magnesium has been investigated in models of focal and global ischemia and in human clinical trials. Despite its theoretical advantages, a recent meta-review of magnesium administration in cerebral ischemia found that some of its neuroprotective properties may have been due to hypothermia as a result of lack of post-ischemic temperature control during some of the experiments . The authors also found that in global ischemia magnesium administration combined with hypothermia increased CA1 survival compared to either normothermic administration of magnesium or hypothermia alone. Because magnesium may only be effective if it is administered promptly after stroke or cardiac arrest, early (pre-hospital) administration of magnesium in stroke victims is currently being investigated in the phase III FAST-MAG trial.
Because magnesium may be beneficial in some of the most upstream events in the ischemic cascade (energy metabolism, excitotoxicity, calcium overload), and it also enhances the neuroprotective effects of hypothermia and reduces the required dosage of propofol to secure general anesthesia, a prominent place for this agent in human cryopreservation stabilization protocol should be considered. Another advantage is its cost. Considering the possibility that an expensive, poorly soluble and unstable molecule like L-kynurenine may only be necessary to suppress cerebro-electrical bursts after resuscitation, a more cost-effective agent with similar properties may be a good substitute. Another advantage is that magnesium can be easily obtained and added to a multimodal resuscitation fluid. One concern about magnesium as a neuroprotective agent is that its optimal use seems to be very dose specific and at least one study has found remarkable neuroprotective differences between the chloride and sulfate cations of magnesium .
Cerebral ischemia produces a rapid decrease in energy substrates and a switch from aerobic to anaerobic glycolysis to generate ATP. As a result, intracellular pH drops and intracellular sodium increases by extruding hydrogen in exchange for sodium through the Na+/H+ exchanger upon reoxygenation. Accumulation of intracellular sodium increases calcium influx through the Na+/Ca+ exchanger. Because intracellular calcium overload is linked to a cascade of cytotoxic events, inhibition of the Na+/H+ exchanger has been investigated as a neuroprotective strategy. A number of potent Na+/H+ exchangers have been identified in models of cardioplegic arrest and cerebral ischemia like methyl isobutyl amiloride, HOE 642 (cariporide) and HOE 694.
Inhibition of the Na+/H+ exchanger seems to have a number of clear advantages. Sodium accumulation induced cellular edema and calcium overload are clear upstream pharmacological targets. Because cryonics stabilization procedures are generally started promptly after pronouncement of legal death, targeting the most upstream events during ischemia is realistic. Na+/H+ inhibition may not only mitigate the severity of downstream events, it also has the advantage of being the only small volume medication that can prevent cellular edema, protect endothelial cells and improve blood flow at an early stage.
Na+/H+ inhibition as a pharmacologic objective raises a number of issues. First, will inhibition of the Na+/H+ exchanger not conflict with restoring physiological pH, by retaining hydrogen in the intracellular space? One answer is that the benefits of Na+/H+ inhibition can be reaped without the disadvantages because cryonics stabilization protocol also includes a buffer agent to treat acidosis. Another possibility is that Na+ /H+ inhibition restores mitochondrial function (or prevents calcium induced mitochondrial dysfunction) and subsequently reduces intracellular acidosis . Second, is Na+/H+ inhibition the most effective exchanger and/or channel inhibition strategy in cerebral ischemia? During ischemia sodium also enters the cell through voltage sensitive sodium channels and ionotropic glutamate receptors and calcium is released by intracellular organelles such as the endoplasmic reticulum. A better understanding of the temporal and relative importance of the different mechanisms that upset ion homeostasis could help in choosing the most potent treatment available for ischemia-induced cell depolarization.
Tacrolimus (FK 506) and cyclosporine are immunosuppressive calcineurin inhibitors that have been shown to be neuroprotective in focal and global models of ischemia. The brain contains higher calcineurin levels than most other studied tissues. Calcineurin induces transcription of interleukin 2 and increases intracellular calcium by dephosphorylating Ca+ channels, NMDA receptors, ryanodine receptors, and IP3 receptors . Calcineurin also regulates iNOS activity in a calcium dependent manner. As a result, inhibition of calcineurin may reduce free radical damage by modulating generation of peroxynitrite. Calcineurin inhibition can also reduce apoptosis by inactivating the pro-apoptotic protein Bad . Both drugs have been associated with inhibition of the opening of the mitochondrial permeability pore, but in the case of Tacrolimus this may be an indirect effect of modulating intracellular calcium levels. A systematic review and meta-analysis of Tacrolimus found substantial efficacy for Tacrolimus in experimental stroke . Tacrolimus is used as an immunosuppressant in liver and kidney transplantation and has also shown potential as a component of (hypothermic) organ preservation solutions. Because cyclosporine cannot cross an intact blood brain barrier, the more potent Tacrolimus seems to be the better candidate for neuroprotection.
High energy consuming organs like the brain and the heart are uniquely vulnerable to short interruptions of blood flow. For obvious reasons most attention in cryonics has been paid to securing viability of the brain. The effects of cardiac arrest and hypothermia on the gastrointestinal tract have never received systemic treatment in cryonics despite the fact that abdominal swelling during washout and whole body cryoprotective perfusion are often observed.
Although administration of the antacid Maalox (aluminium hydroxide and magnesium hydroxide) has been a core component of cryonics stabilization protocol for many years, this fluid has routinely been omitted in recent cryonics cases for a number of reasons. Unlike the other medications and fluids, Maalox needs to be infused directly into the stomach using either a gastric tube or the open-ended lumen of the Combitube. The challenge this presents often leads to omission of Maalox administration altogether. Another reason for routine omission of Maalox administration is limited knowledge about its objective and importance. Audrey U. Smith reviewed the importance of neutralizing hydrochloric acid in the stomach during circulatory arrest and hypothermia in her work on reviving mammals from subzero temperatures. One theory is that during hypothermia induced circulatory arrest the selective permeability of the inner lining of the gastric mucosa is lost and hydrogen and chloride ions will diffuse into the tissues. Smith et al. tested this theory by withdrawing food for 15 hours and by neutralizing the stomach contents with sodium bicarbonate prior to cooling rabbits below zero. Unlike the animals in earlier experiments, animals receiving this treatment showed no sign of gastric hemorrhage. Histological examination showed that the mucous membrane was indistinguishable from normal . These, and similar, observations guided the decision to use cimetidine to inhibit gastric hydrochloric acid production, and Maalox to neutralize acidic stomach contents in cryonics and the Cryovita/Alcor canine washout experiments.
Complications of gastrointestinal perfusion have also been identified in trauma patients, septic- and circulatory shock, and cardiopulmonary bypass (CPB). Low splanchnic blood flow can lead to increased mucosal permeability, endotoxemia and multiple organ failure . Low gastric mucosal pH has been found to have a high specificity for predicting patient survival in critically ill patients . Gastrointestinal complications are believed to result from CPB induced vasoconstriction and systemic inflammation. Interestingly, non-pulsatile flow is associated with reduced gastric intramucosal pH and a higher incidence of gastrointestinal complications .
The gastrointestinal system has been called ‘the motor of multiorgan failure. ’ In light of the fact that many cryonics patients experience many of the conditions associated with gastrointestinal dysfunction (e.g. , trauma, shock, hypovolemia) and are exposed to a number of cryonics procedures that can worsen these complications (e.g. , prolonged low flow CPR, vasoconstriction, hypothermia, CPB), renewed efforts should be made to investigate the effects of gastrointestinal ischemia during stabilization and transport.
Gastrointestinal complications such as hyperpermeability and abdominal swelling are not only limited to stabilization and transport but can also have profound effects on the ability to cryoprotect whole body patients. In the case of cryoprotectant perfusion these challenges may not only be the result of prior hypoperfusion or ischemia, but may be exacerbated by the hypertonicity and toxicity of the vitrification solution, rapid increases in cryoprotective concentration, non-pulsatile flow, and perfusion at high subzero temperatures.
This section on depressed metabolism was originally published as an individual article in Cryonics magazine, 3rd Quarter, 2007.
Human cryopreservation protocol includes two treatment modalities to modulate cerebral metabolism: administration of a general anesthetic and induction of hypothermia. Many general anesthetics reduce cerebral metabolic demand by reducing excitatory brain activity via GABA-A receptor potentiation. It is not surprising that general anesthetics have been investigated as treatments for medical conditions in which cerebral metabolic demand exceeds energy supply, such as focal and global ischemia. Not unlike other neuroprotective strategies explored in ischemia research, results of human clinical trials have been disappointing. This is remarkable because reduction of metabolic demand should be more effective than many other neuroprotective strategies which target only specific parts of the ischemic cascade of harmful biochemical events that follows cardiac arrest.
Perhaps one reason why general anesthetics do not improve outcome is that the agent can only be administered during reperfusion, when blood supply returns to the tissue after a period of ischemia. However, at this point the energy imbalance has already been upset during the ischemic period. Or to state the matter more generally, a neuroprotective agent can only confer benefits if the agent intervenes at events that are initiated at or continued downstream from the time of reperfusion. Ischemia-induced cell membrane depolarization is one of the more upstream events that produces a number of different pathological changes (e.g. , intracellular calcium overload, mitochondrial failure) that can no longer be reversed by just reducing cerebral metabolic demand with a monoagent upon reperfusion.
Given the extremely narrow time window for mitigating ischemia-induced energy imbalance, perhaps a general anesthetic can only provide a benefit in situations where resuscitation is initiated immediately but remains inadequate to provide sufficient cerebral perfusion. This situation may apply to in-hospital cardiac arrest situations and, of course, many human cryonics stabilization cases. Although administration of a general anesthetic in cryonics is complemented by a number of other (downstream) neuroprotective medications and fluids, the search for a more potent inhibitor of cerebral metabolic demand remains elusive.
It is tempting to look for an agent that would drop metabolic demand to zero, but considering the fact that most of the energy of the brain is expended on regulating intra- and extracellular ion gradients, reductions in metabolic demand that would interfere with basic cellular homeostasis without a corresponding drop in metabolic rate will produce ischemic injury themselves. An intriguing question is whether there are broad neuroprotective strategies that are more potent than general anesthetics in reducing metabolic demand but do not upset basic cellular homeostasis or risk damage to the ultrastructural basis of identity and memory. Such strategies would not only include depressing excitatory activity but also inhibiting downstream and intermediate steps such as protein synthesis, protein transcription, “futile” repair cycles (such as PARP activation), and immune function. Some of these strategies, or the more radical variants thereof, may still remain largely unexplored in mainstream biomedical research because they would be detrimental in a short-term ischemia-reperfusion recovery model. Examples of these include antioxidant and free radical interventions that are so thorough that they will negatively influence the free radical mediated parts of the immune system after reperfusion.
One obstacle for identifying such strategies is the lack of proper definitions of terms like metabolic demand, metabolic rate and metabolic inhibition. Reducing metabolic “demand” can mean the inhibition of specific physiological events with the result that a number of downstream biochemical reactions are activated at a lower rate, or not activated at all. This is conceptually different from strategies that modulate the rate of biochemical reactions in general, such as induction of clinical hypothermia.
Induction of hypothermia is an interesting case because it not only reduces the rate of biochemical reactions, but some (patho) physiological are inhibited altogether at specific temperatures. For example, humans typically will experience ventricular fibrillation and asystole (no cardiac electrical activity) between 15 and 25 degrees Celsius, whereas some natural hibernators will continue to lower heart rate at temperatures down to the freezing point of water. One reason why hypothermia may even confer neuroprotective benefits when the temperature is only slightly lowered is because some parts of the ischemic cascade, like excitatory amino acid release (excitotoxicity), are inhibited to a greater degree than predicted by the Q10 value (2.0) that is often associated with induction of hypothermia . Variability in the effects of temperature on protective and pathological reactions may also explain why hypothermia may even be beneficial after the ischemic insult.
Induction of hypothermia presents a number of challenges as a clinical treatment. One challenge is that the human body will attempt to compensate for unnatural drops in temperature by energy-consuming means, such as shivering. In cryonics this may be prevented by administration of a general anesthetic. External cooling also causes peripheral vasoconstriction that further limits heat exchange to the core of the body. But the fundamental limitation of external cooling is that it is a very ineffective method to lower the core temperature of the patient. Potent alternatives for external cooling in cryonics include extracorporeal cooling and cyclic lung lavage (liquid ventilation).
At very low temperatures (profound and ultra-profound hypothermia), induction of hypothermia itself may produce adverse rheological, metabolic, gastrointestinal, and neurological effects because the balance of protective and pathological metabolic modulation changes in favour of the latter. A good example of this is that cold impairs ATP-driven ion pumps, but passive transport continues as ions move down their electrochemical gradients causing membrane depolarization, intracellular calcium overload, and ultimately, cell death. This is one of the reasons for washing out the blood and replacing it with an “intracellular” organ preservation solution during remote cryonics cases with long transport times.
Although small decreases in brain temperature can confer potent neuroprotective benefits, the logistical challenges of external cooling with ice packs are a concern for cryonics organizations. Not only does the patient needs to be enclosed in a portable ice bath to fully benefit from immersion in circulating ice water, effective cooling is also dependent on vigorous cardiopulmonary support and administration of vasoactive medications. Even if cyclic lung lavage can be established promptly as a bridge to extracorporeal cooling, cryonics stabilization could benefit from practical normothermic methods of metabolic depression to complement, or as a temporary substitute for, hypothermia. Recent investigations into anoxia tolerance, estivation, and hibernation may guide cryonics-specific research to develop these technologies.
Producing a hibernating state in humans after cardiac arrest seems to be a formidable challenge considering the complex and multifactorial biochemical changes of hibernating animals during the hibernation cycle. Hibernators prepare for dormancy, or torpor, by increasing food intake and storage and by decreasing physical activity. Entrance into torpor is marked by lowering of the hypothalamic temperature setpoint, depression of metabolic activity, sequestering of leukocytes, and a decrease in body temperature. During torpor, heart rate and respirations are substantially reduced, or in the case of freeze tolerant animals, like the wood frog, heart rate and respiration are stopped completely . Typical changes in metabolic rate can range from 80% to nearly 100% in cryptobiotic animals, whose metabolic activities come to a reversible standstill. Arousal can be rapid and the need for intermittent euthermic arousal from torpor may involve the need to eliminate sleep debt, restore antioxidant defences, replenish carbohydrates, and remove metabolic end products.
A number of general criteria apply to all animals that tolerate long-term hypometabolic suppression: 1) controlled global metabolic rate suppression, (2) storage and alternate energy metabolism and limited production of toxic end products, (3) triggering and signaling transduction mechanisms to coordinate metabolic pathways between cells and organs, (4) reorganization of metabolic priorities and energy expenditure, (5) coordinated up-and-down regulation of genes, and (6) enhanced defense mechanisms such as increased production of antioxidants and stabilization of macromolecules . Hibernating animals prevent hypothermia-induced injury by maintaining membrane ion potentials, decreasing blood clotting, and limiting energy expenditures to basic physiological necessities at the expense of protein synthesis, gene transcription, and cell division. Selective up-and-down regulation of regulatory enzymes and rapid arousal from torpor is achieved by reversible phosphorylation.
Because aspects of hypometabolism have been induced in some non-hibernating animals by injecting them with the plasma of hibernating animals, some researchers have speculated that a “hibernation induction trigger” (HIT) may exist that controls entry into hibernation. If such a molecule (or number of molecules) exists, it is tempting to believe that activation of HIT in humans can produce hibernation on demand. Practical applications would range from stabilization of cardiac arrest and stroke victims to long-term space flight. Current investigations into HIT-like substances indicate involvement of opioid receptors.
The most promising HIT mimetic so far is the synthetic delta-opioid peptide DADLE (D-Ala2,D Leu5enkephalin). Administration of DADLE to a normothermic multiorgan block preparation was able to extend survival of organs to 46 hours, including the heart and liver . Using the same multiorgan block autoperfusion method, successful single canine lung transplantation after 24 to 33 hours was achieved when the lungs were preserved with woodchuck HIT-containing plasma . Hypothermic preservation time of the rat lung has been enhanced by adding DADLE to Euro-Collins solution . Improved function of hearts pretreated with HIT or DADLE after hypothermic storage have been reported for a number of non-hibernating species including rats, rabbits and swine .
Although beneficial effects of DADLE have been reported in cortical neurons, investigation of DADLE as a neuroprotectant in global and forebrain ischemia has been limited to date. A 2006 study didn’t find any improvement for pre-ischemic administration of DADLE in a forebrain ischemia rat model . In 2007 the Safar Center for Resuscitation Research reported that DADLE failed to improve neurological outcome in a deep hypothermic circulatory arrest rat model and even produced worse extracerebral organ injury for the highest dose administered (10 mg/kg). One explanation for these results is poor blood brain barrier (BBB) permeability of DADLE because of its unfavorable hydrophylicity and charge. A series of cyclic prodrugs of DADLE only improved BBB permeability in the presence of a P-glycoprotein inhibitor to prevent P-gP mediated efflux transporter activation. Bioconversion of the parent drug, however, was low .
Alternatively, pre-ischemic cerebroventricular (ICV) administration of DADLE did confer neuroprotective benefits in a rat model of forebrain ischemia . As these results indicate, neuroprotective agents with high treatment potential do not necessarily have privileged access to the brain.
Opioid receptor modulation in cerebral ischemia has proven to be a viable research direction but the results obtained with HIT-like substances do not seem to produce the multi-factorial and coordinated physiological effects of hypometabolism-mediated cytoprotection that can be observed in hibernating animals. Although induction of artificial hypometabolism in humans may be possible by pharmacologic modulation of conserved metabolic pathways shared with natural hibernators, it is doubtful that hibernation on demand will be possible anytime soon. This challenge is not dissimilar to cryobiological research that aspires to protect humans from the extensive injury that results from exposure to low (subzero) temperatures. Ultimately, advances in modulation of hypometabolism are necessary to protect cryonics patients from brain injury during stabilization and the descent from normothermia to cryogenic temperatures for long-term care. The subtle adverse effects of exposure of the human brain to low temperatures as such may turn out to be one of the final obstacles to be overcome to achieve real suspended animation.
Carbon Monoxide and Hydrogen Sulfide
A number of alternative approaches to induce hypometabolism and hypoxia tolerance that have been explored in recent years include administration of carbon monoxide and hydrogen sulfide. The choice of these two gases is remarkable because both are known to be dangerous poisons at supraphysiological levels. For example, high levels of carbon monoxide can displace oxygen at hemoglobin at a rate in excess of 200 times the rate of oxygen, causing an acute drop in oxygen levels to the tissues. At physiological levels, however, both substances are produced endogenously in the human body where they perform a number of regulatory and signaling functions [61, 62]. Therapeutic administration of low concentrations of carbon monoxide and hydrogen sulfide has been investigated in various models of ischemic injury. Low dose carbon monoxide can enhance protection against hypothermic renal injury and improve function of renal grafts . Hydrogen sulfide increases glutathione levels in glutamate-mediated oxidative stress .
Of most interest is administration of carbon monoxide or hydrogen sulfide to produce a state of hypometabolism or hypoxia tolerance. C. elegans can survive mild hypoxia by hypoxia-inducible factor 1 (HIF-1) modulated anaerobic energy production and up-regulation of antioxidants. C. elegans can also survive extreme hypoxia by entering a state of “suspended animation. ” An intermediate level of hypoxia, however, is deadly to the organism. Carbon monoxide-induced hypometabolism can protect C. elegans embryos against this intermediate level of hypoxia, even in the absence of HIF-1 function .
Following this line of research, in a widely publicized series of experiments, hydrogen sulfide has been found to produce hypometabolism and hypoxia tolerance in mice. Although the research to date has not produced much insight into its molecular mechanisms, results presented so far indicate that hydrogen sulfide exposure produces a change in energy utilization and physiological response that is typical of hibernators .
One issue that has been raised is the lack of proper temperature controls in these experiments . Although it is typical for real hibernators that reductions in metabolic rate precede hypothermia, biochemical versus temperature-induced modulation of metabolism are left implicit in the published results so far. Recent research also indicates that different strains of mice use different metabolic strategies to protect themselves from acute hypoxia . C57BL/6J (C57) inbred mice, the strain used in the hydrogen sulfide experiments, were found to be more hypoxia tolerant than CD-1 outbred mice. In 2007 Haouzi et al. reported that hydrogen sulfide was not able to induce hypometabolism in sedated sheep .
Even if hydrogen sulfide is not able to induce profound normothermic hypometabolism, identification of a molecule that could induce a hibernation-like state in humans, or even just confer broad cytoprotection during induction of artificial hypothermia, would be a non-trivial therapeutic breakthrough. The biomedical potential of the gases nitric oxide, carbon monoxide and hydrogen sulfide are currently being investigated by a new biotechnology company called Ikaria, which includes the prominent nitric oxide / PARP researcher Csaba Szabo among its scientific staff.
Advocates of human cryopreservation may find the increasing use of the term suspended animation for therapeutic interventions like whole body profound asanguineous hypothermia and normothermic hypometabolism indicative of a lack of precision. But the increased support for and research in these areas in mainstream biomedical science and the media may produce a more favorable reception of research aimed at reversible human cryopreservation and real suspended animation. Another advantage of increased research efforts in these areas is that cryonics providers can benefit from these findings to enhance their own capabilities and initiate informed research into improved organ preservation solutions and “hibernation mimetics. ”
The large number of medications and fluids in cryonics medication protocol present non-trivial logistical and practical problems. In some cases only a few medications can be administered before the standby team starts transporting the patient from one location to another. As a result, some of the medications are administered at a time when the more upstream ischemic events may have already started running their course. A good example is delayed administration of VitalOxy, an agent that ideally should be given immediately after restarting circulation and ventilation. In addition, the large number of medications increases the probability of errors and omissions. In light of this, it is understandable that there have been repeated suggestions to combine a number of the medications and fluids in one solution.
There are a number of challenges that need to be considered with this approach. The most important question is which of these medications can be combined in one solution and how stable such a solution will be during long term storage. This question is not solely about the risk of adverse interactions between the individual drugs, but also involves more subtle issues such as variability in recommended storage temperatures for the individual components. The problem in cryonics is aggravated by the fact that some of the molecules that are used are not ordinary drugs and have never been investigated for interaction with other drugs, let alone during long term storage in solution. In case a solution like this would be produced in advance by the cryonics organization, a new set of problems is created as a result of having to combine several sterile parentals and non sterile molecules in one solution.
Combining different medications in a solution also limits the ability of a cryonics team to eliminate a drug that is contra-indicated in a specific patient. And if some of the medications in the solution should be given by weight and others should not, the ability to vary the dosage of the weight adjusted medications will be eliminated as well. It will also reduce the flexibility in changing the sequence and total volume of the medications. For example, if neuroprotective molecules are combined with a crystalloid or colloid, the fluid will have to be administered regardless whether the volume status of the patient mandates this course of action.
Moving forward, cryonics organizations can start investigating which drugs can be safely combined shortly before a case. This way the benefits of combining medications and fluids can be reaped without the disadvantages of creating fixed solutions that eliminate flexibility.
If a multi-modal solution needs to be developed for cryonics it should have some, or all, of the following characteristics. The vehicle itself is neuroprotective (e.g. , hypertonic saline) or has anticoagulant properties (e.g. , sodium citrate). The individual components have multiple and independent beneficial effects that reduce the need for a large number of different molecules. The solution should not have adverse hemodynamic effects. The solution should only include components that are rarely contra-indicated in a typical cryonics patient. All the individual molecules can be obtained cost-effectively from chemical suppliers. The solution should be validated for long term storage at different temperatures. A major advantage of formulating such a solution is that it would enable cryonics organizations that currently don not offer standby and stabilization to offer some degree of pharmacological intervention. A fluid like this can be shipped in advance or overnight to funeral directors or local cryonics support groups.
Cryonics stabilization medications protocol has come a long way since the first cryonics pioneers advocated prompt administration of heparin after legal pronouncement of death to improve cryoprotectant perfusion. Mostly due to dedication and hard work by Mike Darwin and Jerry Leaf, cryonics stabilization protocol now includes a large number of medications and fluids to secure cerebral viability. Although some medications were eliminated and others have been added, the core objectives still include reduction of metabolic demand, reversal and inhibition of blood clotting, restoring pH, improving blood pressure and microcirculation, oncotic support, antibiotics, and multi-modal protection against cerebral ischemia and reperfusion injury.
Although some of these agents are routinely used in emergency medicine and critical care medicine, some are experimental molecules that are believed to work in synergy in protecting the brain. As evidenced by the results obtained in the cerebral resuscitation research at CCR, such an outcome driven approach does not only make theoretical sense, it works as well. The most obvious question, however, and one that has been frequently asked, is which drugs are the most important to secure cerebral viability. This may or may not be the same as asking which drugs are the most important to ensure optimal cryoprotective perfusion of the brain.
Although it is possible that the limits of cerebral resuscitation can be pushed even further by adding new or more potent medications, at some point the sheer number of medications becomes too difficult to manage, even with sophisticated technologies to administer them. A related problem is increased possibility of adverse interaction effects or excessive volume. Further increasing the list of medications will make it even harder to identify the relative efficacy of the individual components, let alone persuading the medical establishment to abandon its current approach. After demonstrating “proof of principle” that combinational pharmacotherapy works in normothermic cerebral resuscitation the next step is to outline a research agenda to identify how much of these effects can be obtained with a smaller number of drugs.
A core objective in such an endeavor would be to investigate and validate substances that can achieve a more potent reduction in normothermic cerebral energy utilization than the anesthetics used today. This would require moving in the opposite direction of recent research to develop more selective agents that only act on certain subunits of the GABA-A receptors to produce more controlled and gentle effects (for example anesthesia without hypnosis). Recent advances in the molecular biology of hibernation and inducing “suspended animation” in mammals indicate the potential of such alternatives. One advantage of securing more profound levels of metabolic depression may be that its effects will not be completely offset by a corresponding drop in blood pressure that is often associated with agents with neurodepressive effects.
Reversing and preventing blood coagulation still remains the most important intervention in cryonics stabilization. Whereas most of the neuroprotective agents are believed to confer benefits in terms of securing viability, massive blood clotting could frustrate adequate cryoprotective perfusion and produce ultrastructural damage as a result. Remarkably, the mechanisms of stasis and reperfusion induced blood clotting in cryonics have not been given as much attention as one would expect. In vitro and in vivo simulation of “post-mortem” behavior of blood (and related rheological effects) should be another research priority in cryonics. Ultimately, fibrinolytics, anticoagulants and antiplatelet drugs should be validated in realistic models that have cryoprotectant perfusion of the brain as an end point. These investigations should also include the effects of (profound) hypothermia on the blood and the effect of temperature on the drugs used to modulate blood abnormalities.
The objective to investigate blood coagulation with cryoprotective perfusion as an end point raises a general point about validating cryonics stabilization medications. The medications that are used in cryonics today have been investigated in a canine cerebral resuscitation model. Although it is plausible to assume that these interventions will also contribute to better outcomes in cryoprotective perfusion this research has never been done. Optimizing cryoprotective perfusion does not necessarily lead to the same kind of research priorities and choice of drugs as securing cerebral viability. The potent anticoagulant citrate and some drugs that profoundly depress (cerebral) metabolism may not be the ideal choice in a short term recovery model but could be core components of a protocol aimed at optimizing cryoprotective perfusion of the brain. Securing viability of the brain of most cryonics patients remains elusive but pharmacologic treatment of the patient to optimize complete distribution of the cryoprotective agent in the brain may be within reach for many patients. Other areas that may benefit from such research include the composition of organ preservation solutions and cryoprotectant carrier solutions.
The advantages and disadvantages of most of the agents reviewed in this article require more investigation but some observations may warrant changes to current protocol: a more prominent place for vasopressin (and a reconsideration of epinephrine) and Maalox; a reduction of the number of NSAIDs; a decision to use either PBN or TEMPOL; increased dosage of niacinamide; addition of minocycline, magnesium, FK 506 and an Na+/H+ exchange inhibitor; and substitution of hypertonic saline (dextran) for dextran-40 and/or mannitol. These changes may confer moderate improvements in hemodynamics and neuroprotection without increasing the total number of medications and fluids currently used. Other improvements may be possible with “hibernation mimetics” and opioid agonists, and new agents to modulate -blood coagulation, -blood rheology and the -immune system, provided a systematic effort to identify, investigate and validate such drugs in a realistic model is made.
Currently, not much is known about pharmacodynamics and pharmacokinetics during a cryonics case. Long agonal periods, advanced atherosclerosis, cardiopulmonary support and induction of hypothermia may have unknown effects on the absorption, distribution and biotransformation of many of the drugs. The likelihood of adverse or competitive interactions between these drugs is another area that warrants review.
A realistic model to investigate cryonics stabilization medications that can mimic the typical agonal state of a cryonics patient (such as sepsis, multiple organ failure and prolonged hypoxia), CPS (low flow perfusion) and induction of hypothermia is currently being investigated. This model can also be used to validate the possibility of multi-modal resuscitation fluids that can be used as (low cost) substitutes for a number of drugs and fluids or as a complement to existing therapy. Rapid intranasal delivery of neuroprotectant agents directly to the brain of cryonics patients is another new research direction in cryonics . Intrapulmonary administration of medications also remains largely uninvestigated.
I am greatly indebted to the Life Extension Foundation for financial support to research and write this review. I am also indebted to Chana de Wolf and Mike Darwin, who devoted considerable time to proofread and edit the text. Without Mike Darwin a review of this scope would not have been possible.
3. Doursout MF, Joseph PM, Liang YY, Hartley CJ, Chelly JE. Role of propofol and its solvent, intralipid, in nitric oxide-induced peripheral vasodilatation in dogs. British Journal of Anaesthesia. 2002 Sep;89(3):492-8.
7. Böttiger BW, Motsch J, Böhrer H, Böker T, Aulmann M, Nawroth PP, Martin E. Activation of blood coagulation after cardiac arrest is not balanced adequately by activation of endogenous fibrinolysis. Circulation. 1995 Nov 1;92(9):2572-8.
8. Rijken DC, Seifried E, Barrett-Bergshoeff MM, Dooijewaard G. Plasminogen activation at low temperatures in plasma samples containing therapeutic concentrations of tissue-type plasminogen activator or other thrombolytic agents. Thrombosis and Haemostasis. 1990 Aug 13;64(1):47-52.
9. Juhlin P, Boström PA, Torp A, Bredberg A. Streptokinase antibodies inhibit reperfusion during thrombolytic therapy with streptokinase in acute myocardial infarction. Journal of Internal Medicine. 1999 May;245(5):483-8.
11. Nicolini FA, Lee P, Rios G, Kottke-Marchant K, Topol EJ. Combination of platelet fibrinogen receptor antagonist and direct thrombin inhibitor at low doses markedly improves thrombolysis. Circulation. 1994 Apr;89(4):1802-9.
12. Nakayama M, Uchimura K, Zhu RL, Nagayama T, Rose ME, Stetler RA, Isakson PC, Chen J, Graham SH. Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proceedings of the National Acadamy of Sciences of the United States of America. 1998 Sep 1;95(18):10954-9.
14. Wenzel V, Linder KH, Augenstein S, Prengel AW, Strohmenger HU. Vasopressin combined with epinephrine decreases cerebral perfusion compared with vasopressin alone during cardiopulmonary resuscitation in pigs. Stroke. 1998 Jul;29(7):1462-7.
16. Németh H, Robotka H, Kis Z, Rózsa E, Janáky T, Somlai C, Marosi M, Farkas T, Toldi J, Vécsei L. Kynurenine administered together with probenecid markedly inhibits pentylenetetrazol-induced seizures. An electrophysiological and behavioural study. Neuropharmacology. 2004 Nov;47(6):916-25.
17. Schuck PF, Tonin A, da Costa Ferreira G, Viegas CM, Latini A, Duval Wannmacher CM, de Souza Wyse AT, Dutra-Filho CS, Wajner M. Kynurenines Impair Energy Metabolism in Rat Cerebral Cortex. Cellular and Molecular Neurobiology. 2007 Feb;27(1):147-60.
18. Reiter RJ, Tan DX, Leon J, Kilic U, Kilic E. When melatonin gets on your nerves: its beneficial actions in experimental models of stroke. Experimental Biology and Medicine, 2005 Feb;230(2):104-17.
19. Kuroda S, Tsuchidate R, Smith ML, Maples KR, Siesjo BK. Neuroprotective effects of a novel nitrone, NXY-059, after transient focal cerebral ischemia in the rat. Journal of Cerebral Blood Flow and Metabolism. 1999 Jul;19(7):778-87.
21. Wu D, Bassuk J, Arias J, Kurlansky P, Lozano H, Lamas G, Adams JA. Different roles of nitric oxide synthase isoforms in cardiopulmonary resuscitation in pigs. Resuscitation. 2007 Apr;73(1):144-53.
22. Moroni F, Meli E, Peruginelli F, Chiarugi A, Cozzi A, Picca R, Romagnoli P, Pellicciari R, Pellegrini-Giampietro DE. Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia. Cell Death and Differentation. 2001 Sep;8(9):921-32.
23. Klaidman L, Morales M, Kem S, Yang J, Chang ML, Adams JD Jr. Nicotinamide offers multiple protective mechanisms in stroke as a precursor for NAD+, as a PARP inhibitor and by partial restoration of mitochondrial function. Pharmacology. 2003 Nov;69(3):150-7.
24. Ying W, Wei G, Wang D, Wang Q, Tang X, Shi J, Zhang P, Lu H. Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia. Frontiers in Bioscience. 2007 Jan 1;12:2728-34.
27. Tsai MS, Chiang WC, Lee CC, Hsieh CC, Ko PC, Hsu CY, Su CP, Chen SY, Chang WT, Yuan A, Ma MH, Chen SC, Chen WJ. Infections in the survivors of out-of-hospital cardiac arrest in the first 7 days. Intensive Care Medicine. 2005 May;31(5):621-6.
29. Kraus RL, Pasieczny R, Lariosa-Willingham K, Turner MS, Jiang A, Trauger JW. Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity. Journal of Neurochemistry. 2005 Aug;94(3):819-27.
30. Alano CC, Kauppinen TM, Valls AV, Swanson RA. 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.
31. Ianaro A, Ialenti A, Maffia P, Sautebin L, Rombolà L, Carnuccio R, Iuvone T, D’Acquisto F, Di Rosa M. Anti-inflammatory activity of macrolide antibiotics. Journal of Pharmacology and Experimental Therapeutics. 2000 Jan;292(1):156-63.
38. Ren Z, Ding W, Su Z, Gu X, Huang H, Liu J, Yan Q, Zhang W, Yu X. Mechanisms of brain injury with deep hypothermic circulatory arrest and protective effects of coenzyme Q10. Journal of Thoracic and Cardiovascular Surgery. 1994 Jul;108(1):126-33.
39. Damian MS, Ellenberg D, Gildemeister R, Lauermann J, Simonis G, Sauter W, Georgi C. Coenzyme Q10 combined with mild hypothermia after cardiac arrest: a preliminary study. Circulation, 2004, Nov 9;110(19):3011-6.
42. Miles AN, Majda BT, Meloni BP, Knuckey NW. Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats. Neurosurgery. 2001 Dec;49(6):1443-50
43. Matsumoto Y, Yamamoto S, Suzuki Y, Tsuboi T, Terakawa S, Ohashi N, Umemura K Na+ /H+ Exchange Inhibitor, SM-20220, Is Protective Against Excitotoxicity in Cultured Cortical Neurons. Stroke, 2004; 35: 185-190
44. Kaminska B, Gaweda-Walerych K, Zawadzka M. Molecular mechanisms of neuroprotective action of immunosuppressants–facts and hypotheses. Journal of Cellular and Molecular Medicine. 2004 Jan-Mar;8(1):45-58.
45. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 1999 Apr 9;284(5412):339-43.
46. Macleod MR, O’Collins T, Horky LL, Howells DW, Donnan GA. Systematic review and metaanalysis of the efficacy of FK506 in experimental stroke. Journal of Cerebral Blood Flow and Metabolism. 2005 Jun;25(6):713-21.
49. Doglio GR, Pusajo JF, Egurrola MA, Bonfigli GC, Parra C, Vetere L, Hernandez MS, Fernandez S, Palizas F, Gutierrez G. Gastric mucosal pH as a prognostic index of mortality in critically ill patients. Critical Care Medicine. 1991 Aug;19(8):1037-40
50. Hamulu A, Atay Y, Yağdi T, Dişçigil B, Bakalim T, Büket S, Bilkay O. Hamulu A, Atay Y, Yağdi T, Dişçigil B, Bakalim T, Büket S, Bilkay O. Effects of flow types in cardiopulmonary bypass on gastric intramucosal pH. Perfusion. 1998 Mar;13(2):129-35.
54. Chien S, Oeltgen PR, Diana JN, Salley RK, Su TP. Extension of tissue survival time in multiorgan block preparation with a delta opioid DADLE ([D-Ala2, D-Leu5]-enkephalin). Journal of Thoracic and Cardiovascular Surgery. 1994 Mar;107(3):964-7
58. Iwata M, Inoue S, Kawaguchi M, Kurita N, Horiuchi T, Nakamura M, Konishi N, Furuya H. Delta opioid receptors stimulation with [D-Ala2, D-Leu5] enkephalin does not provide neuroprotection in the hippocampus in rats subjected to forebrain ischemia. Neuroscience Letters. 2007 Mar 13;414(3):242-6.
59. Ohe T, Sato M, Tanaka S, Fujino N, Hata M, Shibata Y, Kanatani A, Fukami T, Yamazaki M, Chiba M, Ishii Y. Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid/plasma concentration ratio. Drug Metababolism and Disposition. 2003 Oct;31(10):1251-4.
60. Su Dian-san, Wang Zhen-hong, Zheng Yong-jun, Zhao Yan-hua and Wang Xiang-rui. Dose-dependent Neuroprotection of Delta Opioid Peptide [D-Ala2, D-Leu5] Enkephalin in Neuronal Death and Retarded Behavior Induced by Forebrain Ischemia in Rats. Neuroscience Letters. 2007, in press.
63. Neto JS, Nakao A, Kimizuka K, Romanosky AJ, Stolz DB, Uchiyama T, Nalesnik MA, Otterbein LE, Murase N. Protection of transplant-induced renal ischemia-reperfusion injury with carbon monoxide. American Journal of Physiology: Renal Physiology. 2004 Nov;287(5):F979-89.
65. Nystul TG, Roth MB. Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 2004 Jun 15;101(24):9133-6.