Summary

The most important objective in cryonics after pronouncement of legal death is to drop metabolism as fast as possible to protect the brain and other tissues from rapid deterioration prior to vitrification. To date, most strategies to reduce metabolic demand are of limited value or not practical as an emergency treatment. General anesthetics may only work if they are administered within a very tight time window. Cooling is a potent strategy to protect the brain but presents a number of practical and clinical challenges, including detrimental effects at lower temperatures.

Studies of hibernating animals provide an interesting perspective on the biological mechanisms involved in depressing metabolism and protection from cold injury. So far, practical applications of these studies have been limited to the identification of molecules involved in hibernation and a number of modest successes in organ preservation and reduction of heart and brain injury. Although hibernation on demand for humans remains elusive, recent research into carbon monoxide and hydrogen sulfide-induced “suspended animation” revives hopes for better protection of cryonics patients during stabilization and transport.

Introduction

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.

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 [1]. 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.

Hibernation

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 [2]. 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 [3]. 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 [4]. 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 [5]. Hypothermic preservation time of the rat lung has been enhanced by adding DADLE to Euro-Collins solution [6]. 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 [7].

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 [8]. 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 [9].

Alternatively, pre-ischemic cerebroventricular (ICV) administration of DADLE did confer neuroprotective benefits in a rat model of forebrain ischemia [10]. 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 [11, 12]. 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 [13]. Hydrogen sulfide increases glutathione levels in glutamate-mediated oxidative stress [14].

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 [15].

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 [16].

One issue that has been raised is the lack of proper temperature controls in these experiments [17]. 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 [18]. 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 [19].

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. ”

Endnotes

1. Nakashima Ken and Todd Michael M. Effects of Hypothermia on the Rate of Excitatory Amino Acid Release After Ischemic Depolarization. Stroke, May 1996; 27: 913 – 918.

2. Layne JR, Lee RE, Heil TL. Freezing-induced changes in the heart rate of wood frogs (Rana sylvatica). American Journal of Physiology. 1989 Nov; 257(5 Pt 2): 1046-9.

3. Storey KB, Storey JM. Tribute to P. L. Lutz: putting life on ‘pause’–molecular regulation of hypometabolism. Journal of Experimental Biology. 2007 May;210(Pt 10):1700-14.

4. 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

5. Oeltgen PR, Horton ND, Bolling SF, Su TP. Extended lung preservation with the use of hibernation trigger factors. Annals of Thoracic Surgery. 1996 May;61(5):1488-93

6. Wu G, Zhang F, Salley RK, Diana JN, Su TP, Chien S. delta Opioid extends hypothermic preservation time of the lung. Journal of Thoracic and Cardiovascular Surgery. 1996 Jan;111(1):259-67.

7. Sigg DC, Coles JA, Gallagher WJ, Oeltgen PR, Iaizzo PA. Opioid preconditioning: myocardial function and energy metabolism. Annals of Thoracic Surgery. 2001 Nov;72(5):1576-82.

8. 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.

9. 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.

10. 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.

11. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiological Reviews. 2006 Apr;86(2):583-650.

12. Łowicka E, Bełtowski J. Hydrogen sulfide (H2S) – the third gas of interest for pharmacologists. Pharmacological Reports. 2007 Jan-Feb;59(1):4-24

13. 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.

14. Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress. FASEB Journal. 2004 Jul;18(10):1165-7.

15. 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.

16. Blackstone E, Roth MB. Suspended animation-like state protects mice from lethal hypoxia. Shock. 2007 Apr;27(4):370-2.

17. Wowk, B. Is Hydrogen Sulfide the Secret to Suspended Animation? Cryonics, July/August 2005

18. Zwemer CF, Song MY, Carello KA, D’Alecy LG. Strain differences in response to acute hypoxia: CD-1 versus C57BL/6J mice. Journal of Applied Physiology. 2007 Jan;102(1):286-93.

19. Haouzi P, Notet V, Chenuel B, Chalon B, Sponne I, Ogier V, Bihain B. H(2)S induced hypometabolism in mice is missing in sedated sheep. Respiratory Physiology & Neurobiology, 2007 Sep 14, in press