“Cryonics does not involve the freezing of dead people. Cryonics involves placing critically ill patients that cannot be treated with contemporary medical technologies in a state of long-term low temperature care to preserve the person until a time when treatments might be available.”
L-Kynurenine (L-KYN) is one of the neuroprotective agents used in cryonics stabilization protocol to limit injury to the brain after cardiac arrest. Administration of L-KYN was perceived to be essential to resuscitate dogs from extended periods (up to 17 minutes) of normothermic ischemia during the Critical Care Research (CCR) cerebral resuscitation experiments in the late 1990s. In cryonics L-KYN has been combined with another neuroprotective agent, niacinamide, to make the compound NiKy.
L-KYN is the precursor of kynurenic acid, the only known endogenous antagonist of the excitatory amino acid receptors. Unlike kynurenic acid, L-KYN can cross the blood brain barrier. Because in cryonics neuroprotective agents are administered to the systemic circulation, the effects of such molecules on blood pressure and cerebral blood flow must be weighed against the benefits as a neuroprotectant.
Katalin Sas et al. investigated the effect of systemic administration of L-Kynurenine on corticocerebral blood flow under normal and ischemic (unilateral carotid artery occlusion) conditions in conscious rabbits. Corticocerebral blood flow (cCBF) was measured using the hydrogen clearance technique (i.e., hydrogen polarography). The investigators observed a significant increase in cCBF for both normal and ischemic animals. In the ischemic animals systemic administration of L-KYN resulted in cCBF that approached or even exceeded the base values measured in the normal animals. Administration of L-KYN did not alter arterial blood pressure or heart rate and its effects were of long duration, peaking between 60 and 240 minutes after administration.
The experiments cannot answer the question of whether L-KYN itself or one of its derivatives (such as kynurenic acid) increases cCBF. Other NMDA receptor antagonists have been found to increase cCBF as well. The authors investigated the possibility that the increase of cCBF might be caused by activation of the ascending cholinergic pathways as a response to NMDA receptor antagonism and found that pre-treatment with atropine prevented the increase of cCBF by L-KYN. The authors also investigated the effect of pre-treatment with the non-specific nitric oxide synthase (NOS) inhibitor L-NAME and found that the increase of cCBF was blocked by this agent as well. This suggests that the L-KYN induced increase in cCBF may be mediated by NMDA antagonist induced NO production. A direct effect of L-KYN on cerebral vessels is doubtful because other studies using either glutumate, NMDA, or agonists and antagonists of the former, failed to affect the tone of isolated cerebral arteries.
If systemic administration of L-KYN enhances cCBF in humans as well, L-KYN might be an attractive agent to treat stroke and cardiac arrest due to its multimodal properties. The beneficial properties of L-KYN on cCBF, instead of (or in addition to) its properties as a neuroprotectant may explain its importance in the CCR cerebral resuscitation experiments. Unlike some neuroprotective agents used in cryonics, such as Propofol and Tempol, L-KYN does not appear to have adverse hemodynamic effects and even improves cerebral blood flow. Although the efficacy of kynurenine as a neuroprotectant in cryonics remains uncertain and investigations into the biochemical and temporal aspects of its metabolism (and the effects of rapid induction of hypothermia on this) are warranted, the drug cannot be ruled out because of adverse effects on blood pressure or cerebral blood flow.
A comprehensive review of cryonics stabilization medications is now published on the Alcor website.
Table of contents:
* Introduction
* General Anesthesia
* Blood Coagulation
* Vasoactive Medications
* Excitotoxity
* Free Radicals
* Nitric Oxide and PARP
* Inflammation
* Antibiotics
* Acidosis
* Hemodilution and Osmotic Therapy
* Coenzyme Q10
* Magnesium
* Na+ /H+ Exchange Inhibition
* Immunosuppressive Drugs
* Gastrointestinal Ischemia
* Depressed Metabolism
* Combining Medications
* Conclusion
Elevation of body temperature occurring as a result of hypothalamic coordination of autonomic, neuroendocrine, and behavioral responses in reaction to physiological injury or invasion is generally known as fever. Traditional thought is that the “febrile response” is beneficial in preventing the proliferation of invading microorganisms, but some caregivers consider fever to be harmful and prescribe antipyretic agents and/or physical cooling methods to suppress fever. In their recent publication, Aiyagari and Diringer summarize the data that exists concerning the efficacy of physical and pharmacological treatments in reducing temperature and improving outcome in a variety of acute neurological disorders including stroke, traumatic brain injury, and cardiac arrest.
Several rationales exist for treating fever, including the relief of discomfort associated with fever, reduction of fever-imposed increase in metabolic demand, reduction in morbidity and mortality, reduction of fever-induced cognitive impairment, and prevention of febrile seizures. Most of these rationales are beneficial in theory, but have not been proven in practice. In the case of morbidity/mortality reduction, treatment with antipyretics has been shown to prolong certain infections; similarly, fever is known to improve survival of patients with community acquired pneumonia, Eschericia coli bacteremia, and Pseudomonas aeruginosa sepsis. Compounding these issues is the fact that traditional methods of lowering temperature in febrile patients are ineffective.
Elevated temperature exacerbates neuronal injury caused by cerebral ischemia or traumatic brain injury (TBI) and, conversely, hypothermia acts as a neuroprotectant in such cases. Well-controlled animal models of global and focal ischemia demonstrate a significantly detrimental effect of hyperthermia on clinical outcome and neuropathological changes. Ginsberg and Busto ( 1998 ) list a number of mechanisms through which hyperthermia worsens outcome in cerebral ischemia: increased neurotransmitter release, increased free radical production, opening of the blood-brain barrier, increased depolarizations within the penumbra, impaired brain metabolism and second messenger inhibition, and cytoskeletal degradation. The authors also note that “the action of otherwise neuroprotective drugs in ischemia may be nullified by mild hyperthermia.” Meticulous brain temperature monitoring and treatment of elevated temperature in patients suffering from neurological insult may, therefore, help prevent secondary injury.
Clinical studies of TBI and survivors of cardiac arrest have demonstrated an independent relationship between fever and poor outcome. Although fever is extremely common in neurological intensive care unit patients, the lack of effective fever treatment options has severely limited the availability of data regarding the benefits of fever reduction in such patients. However, recent advances in surface and intravascular cooling devices have lead to improvements in ability to reduce temperature, especially in patients with neurological injuries. An external cooling device known as the Medivance Arctic Sun temperature management system appears to be quite effective at reducing temperature in febrile patients (75% reduction in fever burden) as compared with more traditional means of fever reduction such as air- or water-circulating cooling blankets. Similarly, a newly-devised catheter-based heat exchange system (Cool Line/Cool Gard) has been tested in patients with subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), cerebral infarction, or TBI, showing a 64% reduction in fever burden as compared to the conventional treatment group (antipyretic, cooling blanket, and ice packs). Unfortunately, no data exists concerning these interventions’ impact on outcome.
As cooling devices and methods are improved and proven to be effective, more data concerning the effect of fever reduction on outcome should be forthcoming. Importantly, as Aiyagari and Diringer point out in the conclusion of their review:
“In the absence of conclusive data, the approach to fever management should be based on the balance between the potential for fever to exacerbate brain insults vs. enhance the ability to fight infections. Fortunately, the risk of ongoing brain injury is usually limited to the early phase in the course of most acute insults while the risk of infection rises as time goes on. Thus it would seem reasonable to aggressively control fever during the first few hours to days following ischemic stroke, intracerebral hemorrhage and head injury. Subsequently, aggressive fever control is less likely to be of help and could be detrimental.”
In cryonics patients, infection exacerbation is less important than protecting the brain from injury and warrants immediate induction of rapid cooling to protect patients from injury due to elevated temperatures. The benefits of treating fever in brain injury also highlights the importance of maintaining normothermia, or even hypothermia, in agonal (hypoxic) patients that present for cryopreservation.
In a widely publicized series of experiments by Blackstone et al., hydrogen sulfide (H2S) was found to induce hypometabolism in mice. These experiments raised interest in whether such “suspended animation” could be achieved in humans. If administration of H2S would be able to reduce metabolic rate in humans to the same extent as observed in mice, the practical applications could range from management of trauma victims to space travel.
Recent work by Haouzi et al. is not encouraging. The researchers were able to induce hypometabolism in mice but did not find any change in metabolic rate in sedated sheep after exposing them to 60 ppm H2S. What the investigators did report, and which was left implicit in previous experiments with mice, is that H2S-induced hypometabolism preceded hypothermia. H2S-induced hypometabolism in mice is not just the result of hypothermia, so the inability of H2S to produce hypometabolism in sheep cannot be attributed to thermal inertia of large animals.
How can H2S induce hypometabolism in mice? The authors state that “the present results have little to offer on the pathways that are responsible for H2S-induced decrease of metabolism.” They raise the point that in small animals such as mice a large portion of metabolism is devoted to heat production instead of ATP production. In contrast, small reductions in oxygen utilization in humans, as produced by H2S exposure, will affect ATP generation. Or as Ikaria‘s Csaba Szabo speculates in “Hydrogen sulphide and its therapeutic potential”, “the window of opportunity to compromise oxidative phosphorylation in a human, therefore, must be smaller than in the mouse.“
The authors do not expect that higher dosages of H2S will produce hypometabolism in large mammals because the 60 ppm that was administered to sheep already exceeds what is known to be toxic in humans.
The search for molecules that induce hypometabolism, let alone hibernation-on-demand, in humans remains elusive. So far most research in this area involves attempts to activate conserved metabolic pathways of hibernating animals in non-hibernating animals instead of direct pharmacological inhibition of high energy consuming physiological activities.
One consequence of the growing understanding of the biochemical pathways involved in brain injury resulting from cardiac arrest, stroke, and brain trauma is that there is an increasing consensus among researchers that combination therapy is the most logical treatment for the multifactorial injury mechanisms responsible for neuronal death. In this context, combination therapy can mean either combining different forms of treatment, such as hypothermia and a neuroprotective agent, or the combination of multiple neuroprotective agents. But despite encouraging results with combination therapy in animal models, and disappointing outcomes for single neuroprotective agents (such as the recent free radical spin trap agent NXY-059) in human clinical trials, there are no indications that the current trend of investigating just one neuroprotective agent will be reversed soon.
One obstacle for successful combination treatment that is not often addressed is that cardiac arrest, stroke, and brain trauma are acute events that do not allow a vocal pro-active role of the patient at the time that this could benefit him. During the immediate post-insult period when the molecular events leading to neuronal death, and even higher brain death, play out, most patients are not able to communicate their wishes, or are in a coma. As a result, the patient is not present at the time when the most important decisions about his survival as a person are being made.
This predicament is different from patients suffering from serious but chronic diseases such as AIDS and cancer. In his book “Surviving Terminal Cancer: Clinical Trials, Drug Cocktails, and Other Treatments Your Oncologist Won’t Tell You About” psychology professor Ben Williams documents how he improved his odds of surviving a glioblastoma multiforme brain tumor by researching and pursuing his own treatment, which consists of a combination of conventional and “alternative” treatments.
Williams’ successful case of personalized combination therapy does not present strict scientific evidence that his treatment is the cause of his remarkable recovery (so far), but it does highlight the general benefits that may be obtained when patients demand some degree of control over their choice of treatments. Williams stresses that patients such as himself may have much to gain, and not much too lose, from pursuing such an experimental “cocktail” approach. A similar situation applies to patients who are at risk of severe brain injury and cannot afford to wait until the mechanisms and comparative efficacies of each individual component of a neuroprotective cocktail have been thoroughly investigated.
How can such an outcome driven treatment of cerebral ischemia gain acceptance? Since the patient will not “be there” to investigate and demand unorthodox experimental treatments, he can only influence his odds by leaving advance directives to medical care givers and relatives to request that such treatments are given to him. Such measures can only have a chance of succeeding, however, if experimental treatment options are documented for these patients.
In contrast, combinational pharmacotherapy and hypothermia have been core components of human cryopreservation stabilization protocol for many years. To date, researchers involved in cryonics have made record achievements in normothermic cerebral resuscitation and (ultra)profound hypothermic resuscitation. The applications of such research should not be limited to minimizing brain injury in cryonics patients but should be shared with the general public to help build a “supply” side of experimental treatments that can be consulted by medical care givers and relatives of the patient.
Individuals who are signed up for cryonics have a personal interest in stimulating such research, its documentation and dissemination because acute insults such as cardiac arrest, stroke, and brain trauma can produce (higher) brain death before the individual will present for human cryopreservation in the future. Indeed, cryonics may offer the only chance of personal survival for patients who are at risk of major brain damage if they are resuscitated and left to live at room temperature.
The best non-invasive indicator of cardiac output and oxygenation during cardiopulmonary support (CPS) is end tidal carbon dioxide (ETCO2). ETCO2 is the partial pressure of carbon dioxide (CO2) at the end of an exhaled breath. Until recently, cryonics standby kits were equipped with disposable colorimetric ETCO2 detectors. Some limitations of the disposable ETCO2 detectors are that they are not quantitative, not continuous, hard to read in the dark, and can give false readings. In 2006 this situation changed when Alcor used the CO2SMO, a sophisticated monitoring device that can give a complete respiratory profile of the patient, during a case.
Although devices like the CO2SMO represent the state of the art in respiratory monitoring, their cost, size and complexity may limit routine use of this equipment in remote cases. In August 2007 the cryonics company Suspended Animation added the Capnocheck to its standby equipment. The Capnocheck is similar in size to the older colorimetric ETCO2 detectors but gives quantitative and digital readings for ETCO2 and respiratory rates using infrared technology. ETCO2 readings are given in mmHg and the respiratory rate is given in breaths per minute. Some models come with an alarm that can be set for high and low ETCO2 readings.
ETCO2 can be used to evaluate the effectiveness of chest compressions and as a predictor of outcome during cardiopulmonary resuscitation. Studies have found that patients with restoration of spontaneous circulation (ROSC) have higher ETCO2 levels than patients that could not be resuscitated (levels <10 mmHg). Normal ETCO2 levels are between 35 and 45 mmHg. Because numeric readings of ETCO2 have rarely been obtained and analyzed in cryonics, knowledge about what ETCO2 levels to expect and not to expect are unknown. At this point in time, meticulous note taking of ETCO2 levels during CPS is essential to generate a series of data for cryonics patients.
Another important use of ETCO2 monitoring is that it can be used to validate correct placement of the endotracheal tube (or Combitube). If the endotracheal tube has been placed in the esophagus, or has become dislodged, one would expect to see negligible ETCO2 readings. Another issue that needs to be taken into account is the effect of stabilization medications on ETCO2. For example, administration of the vasopressor epinephrine will decrease ETCO2 readings although cerebral blood flow may be improved. Some cryonics technologies such as liquid ventilation appear to be incompatible with ETCO2 monitoring altogether.
ETCO2 monitoring does not give direct information on how well the brain of a cryonics patient is being perfused. New non-invasive technologies that can do this will be reviewed in the future.
As discussed by R. Michael Perry in his recent contribution to Cryonics Magazine, “Alternatives to Cryonics: A Very Preliminary Study,” (3rd Quarter 2007) chemical fixation of the brain may be a substitute for cryopreservation in circumstances where cryonics is not feasible or affordable. Several issues come into play when attempting to determine whether chemical fixation results in acceptable preservation of ultrastructure. An important question is whether chemical fixatives are uniformly distributed throughout the brain, preventing the occurrence of islands of tissue decomposition due to inadequate fixation.
In their 2002 paper, Knudsen et al. provide some preliminary answers to this question. Due to the difficulty in obtaining fresh brains for study from large aquatic mammals, the researchers developed a novel method for in situ fixation of (minke) whale brains. The procedure involved cutting a triangular opening in skull and pouring 8% formalin solution into the epidural and subdural space until it leaked out through the foramen magnum. The foramen magnum was then plugged, the triangular bone piece replaced to reduce loss of fixative, and the fixative level was checked and refilled every 4 hours, if necessary. Pilot studies indicated that a diffusion period of at least 60 hours is required for adequate fixation of large volume (average = 2201 g) minke whale brains, so the researchers set the in situ fixation period at 72 hours “to ensure that even the largest brains were sufficiently fixed prior to excision.” The brains were then excised and stored in formalin for at least 2 months prior to gross and microscopic examination.
Gross examination revealed that “there were no cases where the brain tissue was liquefied or smelled sour due to post mortem bacterial growth and the occurrence of artifacts and autolytic changes due to incomplete fixation was generally low.” Microscopic examination showed well-preserved cells and myelin in all parts of the brain. Specifically, histological evaluation categorized 97.3 to 100% of samples taken from different sites (brain stem and spinal cord, cerebellum, and cerebrum) as ‘good’ fixation (occurrence of mild autolytic changes) or ‘excellent’ fixation (without autolytic changes). “Swiss cheese” artifacts, caused by the invasion of gas-forming anaerobic bacteria, were observed in restricted (central) parts of the brain in 22 of 38 brains, especially in the thalamus, brain stem, and cerebellar vermis. White matter vacuolation was also observed in some of the brains, again in the thalamus and cerebellar vermis. However, in every case, the vacuoles were few (1 to 5) and small (1 to 3 mm).
The authors conclude that “the subsequent histological examination showed that these brains were, in many ways, better preserved than the routine autopsy brains of human and veterinary medicine. We regard the time span from death to start of fixation as the most decisive or crucial factor for this successful result.” They indicate that, although ≈75% of the fixations began within 2 hours post mortem, there were some instances where fixation started later (up to 6 hours), and that variations in fixation quality are likely due to the occurrence of autolytic changes. Importantly, it was noted that even careful handling of fresh brains always results in compression damage, and that fixation of the brain in situ was an excellent remedy for this problem.
In situ whale brain diffusion fixation appears to produce good preservation of the structure of the whole brain, especially in cases where fixation is begun soon after death. If such results can be achieved by passive diffusion of vastly larger brains than the human brain, investigation of the feasibility of reproducable uniform chemical fixation of complete humans brains as a method of biopreservation is warranted.
In their paper “The role of leukocytes following cerebral ischemia: pathogenic variable or bystander reaction to emerging infarct?” D.F. Emerich et al. review the literature on the involvement of neutrophils in cerebral ischemia:
“We reasoned that if neutrophils play an important pathogenic (i.e., cause-effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia.”
The authors did not find much evidence for any of the above and speculate that neutrophil recruitment may not be a cause of injury, but rather a response to postischemic necrosis.
Knowledge of the causal and temporal aspects of cerebral ischemia is important to select the right agents to minimize brain injury of cryonics patients. Neuroprotective agents that confer benefits to cardiac arrest and stroke victims may not necessarily offer additional protection during stabilization of cryonics patients if the targets of these interventions are non-causal and/or delayed events in the ischemic cascade.
An ongoing quest in cryonics is the successful demonstration of memory sustainment after cryopreservation of the brain and rewarming from cryogenic temperatures. To that end, landmark experiments were performed by Pichugin, et al. (2006) on rat hippocampal brain slices which indicate that the hippocampus retains excellent structural integrity and viability (as measured by Na+/K+ ion pump recovery) after vitrification, rewarming, cryoprotectant removal, and exposure to 35°C for over an hour. To address the question of memory itself, investigations into the maintenance of long-term potentiation (LTP) after vitrification of the brain are currently in progress. But even successful observation of LTP after cryopreservation provides only indirect evidence for memory maintenance.
Alternatively, post-burst afterhyperpolarization (AHP) of hippocampal CA1 neurons may be characterized after cryopreservation of animals that have successfully acquired a hippocampus-dependent task. CA1 pyramidal neurons show decreased post-burst AHPs and less accommodation (i.e., increased firing frequency) following learning of such hippocampus-dependent tasks as trace eyeblink conditioning (Moyer et al., 1996, 2000; Thompson et al., 1996) and spatial watermaze training (Oh et al., 2003) with a time course appropriate to support memory consolidation. Furthermore, CA1 neurons of aging animals (i.e., animals at ages that exhibit learning deficits) show greater AHPs and more accommodation than those of young animals (Landfield & Pitler, 1984; Moyer et al., 1992, 2000), indicating an age-related decrease in neuronal excitability in the hippocampus that may underlie learning deficits related to aging.
A carefully designed experiment demonstrating reduced afterhyperpolarization and accommodation in hippocampal CA1 neurons after acquisition of a hippocampus-dependent task and subsequent cryopreservation of the brain would be a huge step in the direction of proving that memories can be cryopreserved.
