January 30 , 2007
A shorter version of this article appeared in Cryonics, Spring 2006.
by Aschwin de Wolf
The goal of human cryopreservation standby and stabilization procedures is to preserve the structure and viability of the brain after medico-legal pronouncement of death. To achieve this goal we employ three different techniques: cardiopulmonary support (CPS), rapid induction of cooling (hypothermia), and pharmacological intervention.
The primary purpose of the medication protocol is to reduce or eliminate injury from cerebral ischemia. Ischemia is interruption of the delivery of adequate amounts of both oxygen and nutrients to the brain. The better we protect the brain from ischemic injury, the better the patient’s chances of future revival. This brief introduction will familiarize the reader with the different classes of medications we use, and some of the issues associated with administering them.
Although virtually all the medications ultimately are given to help mitigate ischemic injury, many of them are pharmaceuticals with other uses in mainstream medicine. Therefore, EMT’s, paramedics and nurses may be familiar with many of them. The most important differences are the number used, the different context and sometimes rationale of use, and different dosages than are common in orthodox medical practice.
Although the human brain accounts for only 2% of total body mass it accounts for about 20-25% of total oxygen consumption. Therefore, the first priority is to reduce cerebral oxygen consumption to make the brain more tolerant to the limited blood flow CPS produces. This can be achieved by inducing deep anesthesia. Because we prefer to use medications that are not scheduled drugs and which also confer anti-ischemic benefits, the current anesthetic of choice is propofol (Diprivan). Naturally, this medication should be given just before, or immediately after, starting CPS. The choice of diprivan is a typical example of the sort of trade off that sometimes needs to be made in human cryopreservation. Propofol produces transient hypotension which is undesirable in the context of trying to restore optimal cerebral blood flow.
Anticoagulants, Antiplatelets and Fibrinolytics
The formation of blood clots during human cryopreservation cases is problematic for a variety of reasons. It may frustrate our attempt to provide adequate CPS, cause serious problems during blood washout, or complicate perfusion with a vitrification solution (in cases without blood washout). Because anticoagulant and antiplatelet agents like heparin and aspirin only prevent blood clotting, a fibrinolytic, streptokinase, is given to dissolve existing blood clots.
In human cryopreservation vasopressors (pressors) are used to increase blood pressure and selectively shift blood flow to the vital organs (including the brain). The current vasopressors of choice are epinephrine and vasopressin. Because avoiding some of the side effects of these medications is not as high a priority in human cryopreservation as in conventional medicine, protocol and dosages may differ somewhat from current practice in cardiopulmonary resuscitation. It is hard to overestimate the importance of restoring adequate cerebral blood flow in the human cryopreservation patient. It is also important to note that epinephrine needs to be given intermittently at short intervals, or continuously infused, instead of administering just one single large bolus . This is done because epinephrine has a short half-life and is rapidly metabolized. Ideally, cardiac output and oxygenation of the brain are measured to validate stabilization procedures in general, and to make informed decisions about the use of vasoactive medications, in particular.
In the ideal case, circulation and ventilation are restored immediately after pronouncement of medico-legal death, in conjunction with the administration of medications and induction of (surface) cooling. Although this protocol is fairly aggressive compared to that which is usually employed by paramedics in out-of-the-hospital resuscitation from cardiac arrest, it is usually inadequate to meet the metabolic demands of the patient. This is especially true if the patient has already experienced some ischemic injury prior to pronouncement and/or the standby team is not able to start the stabilization protocol immediately, or if the patient is febrile at the time of pronouncement.
No (or inadequate) blood flow fails to provide (enough) energy to maintain ion gradients across cell membranes, leading to depolarization. The depolarization of presynaptic membranes overactivates the neurotransmitter glutamate, causing increased calcium ion (Ca++) influx. In the absence of adequate energy production, excessive Ca++ leads to a cascade of damaging events including pathological activation of various enzymes, inflammatory mediators, generation of harmful free radicals and apoptosis (programmed cell death), producing an explosive positive feedback-loop in which one event amplifies and accelerates others .
The agents that are used in human cryopreservation to mitigate ischemia and reperfusion injury (the largely free radical induced damage caused by restoring circulation and ventilation) include a variety of antioxidants, excitotoxicity-inhibitors and inducible nitric oxide synthase-inhibitors to target different parts of the damaging cascade of events resulting from inadequate blood flow.
Administration of kynurenine increases production of kynurenic acid, an endogenous antagonist of excitatory amino acid induced excitotoxicity, one of the upstream events in cerebral ischemia. S-methylthiourea (SMT), an inducible nitric oxide synthase inhibitor, is primarily used to mitigate inducible nitric oxide and associated formation of the peroxynitrite radical. SMT also increases mean arterial pressure.
Ischemia-reperfusion induced free radical generation is further mitigated by a proprietary antioxidant cocktail called Vital-Oxy. Vital-Oxy contains antioxidants like D-alpha tocopherol (Vitamin E), melatonin and the free radical spin trapping agent alpha Phenyl t-Butyl Nitrone (PBN). Vital-Oxy also includes the anti-inflammatory drug carprofen. Another antioxidant administered in cryonics is the low molecular weight superoxide scavenger 4-Hydroxy-Tempo (TEMPOL).
This multi-modal approach in treating cerebral ischemia has been developed and proven to be effective in recovering dogs after 17 minutes of normothermic cardiac arrest at Critical Care Research, a California-based resuscitation research company.
A relatively recent addition to this multi-modal treatment of ischemia is a class of agents called PARP-inhibitors. The overactivation of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) during ischemia leads to a rapid depletion of the major energy sources of the cell. Studies of PARP-inhibitors in animal models and “knock-out” mice (mice with inactivated PARP genes) indicate the potential of PARP inhibition in mitigating cerebral ischemia. One advantage of PARP inhibitors is that PARP activation is a final common pathway in many of the events in the ischemic cascade, potentially offering a greater degree of protection and providing a longer window of opportunity in mitigating cerebral ischemia. The current PARP inhibitor of choice in cryonics is niacinamide, better known as vitamin B3.
Although human blood normally has a pH of 7.4, which is kept in a very tight range in a healthy human being, after a (prolonged) period of ischemia, and/or inadequate circulation and ventilation, the typical patient becomes acidotic. This is a serious concern because this condition damages cells, accelerates blood clotting, induces clumping of red blood cells (agglutination) and exacerbates cold agglutination. Acidosis also renders epinephrine and heparin inactive because, as in the case of epinephrine, the drug is effective only within a certain pH range, or in the case of heparin, acidosis degrades the drug and inactivates it. To prevent and treat acidosis a buffer is given. The current buffer of choice in human cryopreservation is tromethamine (THAM) because it does not have some of the side effects (like cell swelling) of sodium bicarbonate. In the ideal human cryopreservation case, pH is meticulously monitored and additional buffer is administered if acidosis is observed.
Volume Expanders and Oncotic Agents
As indicated above, intravenous access is not only necessary to administer medications but also to administer fluids to address electrolyte imbalances and replace volume (in the dehydrated patient). A fluid like dextran-40 is not only a volume expander but also improves microcirculation and somewhat inhibits hypothermia induced cold agglutination. Another medication employed in fluid resuscitation is mannitol. Mannitol has been proven effective in ischemia induced cerebral edema by promoting movement of fluid from the cells to the vascular space. Other advantages of mannitol are reduction of blood viscosity (improving perfusion) and its free radical scavenging properties. Both of these fluids are given in fairly large volumes (compared to most of the medications), so a basic understanding of fluid balance and electrolytes is desirable to make informed decisions for the patient.
Microbial overgrowth can be an issue during long (normothermic) transport times. The aminoglycoside gentamicin kills (aerobic) bacteria by irreversibly binding to bacterial ribosome, causing the production of faulty proteins. Administration of antibiotics and the use of sterile technique have sometimes been perceived as redundant and expensive for treating cryonics patients. One answer to this objection is that the guiding philosophy of stabilization is to provide a level of care and commitment at least equal to, or better than, the care the patient received before pronouncement of legal death. It’s also important to note that antibiotics and sterile technique are not only used to treat the patient, but also to protect the stabilization team members from infection.
The fairly large number and volume of the medications raise the obvious question of what the preferred sequence should be. The most important consideration is that the sequence should reflect medical priorities. For example, propofol is administered as the first medication to reduce cerebral metabolic demand. The second consideration is to give the small volume medications first, and the larger volume medications later, so that most of the medications can be given in the shortest period of time. Naturally, there can be a conflict between the two. When not desirable to delay the administration of a drug, a small portion of the total volume can be given rapidly and the rest can be administered (as a drip) later.
Although the medications currently used in human cryopreservation reflect years of experience and research, it needs to be stressed that this does not completely release the cryopreservation technician from using medical common sense. For example, considering the total volume of medications in our protocol, a normally hydrated infant may have different fluid (and medical) needs than a severely dehydrated large adult. A patient may have already been heavily pre-medicated with some of the medications in our protocol (aspirin for example). If prompt cardiopulmonary support is not possible, it may be questionable to administer medications to mitigate early ischemic injury.
These kinds of issues stress the importance of comprehensive data collection, detailed reporting and systematic analysis. The more we learn about the different effects of our protocol in different situations, the better we may be able to refine it to suit a particular patient’s needs. In this respect human cryopreservation is not unlike conventional medicine; one size doesn’t fit all.
1. Jakob Johansson (2004). Cardiopulmonary Resuscitation: Pharmacological Interventions for Augmentation of Cerebral Blood Flow, Dissertation, Uppsala University Sweden
2. Paul Morley , Joseph S. Tauskela, and Antoine M. Hakim (1999). “Calcium Overload” in Cerebral Ischemia: Molecular and Cellular Pathophysiology (ed. Wolfgang Walz), pp. 69-105