ISCHEMIA AND REPERFUSION INJURY IN CRYONICS

by Ben Best

Ischemia is the condition suffered by tissues & organs when deprived of blood flow -- mostly the effects of inadequate nutrient & oxygen. Reperfusion injury refers to the tissue damage inflicted when blood flow is restored after an ischemic period of more than about ten minutes. Cryonics patients frequently experience ischemic & reperfusion injury between the time when the heart stops and cryostorage begins.

In this article I attempt to evaluate the nature & extent of ischemic & reperfusion injury in cryonics. I also attempt to assess what can be done to minimize such damage. I focus my attention on ischemic/reperfusion injury to the brain. I rely on peer-reviewed journal articles for information. The single most comprehensive article I have found on ischemic and reperfusion injury is "Ischemic Cell Death in Brain Neurons" by Peter Lipton [PHYSIOLOGICAL REVIEWS 79(4):1431-1568 (1999)]. Most

unreferenced factual statements I make are based on Lipton's review.

Most of the metabolic energy of neurons is expended on maintaining ion gradients across the cell membrane. A sodium potassium (Na+/K+) pump keeps extracellular potassium low and extracellular sodium high compared to intracellular concentrations. This pump is driven by the energy stored in ATP molecules manufactured in the mitochondria.

Within two minutes without blood flow (due to heart stoppage or blood vessel occlusion) neurons lack the energy to power the sodium/potassium pump. Potassium ions rush out of the cell while sodium & chloride ions rush

inward as the cell membranes depolarize. Neurons attempt to produce ATP by anaerobic glycolysis, resulting in lactic acid. Accumulation of carbon dioxide results in carbonic acid (H2CO3), which further increases acidity. Within two minutes of ischemia, extracellular pH can drop from about 7.3 to about 6.7.

Another ATP-driven pump helps keep extracellular calcium ions (Ca2+) 10,000 times more concentrated than within the cytoplasm. Voltage-gated ion channels and ion-exchangers in the cell membrane also regulate ion concentrations.

Depolarization of presynaptic membranes results in release of the neurotransmitter glutamate. Postsynaptic membranes contain several types of glutamate receptors, notably NMDA & AMPA receptors, which allow calcium ion entry. Postsynaptic membranes contain two voltage-gated calcium channels (L-type & T-type) as well as a sodium calcium exchanger, but the NMDA channel is particularly adept at allowing large amounts of calcium ion to enter the cell.

High levels of intracellular calcium ion activate enzymes (known as calpains) that break down many cell proteins, particularly those in the cytoskeleton. Calcium ion also activates the enzyme phospholipase A2, which attacks cell membrane phospholipids causing the release of arachidonic acid. Activated platelets also release arachidonic acid.

Rupturing of cell membranes can result in leakage of enzymes, notably Lactate DeHydrogenase (LDH). Blood or tissue levels of LDH have often been used as an indicator of cell damage due to ischemic/reperfusion injury.

Restarting blood flow after more than about ten minutes of ischemia is typically more damaging than the ischemia itself because the ischemia sets the stage for oxygen to generate free radicals. Arachidonic acid conversion to eicosanoids can lead to production of the superoxide (O2-.) radical when oxygen is available. Lipid peroxidation chain reactions occur in the membranes of neurons as well as of astrocytes.

Free radical damage to blood vessels is particularly severe. Ischemia results in large amounts of ATP being broken-down to xanthine. Reperfusion allows the endothelial enzyme xanthine oxidase to convert xanthine plus oxygen to superoxide & uric acid. Reperfusion increases the amount of nitric oxide produced by the endothelial cells. The nitric oxide can combine with superoxide to produce peroxynitrite (ONOO-), which is nearly as damaging as the hydroxyl radical. Liberated iron & zinc ions further increase free radical damage.

Neutrophils which have accumulated in blood vessels due to the ischemia can release oxygen-rich free radicals with the availability of oxygen from reperfusion -- thereby attracting more neutrophils. The explosive endothelial cell swelling combined with accumulated leukocytes & blood clots plugging capillaries can result in a "no-reflow" phenomenon -- blocked blood flow.

There is a linear correlation between the amount of reperfusion injury and disruption of the blood-brain barrier. Water flow into the brain leads to edema.

Can drugs help prevent ischemic damage in cryonics patients? A study of the literature on stroke therapy is instructive.

One might think that drugs blocking calcium ion entry via NMDA receptors would be beneficial for stroke, but clinical trials with these substances have  been a failure. Although animal studies show NMDA-blockers to be effective for the first 4 minutes, after 8 minutes intracellular levels of calcium ion are  the same whether NMDA-blockers are used or not. L-channel blockers (like nimodipine) make no difference.

There are plausible reasons why NMDA-blockers -- even when combined with L-channel blockers -- are of limited usefulness in preventing calcium entry into ischemic cells. Low levels of ATP mean reduced capacity of the calcium-ATP pump to keep calcium out of the cell. High cytoplasmic sodium means high activity of the membrane s dium/calcium exchangers -- particularly those on mitochondrial membranes. Blockage of L-channels leaves T-channels unblocked. And phospholipase breakdown products help to release large amounts of calcium ion which has been bound to the

endoplasmic reticulum.

Animal studies have shown benefit from antioxidants such as Vitamin E

[BRAIN RESEARCH 510:335-338 (1990), melatonin & nifedipine [JOURNAL OF PINEAL RESEARCH 33:87-94 (2002)] and PBN [PNAS 92(11):5057-5061 (1995)] as well as from other neuroprotective agents. But all have thus far

failed to pass clinical trials and be accepted as therapeutic agents. Currently,

the only accepted drugs used for stroke therapy are thrombolytics, anticoagulants and antiplatelet drugs.

Tissue plasminogen activator (tPA) is useful for breaking-up blood clots, but only when given within 3 hours of the onset of stroke. When given within 90 minutes of stroke, tPA more than doubles the 3-month survival of stroke patients [NEUROLOGY 55(11):1649-1655 (2000)]. Prior to tPA, streptokinase & urokinase were the most efficacious thrombolytics. The anticoagulant heparin is given in the hospital and warfarin is used for long-term maintenance.

Aspirin may be used as an antiplatelet agent. These therapies cannot be used for hemorrhagic stroke because they worsen that condition. 

ORGAN TRANSPLANTATION SOLUTION

Some neuroprotective agents that have not passed clinical trials for stroke therapy have shown to be of demonstrable benefit in preservation of organs for transplant. Explanations for the benefits of the ingredients used in the organ preservation solution Viaspan can be found on the Viaspan website (www.viaspan.com).

Allopurinol inhibits xanthine oxidase, blocking the conversion of xanthine & oxygen to superoxide & uric acid. Glutathione is used as an antioxidant with membrane-stabilizing properties. Dexamethasone also stabilizes membranes. Magnesium seems to counteract some of the effects of intracelluar calcium and the sulfate ion resists cell swelling because it is relatively impermeable to cell membranes.

To counteract loss of ATP (Adenosine TriPhosphate), adenosine is added to provide more substrate for ATP synthesis. Monobasic potassium phosphate also supplies substrate for ATP synthesis while opposing acidification and potassium-leakage. Potassium hydroxide also maintains a high pH while opposing potassium-leak.

By using a cocktail of agents Mike Darwin and Dr. Steve Harris of Critical Care Research extended the period dogs can tolerate warm (room-temperature) ischemia to 17 minutes. A cocktail of such agents reportedly could never pass FDA approval for stroke therapy or cardiac arrest treatment, hence it did not receive widespread interest or application in conventional medicine. Dogs have a higher heart rate and metabolic rate than do humans. The ischemic tolerance for humans is estimated to be as high as 20 minutes [CRITICAL CARE MEDICINE 16(10):923-941 (1988)].

Under ideal circumstances, however, a cryonics patient experiences little room-temperature ischemia. If cardiopulmonary support and cooling are begun immediately ischemia can be minimized. Under non-ideal circumstances room-temperature ischemia is often considerably more than 17 minutes. It is commonly noted that metabolic rate is halved for every 10ºC drop in temperature. But reducing temperature has a protective effect on the brain which exceeds reduction of metabolism. Experiments on gerbils indicate that a drop in temperature from 37ºC to 31ºC nearly triples the amount of time that neurons can tolerate ischemia [CRITICAL CARE MEDICINE 31(1):255-260 (2003)]. Dogs cooled to 20oC can withstand 60 minutes of ischemia and can withstand 120 minutes of ischemia at 10ºC [CRITICAL CARE MEDICINE 31(5):1523-1531 (2003)].

But if a cryonics patient is given immediate cardiopulmonary support, ischemia can be greatly reduced, if not eliminated. Normal physiologic cerebral blood flow is about 50mL per 100 grams of brain tissue per minute. Good cardiopulmonary support can maintain cerebral blood flow not much higher than 15mL (and usually lower). This is critically close to the 10mL associated with the beginning of irreversible cell damage if such a flow rate is maintained for an extended period [JOURNAL OF NEUROSURGERY 77:169-184 (1992)]. But with effective cooling the flow provided even with

moderately-effective CPR may be adequate to maintain brain structure. Temperature drop is most rapid upon initial application of cooling and there is a natural drop in brain temperature associated with reduced blood flow. Under these circumstances the added benefit of anti-ischemic agents may not be great.

These facts should provide some comfort for those who feel they cannot afford to supplement the cooling and cardiopulmonary support of cryonics rescue with expensive anti-ischemic cocktails. Nonetheless, pretreatment of the patient with aspirin, vitamin E and other anti oxidants is an inexpensive means of reducing ischemia after the heart stops. Such pretreatment may give better antioxidant tissue levels than infusing them after deanimation. Epinephrine has commonly been used to maintain blood pressure and supplement CPR by maintaining blood pressure, although vasopressin may also be used [CRITICAL CARE MEDICINE 30(supplement 4):S157-S161 (2002)].

Pronouncement of death may occur soon after the heart stops. In a do-not resuscitate (DNR) situation rapid application of CPR could easily cause the legally dead person to regain consciousness. It is also seems possible that the heart could restart, but if ischemia has elevated extracellular & plasma potassium levels, the heart is unlikely to restart. The heart rarely restarts without an electonic defibrillation except in young children.

Regaining of consciousness by a cryonics patient would provide reassurance of the effectiveness of the cardiopulmonary support, but it would be traumatic for all concerned -- and a "political" disaster. Barbiturates would be an effective means of maintaining unconsciousness, but as a narcotic its use can be both a political & legal hazard. Fortunately, propofal is not a controlled substance and can keep the patient unconscious. If a funeral director, medical professional or other person can administer heparin, he or she should also be able to administer epinephrine, propofol, a thrombolytic, antioxidants and other agents to combat acidosis.

Nanotechnology may be able to repair freezing damage, but for a brain that has experienced many days of warm ischemia the prospects are not so good. Unlike freezing damage, warm ischemia eventually leads to dissolution of brain tissue into a structureless soup.

On the other hand, claims that a few hours of warm ischemia means

certain loss of personal identity cannot be supported. Even after two hours of warm ischemia (without reperfusion) lysosomal membranes in cat brain cells remain intact [VIRCHOWS ARCHIV B 25:207-220 (1977)]. Monkey brains subjected to an hour of warm ischemia and protected from reperfusion injury show short-term recovery [JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 6(1):15-33 (1986)]. Post-mortem mouse brains subjected to 6 hours of room temperature and another 18 hours at 4oC show half the neurons to be morphologically intact [VIRCHOWS ARCHIV B 63:331-334 (1993)].

The CA1 pyramidal neurons of the hippocampus are often regarded to be the most sensitive to ischemic injury of all neurons. After 30 minutes of ischemia followed by reperfusion, the CA1 neurons invariably die after 2 or 3 days whereas the reputedly resistant striatal neurons begin to die after several hours [ANNALS OF NEUROLOGY 11:491-498 (1982)]. In either case, a cryonics patient should be in a low-temperature condition well before that time.

Cell death by apoptosis ("cell suicide") is a controlled process requiring DNA transcription, new protein synthesis and usually many hours. Apoptosis is probably no ultimate hazard for cryonics patients who deanimate without pre-mortem ischemic damage and who receive prompt cardiopulmonary support & cooldown. Just as future technology may reverse "death" in whole persons, future technology should also be able to reverse much of what passes for irreversible death of cells. Certainly we should expect reversibility from the early stages of apoptosis. Cell death by necrosis is another matter.