(For a more quantitative approach less concerned with molecular biology, see Quantifying Ischemic Damage for Cryonics Rescue.)

1. INTRODUCTORY REMARKS
2. ISCHEMIC INJURY IN BRAIN AND ELSEWHERE
3. REPERFUSION INJURY AND "NO REFLOW"
4. STROKE THERAPY
5. HIBERNATION AND ESTIVATION
6. ORGAN TRANSPLANTATION SOLUTION
7. BRAIN DAMAGE DUE TO ISCHEMIA/REPERFUSION
8. PREVENTING ISCHEMIC/REPERFUSION INJURY IN CRYONICS
9. PRE-TREATMENT FOR CRYONICS PATIENTS
10. CONCLUSIONS

I. INTRODUCTORY REMARKS

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. Ischemia and reperfusion can cause serious brain damage in stroke or cardiac arrest. 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 -- primarily focused on the impact for cryonics (although certainly relevant to stroke and cardiac arrest). 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.

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PhosphoCreatine replenishes ATP

II. ISCHEMIC INJURY IN BRAIN AND ELSEWHERE

Depolarization of presynaptic membranes results in release of the neurotransmitter glutamate (glutamic acid). 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. Excessive glutamate release resulting in excessive Ca⁺² entry into cells is the excitotoxicity which initiates the brain ischemic damage seen in stroke and cardiac arrest.

In times of high metabolic demand and adequate availability of oxygen, elevated calcium in mitochondria can increase ATP production by stimulation of three enzymes in the Krebs citric acid cycle: pyruvate dehydrogenase, alpha-ketoglutarate and isocitrate dehydrogenase. But when oxygen is not available in adequate amounts to accept electrons (hydrogen atoms) from NADH, the excess electrons form superoxide from the residual oxygen. Countering NADH production, calcium action on the mitochondrial permeability transition pores increases inner membrane permeability thereby reducing proton potential, causing the matrix to swell and ultimately releasing cytochrome c (an initiator of apoptosis).

High levels of intracellular calcium ion activate proteolytic enzymes (known as calpains) that break down many cell proteins, particularly those in the cytoskeleton of neurons (spectrin, neurofilament and microtubule-associated protein). The fact that Alzheimer's Disease patients have triple the normal levels of calpain in their prefrontal cortex could indicate a role of ischemia as a cause of the disease [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); 90(7):2628-2632 (1993)]. Calcium-activated nuclear endonucleases can cleave chromatin and begin the process of apoptosis ("cell suicide").

Calcium ions also activate phospholipase enzymes which attack cell membrane phospholipids causing the release of arachidonic acid. Inhibitors of the enzymes lipoxygenase & cyclo-oxygenase (which break down arachidonic acid into eicosanoids such as prostaglandin) can reduce cerebral deficits caused by ischemia [CRITICAL REVIEWS OF NEUROBIOLOGY 15(1):61-90 (2003)]. (For more information about phospholipase, eicosanoids, etc., see Essential Fatty Acids in Cell Membranes.)

Most ischemic brain damage is to the lipid portion of cell membranes through lipid peroxidation and phospholipase activity. Cerebral ischemia results in rapid release of fatty acids (especially arachidonic acid) due to phospholipase enzymes. Calcium-dependent cytoplasmic PhospoLipase A₂ (cPLA₂) is activated by Ca⁺² entry into cells after a few minutes of ischemia. cPLA₂ preferentially releases oxidized arachidonic acid (which is present in large quantities in neural membranes). Lipoxgenase enzymes form lipid hydroperoxides (ROOH) which can lead to lipid peroxidation by Fenton-like reactions [BIOLOGICAL CHEMISTRY 383:365-374 (2002)]. Arachidonic acid itself has an uncoupling effect on mitochondria in addition to its direct inhibition of mitochondrial respiratory enzymes and promotion of free-radical formation [FREE RADICAL BIOLOGY AND MEDICINE 27(1-2):51-59 (1999)].

Low cell energy and damaged membranes reduce glutamate uptake worsening excitotoxicity. Soon neuron membrane damage is so great that the major mechanism of glutamate release is direct leakage through cell membranes [BRAIN RESEARCH BULLETIN 34(5):457-466 (1994)]. The large (molecular weight 140,000) enzyme Lactate DeHydrogenase (LDH) is soon seen leaking through ischemia-damaged membranes. Blood or tissue levels of LDH have often been used as an indicator of cell damage due to ischemic/reperfusion injury. LDH is very suitable as an assay for cell lysis because it exists in relatively high concentration in all cells, and is stable.

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III. REPERFUSION INJURY AND "NO REFLOW"

Prompt restarting of circulation following ischemia can prevent tissue damage. 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 rather than to contribute to cellular energy production [CARDIOVASCULAR RESEARCH; Zweier,JL; 70(2):181-190 (2006)]. In addition to oxygen-generated free radicals, cytokines can be a significant source of reperfusion injury [EMEDICINE; Elzawahry,H; (June 24,2009)]. Two to six hours of ischemia followed by 24 hours of reperfusion more than triples infarct volume [JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM; Aronowski,J; 17(10):1048-1056 (1997)]. A historical review of oxygen injury due to delayed reperfusion following ischemia can be found in section one of [CARDIOVASCULAR RESEARCH; Zweier,JL; 70(2):181-190 (2006)].

The acidity produced by ischemia greatly reduces the release of arachidonic acid from cell membranes by phospholipases, so phospholipase activity isoeicosanoid products of arachidonic acid oxidation show distinctly high urine levels following reperfusion of myocardial infarction patients [JOURNAL OF BIOLOGICAL CHEMISTRY; CIRCULATION;96(10):3314-3320 (1997)]. (For more information about isoeicosanoids, see Essential Fatty Acids in Cell Membranes.)

During the ischemic period there is an accumulation of lactic acid which lowers cellular pH. Cells use Na⁺/H⁺ exchange to eliminate excess protons, but in the process accumulate excess Na⁺ which cannot be exported with the sodium pump (Na-K-ATPase) due to ATP deficiency. As a consequence cells use the Na⁺/Ca⁺² exchange, which loads cells with Ca⁺². Upon reperfusion Ca⁺² enters the mitochondria, but the Mitochondrial Permeability Transition Pore (MPTP) remains closed because the acidity maintains MPTP closure. Elevation of pH with reperfusion can open the MPTP [BIOCHEMICAL SOCIETY TRANSACTION; Halestrap,AP; 34(Pt 2):232-237 (2006)]. If the MPTP can close or if ATP can otherwise be generated cells will die by apoptosis. Without sufficient ATP, MPTP opening results in necrosis [BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS; Kim,J; 304(3):463-470 (2003)].

NAD(P)H oxidase in reperfusion reacts with newly introduced oxygen to produce superoxide [STROKE; Kahles,T; 38(11):3000-3006 (2007)]. Superoxide reacts with iron-sulfur proteins, decreasing their activity and liberating free iron -- which causes hydroxyl radical formation. Nitric oxide in mitochondria reacts with superoxide three times faster than SuperOxide Dismutase (SOD). Superoxide reacts with nitric oxide more efficiently than with any other molecule, rapidly consuming the nitric oxide to form the potent free radical peroxynitrite [JOURNAL OF APPLIED PHYSIOLOGY; Faraci,FM; 100(2):739-743 (2006)]. Peroxynitrite irreversibly inactivates not only SOD, but complexes I and II of the mitochondrial respiratory chain.

In reperfusion there is considerable membrane damage to endothelial cells as well as platelets, leucocytes and other cells in the blood stream. Activated neutrophils produce superoxide, which can be dismutated into hydrogen peroxide. Neutrophil myeloperoxidase enzyme converts hydrogen peroxide to hypochlorous acid. Hypochlorous acid reacting with superoxide can produce hydroxyl radicals. Red blood cell aggregation near the exit of capillaries pushes leukocytes against endothelial cells, thereby increasing leukocyte adhesion [AMERICAN JOURNAL OF PHYSIOLOGY; Pearson,MJ; 279(4):H1460-H1471 (2000)]. Leukocyte adhesion (and reperfusion damage) is higher in older animals [MICROCIRCULATION; Ritter,L; 15(4):297-310 (2008)].

Eicosanoids generated by arachidonic acid (especially leukotrienes) greatly increase the adhesion of leukocytes & platelets to capillary walls — plugging them up. Superoxide also increases the adhesion of leucocytes to vessel walls. Leukocyte adhesion is also potentiated by InterCellular Adhesion Molecule 1 (ICAM−1) protein released from endothelial cell and leucocyte membranes by cytokines during reperfusion (an effect attenuated by hypothermia) [STROKE; Ishikawa,M; 30(8):1679-1686 (1999)]. Eicosanoids (leukotrienes & prostaglandins) and associated oxygen free-radicals make capillary walls more "leaky", causing edema which narrows the channels. ATP depletion significantly reduces the ability of erythrocytes to deform [THE JOURNAL OF CLINICAL INVESTIGATION; Weed,RI; 48(5):795-809 (1969)]. These effects quickly become pronounced enough in reperfusion to block capillaries entirely — the no-reflow phenomenon. Experimental middle cerebral artery occlusion has shown blood flow reduction to 71% of control after a one hour occlusion and reduction to 22% of control after a four hour occlusion [BRAIN RESEARCH; Dawson,DA; 749:200-208 (1997)]. The cerebral cortex, the part of the brain in which consciousness is presumed to reside, is fortunately less vulnerable to no-reflow than other areas of the brain. More than 50% of blood vessels have been shown to be occluded in the thalamus and basal ganglia after 30 minutes of ischemia, but less than 15% of vessels in the cerebral cortex are occluded [STROKE; Fischer,EG; 3(5):538-542 (1972)].

But no-reflow can occur even without blood cells. Free-radical and other membrane damage can loosen or dislodge atherosclerotic plaque causing emboli upon reperfusion.

"NO REFLOW" MECHANISMS

Nitric oxide normally functions to not only reduce platelet aggregation & leukocyte adhesion to the endothelium, but to promote vascular smooth muscle relaxation and reduce endothelial cell cytokine production. Nitric oxide concentrates in lipophilic cellular regions with a partition coefficient of 8:1, and can inhibit lipid peroxidation a thousand times more potently than alpha-tocopherol [JOURNAL OF BIOLOGICAL CHEMISTRY; Rubbo,H; 269(42):26066-26075 (1994)]. Nitric oxide potentiates transcription of phase 2 detoxification enzymes (including antioxidant enzymes) [JOURNAL OF BIOLOGICAL CHEMISTRY;Dhakshinamoorthy,S; 279(19):20096-20107 (2004)]. Nitric oxide inhibits the expression of pro-inflammatory genes by transcription factor NF-kappaB [TRANSPLANTATION PROCEEDINGS 30:4239-4243 (1998)]. NF-kappaB activates the cytokine TNF−α to increase expression of cell adhesion molecules. Nitric oxide inhibits apoptosis by inhibition of caspase-3 enzyme [JOURNAL OF BIOLOGICAL CHEMISTRY; Rossig,L; 274(11):6823-6826 (1999)]. But these beneficial actions of nitric oxide are seen in the absence of ischemia/reperfusion — which converts nitric oxide into a toxin.

Elevated blood levels of the pro-inflammatory cytokine TNF−α induces apoptosis [BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS; Bajaj,G; 345(4):1558-1564 (2006)]. In inflammatory conditions, such as occurs in reperfusion, inducible nitric oxide synthetase can increase nitric oxide concentration to thousands of times normal levels [FREE RADICAL BIOLOGY & MEDICINE; Brown,GC; 33(11):1440-1450 (2002)]. During reperfusion, abnormally high amounts of superoxide converts almost all available nitric oxide to perxoynitrite — regarded as the agent causing most of the damage to brain capillary endothelial cells [NEUROSURGERY 43(3):577-584 (1998)]. In one study, inhibition of reactive peroxynitrite resulting from reperfusion after 30 minutes of warm ischemia doubled recovery of contractile function [JOURNAL OF BIOLOGICAL CHEMISTRY; Wang,P; 271(46):29223-29230 (1996)]. Damage to the endothelium not only increases edema (tissue swelling due to "leakiness"), but causes endothelial protrusions ("blebs") which can block capillaries.

Ischemia in tissues and blood vessels 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. Liberated iron & zinc ions further increase free radical damage. In contrast to the vasculature, mitochondria in tissues rather than xanthine oxidase are the primary source of oxygen free radicals during reperfusion injury [JOURNAL OF CLINICAL INVESTIGATION; 91(2):456-464 (1993)]. But xanthine oxidase-produced superoxide (and resulting peroxynitrite) damage to endothelial cells may be the primary mode of reperfusion damage, with far less damage to parenchymal cells, and far less injury due to neutrophils [SURGERY; Ratych,RE; 102(2):122-131 (1987)].

There is a linear correlation between the amount of reperfusion injury and disruption of the blood-brain barrier (BBB). Water flow into the brain due to BBB disruption can lead to edema. Further BBB damage can transform an ischemic stroke into a hemorrhagic stroke. Proteases (enzymes that degrade proteins) are released in ischemia [STROKE; Fukuda,S; 35(4):998-1004 (2004)]. Matrix MetalloProteinase−13 (MMP−13, a collagenase) originating from an unknown source early in ischemia exerts a corrosive effect on the blood-brain barrier [STROKE; Rosell,A; 36(7):1415-1420 (2005)], but unlike other MMPs does not continue to increase in quantity with time [STROKE; Horstmann,S; 34(9):2165-2170 (2003)]. Leukocytes (neutrophils, probably) activated by ischemic inflammation release increasing amounts of MMP−9 (gelatinase−B) which also degrades the blood-brain barrier [AMERICAN JOURNAL OF PHYSIOLOGY; Gidday,JM; 289(2):H558-H568 (2005)]. Prior to activation of ischemic inflammatory processes, however, reperfusion can activate gelatinase A (MMP−2), which increases capillary permeability and hemorrhage, in addition to opening the blood-brain barrier [STROKE; Rosenberg,GA; 29(10):2189-2195 (1998)].

(For more on "No-reflow", see Reducing "No-reflow". For more on ischemia/reperfusion damage to the blood-brain barrier leading to edema, see Edema in Cryonics.)

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IV. STROKE THERAPY

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)], resveratrol [BRAIN RESEARCH 958:439-447 (2002)], deprenyl [JOURNAL OF NEURAL TRANSMISSION 107:779-786 (2000)], and PBN [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); 92(11):5057-5061 (1995)]. Local anaesthetics have the potential to reduce ischemic damage to brain tissue by blocking sodium (Na⁺) channels — reducing electrical activity & metabolic rate beyond what can be achieved with barbiturates [PHARMACOLOGICAL REVIEWS 48(1):21-67 (1996)]. But all these agents have 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.

Degradation of the fibrin in blood clots by the protease (protein-digesting enzyme) plasmin requires conversion of plasminogen to plasmin by tissue plasminogen activator (tPA). Administering 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 can more than double the 3-month survival of stroke patients [NEUROLOGY 55(11):1649-1655 (2000)]. Because of the risk of reperfusion injury or hemorrhage, thrombolytics are also avoided on patients with severe hypertension, of advanced age or with evidence of cerebral edema. Mannitol has been used to reduce cerebral edema, but not in stroke [PROGRESS IN CARDIOVASCULAR DISEASES 42(3):209-216 (1999)].

Because the plasmin produced by tPA is a non-specific protease it not only dissolves clots, it contributes to vascular degradation and opening of the blood-brain barrier by Matrix MetalloProteinases (MMPs) [STROKE; Pfefferkorn,T; 34(8):2025-2030 (2003)], and can thereby worsen damage from reperfusion injury if given in delayed reperfusion. Treatment with tPA is generally deemed to do more harm than good if given more than 3 hours after a stroke [STROKE; Clark,WM; 31(4):811-816 (2000)] and at any time if the stroke affects a large area of the brain [JOURNAL OF NEUROLOGY, NEUROSURGERY, AND PSYCHIATRY; 65(1):1-9 (1998)]. Tetracyclines, particularly minocycline, have been shown to not only reduce ischemia-associated inflammation [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Yrjanheikki,J; 96(23):13496-13500 (1999)], but to inhibit MMPs [BMC NEUROSCIENCE; Machado,LS; 7:56 (2006)]. Inhibition of the inflammatory cytokine IL−1β can significantly reduce stroke infarct volume [JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM; 65(1):1-9 (1998)].

In many cases strokes can resolve spontaneously within a matter of days, but the cause of this "recanalization" is uncertain [STROKE; Molina,CA; 32(5):1079-1084 (2001) and ARCHIVES OF NEUROLOGY; Kassem-Moussa,H; 59(12):1870-1873 (2002)].

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. It is common practice for low molecular weight heparins to be given in hospitals as prophylaxis against deep vein thrombosis, as for chronically bedridden cancer patients [JOURNAL OF ONCOLOGY PHARMACY PRACTICE; Nishioka,J; 13(2):85-97 (2007)]. Aspirin may be used as an antiplatelet agent. These therapies cannot be used for hemorrhagic stroke because they worsen that condition.

For cryonics purposes streptokinase is the thrombolytic of choice because a dose of tPA costs thousands of dollars, whereas streptokinase costs a few hundred dollars. Steptokinase can be ordered from Sigma-Aldrich (CAS Number 9002-01-1) or other suppliers of medicine.

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V. HIBERNATION AND ESTIVATION

Animals that hibernate or estivate are able to avoid or significantly reduce ischemic damage by reducing their metabolism. The protective mechanisms used by estivators & hibernators can provide insight into the nature of ischemic damage and possibly into means to prevent such damage. Estivation is a state of aerobic hypometabolism that protects animals from dry (often hot) conditions. Alterations in metabolism associated with estivation include water retention, greatly reduced protein synthesis, reduced ion pumping, urea accumulation, and reliance on lipid oxidation (rather than glycolysis) for energy — associated with greatly reduced cytochrome c oxidase activity in mitochondria [COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY PART A; Storey,KB; 133:733-754 (2002)]. Cardiolipin is a phospholipid that is synthesized exclusively in the mitochondria and is required for maximal electron transport activity. Cardiolipin content of mitochondria from estivating snails is reduced 80%, associated with a similar reduction of cytochrome c oxidase activity [AMERICAN JOURNAL OF PHYSIOLOGY; Stuart,JA; 275(6Pt2):R1977-R1982 (1998)]. Toxic ammonia accumulation is prevented by increased urea synthesis, despite the fact that this requires energy [JOURNAL OF EXPERIMENTAL BIOLOGY; Chew,SF; 207:777-786 (2004)].

In hibernating arctic squirrels the leucocyte count drops up to 100-fold, which protects against the "no-reflow" leukocyte adhesion phenomenon associated with disrupted or greatly reduced blood flow [FREE RADICAL BIOLOGY & MEDICINE; Drew,KL; 31(5):563-573 (2001)]. Heart rate may be reduced 100-fold, metabolic rate reduced to less than 5% of normal and body temperature can drop to near 0ºC for small mammalian hibernators. (In non-hibernating mammals temperatures of 10ºC to 20ºC will stop the heart.) Passive efflux of K⁺ and passive influx of Na⁺ is reduced. Ca²⁺ sequestering is enhanced. Reliance on lipid hydrolysis as the primary source of energy results in ketones bodies which may protect the brain against hypoxia damage [PHYSIOLOGICAL REVIEWS; Carey,HV; 83:1153-1181 (2003)]. Changes in expression of the transcription factor protein Hypoxia Inducible Factor (HIF-1) may induce expression of hibernation-regulatory genes [BIOCHEMICA BIOPHYSICA ACT; Morin,P; 1729(1):32-40 (2005)].

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VI. ORGAN TRANSPLANTATION SOLUTION

Cold ischemia, such as is experienced by some hibernators and by transplantable organs being preserved at low temperatures, has unique characteristics distinguishing it from warm ischemia. Unlike cold ischemia, warm ischemia inhibits nitric oxide synthase and results in production of eicosanoid vasoconstrictors during reperfusion [TRANSPLANTATION PROCEEDINGS; Hansen,TN; 32:15-18 (2000)]. Although cold temperature can reduce ischemia, it can introduce new forms of damage, such as chilling injury. Unlike warm ischemia, cold ischemia is also to associated with an increase in chelatable iron which opens the Mitochondrial Permeability Transition Pore (MPTP), usually leading to apoptosis or (more often) necrosis. This phenomenon has been demonstrated in the absence of increased superoxide or hydrogen peroxide for liver endothelial cells, particularly, but also for other tissues [JOURNAL OF HEPATOLOGY; Rauen,U; 40(4):607-615 (2004)].

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® (developed as UW Solution — University of Wisconsin) can be found on the Viaspan® website or in [TRANSPLANTATION; Belzer,FO; 45(4):673-676 (1988)].

Allopurinol inhibits xanthine oxidase, blocking the conversion of xanthine & oxygen to superoxide & uric acid. Glutathione is used as an antioxidant with membrane-stabilizing properties. Hypothermia may actually increase permeability of cells to glutathione [CRYOBIOLOGY; Vreugdenhil,PK; 28:143-149 (1991)]. Dexamethasone can also stabilize membranes, but its actual benefit in Viaspan is dubious. 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.

Adenosine	ATP, ADP and AMP

ATP (Adenosine TriPhosphate) rapidly degrades to adenosine, inosine and hypoxanthine, all of which easily cross cell membranes and can be lost by diffusion. To counteract loss of ATP, adenosine (adenine connected to ribose) is added to provide more substrate for ATP synthesis. Adenosine also reduces adherence of neutrophils to endothelium as well as inhibiting neutrophil production of reactive oxygen species [AMERICAN JOURNAL OF PHYSIOLOGY 257(2 Pt 2):H1334-H1339 (1989)]. Monobasic potassium phosphate also supplies substrate for ATP synthesis while opposing acidification (from anaerobic glycolysis & lactic acid production) and potassium-leakage. Potassium hydroxide also maintains a high pH while opposing potassium-leak.

HydroxyEthyl Starch (HES) is added to UW Solution for oncotic support, ie, to prevent edema in the interstitial space by keeping more fluid in the blood vessels (a role normally played by blood albumin). HES reduces leucocyte adhesion to blood vessels during reperfusion [STROKE; Kaplan,SS; 31(9):2218-2223 (2000)]. Although HES is of most value for perfusion, it has been shown to be of benefit for improved cold storage of organs [TRANSPLANTATION; Southard,JH; 49(2):251-257 (1990)]. Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used as a replacement for HES with good results [THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY; Franco-Gou,R; 22(7):1120-1126 (2007)].

Dextran−40 (molecular weight 40 kilodaltons) inhibits cell clumping and can replace HES as a less viscous oncotic agent which is readily excreted by the kidneys.

HEPES is a zwitterion buffer which is large enough (238 daltons) to provide extracellular osmotic support. The ionization constant of water decreases (pK_w increases) as temperature decreases, which means that the pH will rise with temperature decline. The pK of phosphate and bicarbonate buffers do not change much with temperature, but the pK of HEPES buffer rises with falling temperature, thereby compensating for the rising pK of water. Thus HEPES is a better buffer than phosphate or bicarbonate for maintaining protein (enzyme) structure and function in hypothermia [CRYOBIOLOGY; Baicu,SC; 45(1):33-48 (2002)].

Lactobionate and raffinose are large molecules added for osmotic support and to prevent cell swelling which would result from reduced sodium pump activity. Lactobionate is a strong chelator of calcium and iron ions. Calcium can worsten ischemic damage, but a calcium-free solution will increase membrane permeability to calcium, thereby worsening the effects of subsequent calcium exposure (the "calcium paradox"). Only very small amounts of calcium are necessary to prevent the calcium paradox [CIRCULATION; Marban,E; 80(6 Suppl):IV17-22 (1989)].

The Penicillin in UW Solution can prevent bacterial growth. Insulin can increase glucose uptake by cells, but glucose is omitted from UW Solution in order to reduce cellular acidosis (lactic acid production by glycolysis).

Viaspan® (UW solution) has been reported to be contaminated with iron and to lose glutathione prior to use [TRANSPLANTATION; Salahadeen,AK; 70(10):1424-1431 (2000)]. Viaspan does not reduce the extreme loss of mitochondrial and cellular calcium by unknown causes associated with hypothermia [TRANSPLANTATION; Kim,J; 65(3):369-375 (1998)].

A number of new additives have been proposed for organ transplantation solutions to prevent cold ischemic injury. Dopamine, for example, reduces cold-ischemic oxidation [AMERICAN JOURNAL OF TRANSPLANTATION; Yard,B; 4:22-30 (2004)]. But free-radical damage associated with cold ischemia is evidently primarily due to a hypothermic release of iron. It would therefore be far more effective to eliminate the source of free radicals by the use of an iron chelator [JOURNAL OF INVESTIGATIVE MEDICINE; Rauen,U; 52(5):299-309 (2004)]. Deferoxamine has been used for this purpose, but a novel tetraazaannulene derivative (TAA−1) has been shown to completely inhibit cold-induced injury resulting from chelatable iron release [FREE RADICAL BIOLOGY & MEDICINE; Rauen,U; 37(9):1369-1383 (2004)].

Glycine reduces hypoxic injury by reducing ion fluxes through the plasma membrane of Na⁺ & Ca²⁺ [JOURNAL OF HEPATOLOGY; Frank,A; 32:58-66 (2000)]. The ability of glycine to affect Cl^- flux is not relevant for this protective effect. Glutamine inhibits proteolysis and can activate heat-shock protein, while the addition of other amino acids can have a nutritional benefit [LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)]. Carbon monoxide releasing compounds have a protective vasodilatory effect and increases mitochondrial respiration after cold ischemia and reperfusion [KIDNEY INTERNATIONAL; Sandouka,A; 69(2):239-247 (2006)].

Although Viaspan® was treated as a univeral hypothermic preservation solution for nearly a decade, in the mid-1990s "intracellular-type" solutions with high potassium such as Hypothermosol® proved to be superior for preserving hearts & lungs, as well as other cells and tissues.

(For more on organ preservation solutions, see Blood Washout & Replacement and Reducing Ischemic Damage by Cooling.)

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VII. BRAIN DAMAGE DUE TO ISCHEMIA/REPERFUSION

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)]. Neurons in brain tissue extracted from humans postmortem for 3 to 6 hours have been shown to recover oxidative metabolism and axon transport after suitable in-vitro treatment [THE LANCET 351:499-500 (1998)]. Adult rats subjected to cerebral ischemia showed no signs of neuron necrosis for 2 hours, and only by 6 hours did more than 15% of neurons appear necrotic [STROKE; 26(4):636-643 (1995)]. Similar results have been seen for humans [ANNALS OF NEUROLOGY; 2:206-210 (1977)].

The CA1 pyramidal neurons of the hippocampus are often regarded to be the most sensitive to ischemic injury of all neurons. Following 30 minutes of ischemia and subsequent 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 by which cells die in a slow and orderly manner so as to be removed by macrophages. Necrosis, by contrast, is more rapid — leading to cell membrane rupture, spilling of cell contents and inflammation. Apoptosis requires DNA transcription, new protein synthesis — a process requiring many hours, if not days.

The rapidity & form of cell death has been shown to be a function of the degree of ATP depletion. Mouse kidney cells in which ATP levels were 15% or less than normal (less than control) died by necrosis over a period between 2 and 4 hours. Cells with ATP levels 25% of normal remained viable for at least 6 hours, but had all experienced apoptotic death by 48 hours [AMERICAN JOURNAL OF PHYSIOLOGY; 274(2 Pt 2):F315-F327 (1998)].

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 should be of much more concern than apoptosis in cryonics.

The most damaging effect of ischemia within the first hour or two is to the capacity for cerebral blood flow [BRAIN RESEARCH 81:59-74 (1974)]. Lactic acidosis causes endothelial cells to swell [ACTA NEUROPATHOLOGIA 60:232-240 (1983)]. Blood cells stiffen & agglutinate. The longer the ischemia, the worse is the reperfusion injury to blood vessels due to free-radicals & hemorrhage — and the greater the chance of "no reflow" (impeded circulation). Without circulation there can be no cardiopulmonary support or cryoprotectant perfusion.

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VIII. PREVENTING ISCHEMIC/REPERFUSION INJURY IN CRYONICS

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), but only with the assistance of epinephrine [CIRCULATION 69(4):822-835 (1984)]. 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)]. Active compression-decompression and interposed abdominal compression can improve CPR perfusion considerably [CIRCULATION;100(21):2146-2152 (1999)] — as can mechanical devices (see below).

With effective cooling the flow provided even with moderately-effective CPR may be adequate to maintain brain structure. Newton's law of cooling dictates that 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. (For further discussion of cooling rates, see my essay Physical Parameters of Cooling in Cryonics.)

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. because adenosine inhibits glutamate release, coffee & tea consumption immediately prior to deanimation is contraindicated.

High levels of PARP−1 due to high levels of DNA damage can thus reduce the NAD+ needed for ATP synthesis, leading to ATP depletion and cell death by necrosis. Or PARP−1 may induce apoptosis by p53 stabilization and/or by translocation of Apoptosis-Inducing Factor (AIF) to the nucleus [EXPERIMENTAL HEMATOLOGY 31:446-454 (2003)]. PARP−1 inhibitors have been proposed to protect neurons from excitotoxicity and ischemic damage.

Zinc (Zn²⁺) contributes significantly to neuron death in ischemia, but pre-treatment with EDTA 30 minutes prior to the ischemic event robustly protects neurons [THE JOURNAL OF NEUROSCIENCE; Calderone,A; 24(44):9903-9913 (2004)]. Iron and copper can contribute significantly to free radical damage in ischemia, particularly iron in cold ischemia because cold ischemia releases iron within cells. Endothelial cells are significantly more damaged by reperfusion following cold ischemia than following warm ischemia [TRANSPLANTATION PROCEEDINGS; de Groot,H; 39(2):481-484 (2007)]. The metal chelator deferoxamine has shown signifant benefit against iron-catalyzed ischemic damage, but deferoxamine does not chelate copper [JOURNAL OF EXPERIMENTAL BIOLOGY; Warner,DS; 207(18):3221-3231 (2004)]. Other iron chelators have also been shown to be protective [FREE RADICAL BIOLOGY & MEDICINE; Rauen,U; 37(9):1369-1383 (2004)]. Insofar as blood cells (leukocytes and erythrocytes) are sources of reperfusion injury damage (cytokines, free radicals and other toxins), removal of blood cells prior to cold ischemia (shipment of a cryonics patient on ice) can considerably reduce reperfusion injury (associated with cryoprotective perfusion) [STROKE; Ding,Y; 33(10):2492-2498 (2002)].

At least two studies have shown that deprenyl could be of value in reducing ischemic damage in the brain. A study [STROKE 26:1883-1887 (1995)] involving 14 days of deprenyl on rats and 20 minutes of hypoxia/ischemia showed reduction of area of damage of 75% in the forebrain and about 20% in the cortex. For the hippocampus, 30-38% of the area was damaged in controls, but no damage was seen in the depenyl-treated rats. A similar study on gerbils [JOURNAL OF NEURAL TRANSMISSION 107:779-789 (2000)] showed reduced damage to the CA1 area of the hippocampus for deprenyl given more than a week before, immediately after and more than a week after ischemia due to vessel occlusion. Cell cultures exposed to peroxynitrite have been protected from apoptotic DNA damage by deprenyl [MECHANISMS OF AGING AND DEVELOPMENT 111:189-200 (1999)].

Minocycline can reduce inflammation, edema and damage to the blood-brain barrier, especially when tissue plasminogen activator (tPA) is being used. Activation of MMP−9 by tPA can be countered by the use of hypothermia [STROKE; Horstmann,S; 34(9):2165-2170 (2003)]. Although opening the blood-brain barrier is valuable in stroke treatment it may or may not be valuable in cryonics insofar as opening the blood-brain barrier can assist in getting cryoprotectants into the brain. (See the earlier sections on reperfusion injury and stroke therapy.)

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)]. Epinephrine, heparin (anti-coagulant), tPA and even cardiopulmonary support could be counterproductive for a cryonics patient who has a hemorrhagic stroke [STROKE; Steiner,T; 37(1):256-262 (2006)].

In hospitals, epinephrine is usually standard for ACLS (Advanced Cardiac Life Support). ACLS invariably uses manual CPR, despite the better blood delivery from mechanical devices. Mechanical devices are superior to manual CPR because (1) manual CPR quickly becomes less efficient because it is much more tiring and (2) manual CPR cannot deliver as much blood volume in the best of cases because a mechanical device can deliver a faster high-impulse square-wave compression.

Pronouncement of death may occur soon after the heart stops. In a Do-Not-Resuscitate (DNR) situation rapid application of CPR could cause the legally dead person to regain consciousness. It is unlikely that the heart could restart in an adult — especially if ischemia has elevated extracellular & plasma potassium levels. The heart rarely restarts without 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, propofol is not a controlled substance and can keep the patient unconscious. Fortuitously, propofol has also been shown to inhibit the neural cell apoptosis that can occur as a consequence of ischemia/reperfusion injury [THE JOURNAL OF NEUROSCIENCE; Polster,BM; 23(7):2735-2743 (2003)]. Propofol inhibits the opening of the Mitochondrial Permeability Transition Pore (MPTP) [CARDIOVASCULAR RESEARCH; Javadov,SA; 45(2):360-369 (2000)]. 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.

(For details on more advanced post-mortem changes, see Postmortem Changes or Chemistry of Decomposition.)

(For a more in-depth review of cryonics medications, see Future Directions in Human Cryopreservation Combinational Pharmacotherapy.)

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IX. PRE-TREATMENT FOR CRYONICS PATIENTS

For the terminal cryonics patient it can be asked, why wait until after declaration of legal death before using antioxidants or other agents that can reduce ischemic damage? Higher blood and tissue levels of some antioxidants can be achieved if administered in the days or weeks before legal death than if administered after the event. For antioxidants that are legal and safe, a pre-treatment protocol makes a great deal of sense, although there have been few controlled studies on such pre-treatment by cryonics researchers or anyone else. Relevant experiments in the literature generally involve pre-treatment within one hour prior to induction of ischemia.

Intravenous injection of the alpha-tocopherol form of Vitamin E (20 mg/kg or 9 mg/pound) 30 minutes prior to ischemia has been shown to significantly reduce lipid peroxidation and neurological damage [STROKE 14(6):977-982 (1983)]. A better experiment would have included both alpha-tocopherol and gamma-tocopherol because gamma-tocopherol removes peroxynitrite whereas alpha-tocopherol does not [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); 94(7):3217-3222 (1997)].

Vitamin E pretreatment for cryonics patients has the additional advantage of reducing blood clotting — and does not have the risk of gastric bleeding associated with aspirin. Many fish oils (especially salmon oil) afford the same benefit, in addition to reducing the risk of cardiac arrest [MOLECULAR AND CELLULAR BIOCHEMISTRY 116(1-2):19-25 (1992)]. Reduced clotting in a cryonics patient is a great benefit — and is reason for heparin injection after legal death. For patients undergoing surgery, however, Vitamin E and fish oils may be prohibited because clotting is desired.

Unlike Vitamin E, melatonin acts as an antioxidant through endogenous electron donation, which does not have the same potential for a pro-oxidant side effect [JOURNAL OF PINEAL RESEARCH 32:135-142 (2002)]. The capacity of melatonin to scavenge hydroxyl radicals is three orders of magnitude greater than Vitamin E [JOURNAL OF BIOLOGICAL CHEMISTRY; 274(31):21937-21942 (1999)]. Pretreatment of gerbils with melatonin (10 mg/kg or 4.5 mg/pound) 30 minutes before reperfusion significantly reduced ischemic brain injury [JOURNAL OF PINEAL RESEARCH 29:217-227 (2000)]. Similar effects were achieved with rats, but 5mg/kg showed a greater benefit than a higher or lower dose [JOURNAL OF PINEAL RESEARCH 34:110-118 (2003)]. Melatonin can also protect against ischemia-reperfusion injury by inhibiting inducible nitric oxide production, at least partially by means of inhibiting activation of the pro-inflammatory transcription factor NF-κB and blockage of NF-κB binding to DNA [THE FASEB JOURNAL; Gilad,E; 12(9):685-693 (1998)]. Nitric oxide has been shown to exacerbate apoptosis due to calcium release from the mitochondrial pool and activation of the Mitochondrial Permeability Transition Pore (MPTP) [THE FASEB JOURNAL; Horn,TFW; 16(12):1611-1622 (2002)].

Lipoic acid is beneficial in reducing ischemic-reperfusion injury by direct action as well as by glutathione protection and xanthine oxidase inhibition [FREE RADICAL BIOLOGY & MEDICINE; Packer, L.; 19(2):227-250 (1995)]. Protection against peroxynitrite damage by lipoic acid is highly dependent upon the target molecule (some molecules are protected more than others) [JOURNAL OF BIOLOGICAL CHEMISTRY; Rezk,BM; 279(11):9693-9697 (2004)]. Protection of neurons from glutamate excitotoxicity is equally effective by the R-form and S-form [FREE RADICAL BIOLOGY & MEDICINE; Tirash,O; 26(11/12):1415-1426 (1999)].

CoEnzyme Q₁₀ has been shown to protect rat endothelial cells from ischemia-reperfusion injury [SURGERY; Yokoyama,H; 120(2):189-196 (1996)]. Human cardiac arrest patients admitted to a hospital within 6 hours of cardiac arrest given a 250 mg loading dose of CoQ₁₀ showed 68% survival compared to 30% of controls. Of the survivors, 36% of the CoQ₁₀ group had good neurological outcome, in contrast to 20% of controls [CIRCULATION; Damian,MS; 110(19):3011-3016 (2004)].

Pretreatment of gerbils with deprenyl (0.25 mg/kg or 0.11 mg/pound) two weeks before ischemia reduced damage to neurons in the hippocampus [JOURNAL OF NEURAL TRANSMISSION 107:779-786 (2000)]. N-acetylcysteine (15 grams) infused in human myocardial infarction patients over a 24-hour period significantly reduced ischemic damage [CIRCULATION 92(10):2855-2862 (1995)].

The phytochemical curcumin (which gives curry its yellow color) is a powerful antioxidant which is several times more potent than Vitamin E [THE JOURNAL OF NEUROSCIENCE 21(21):8370-8377 (2001)]. Unlike alpha-tocopherol, curcumin can scavenge peroxynitrite and inhibit inducible nitric oxide synthetase [CARCINOGENESIS; Rao,CV; 20(4):641-644 (1999)] — which has the potential to significantly reduce peroxynitrite damage during reperfusion.

Vitamin C should not be used for ischemia-reperfusion pretreatment. Vitamin C is an antioxidant in the absence of metal ions, but in the presence of metal ions — which are released in large quantities from ischemic brain tissue — Vitamin C becomes a powerful pro-oxidant. (For discussion, see Antioxidant Molecules.)

In sum, a pre-treatment regimen for a terminal cryonics patient weighing 100 kilograms (220 pounds) should at least contain about 600 mg per day of alpha lipoic acid (1000 mg per day if racemic rather than R form), 500 mg per day of CoEnzyme Q₁₀, and 2,000 IU (mg) per day of mixed tocopherol (equal amounts of alpha and gamma). If the moment of deanimation (death) can be predicted (as with the removal of life support) then 50 mg of melatonin should be administered 30 minutes before the removal of life support. Melatonin is quickly metabolized (not stored in tissues) so its value for extended pretreatment could be debatable. In favor of its use for pretreatment, however, is the ischemic injury suffered by terminal patients during the dying process (although if the antioxidants delay the death, the net damage may be the same in the end). Curcumin use would also be advised, although there is no suggested dose.

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X. CONCLUSIONS

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