The Pathophysiology of Ischemic Injury

by Mike Darwin, BioPreservation, Inc. (1995)


In 1960 Kouwenhoven, Jude, and Knickerbocker reported the use of closed-chest cardiopulmonary resuscitation (CC-CPR) in 20 patients with a 70% overall survival rate [1]. In the decades that followed, an international program of enormous scope and cost was launched to implement CC-CPR at every level of emergency care, including the instruction of millions of laypersons in the technique.

In the intervening three decades since CC-CPR was first introduced with the enthusiastic statement by Kouwenhoven, et al. that "Anyone, anywhere, can now initiate cardiac resuscitation procedures. All that is needed are two hands" [2], many studies have been published documenting its ineffectiveness (i.e., survival rates under 20%) in maintaining cerebral viability in cases of cardiac arrest both in the hospital [3,4] and in the field [5,6,7]. Indeed, there is evidence that the survival rate of patients experiencing in-hospital cardiac arrest has declined since CC-CPR replaced open chest CPR (OC-CPR) in the 1960's [8]. In the thirty years since its implementation there has never been a formal, organized assessment of the utility of this technique in terms of cost vs. benefit either financially or medically.

In patients who survive following resuscitation with CC-CPR, the incidence of both transient and permanent neurological deficits and reduced quality of life are high [9,10,11,12].

In recent years there has been a growing awareness of the inadequacy of CC-CPR, with a call by some to return to OC-CPR [13] and vigorous research by others to optimize CC-CPR to address the dismal survival rates and usually poor neurological outcome. Increasingly, public healthcare policy is coming to reflect the reality that neurologists, cardiologists and intensivists have long understood: "CC-CPR doesn't work.". This is reflected in the recent policy change by the American Red Cross, wherein bystanders to cardiac arrest patients are now urged to activate the Emergency Medical System (EMS) first and start CPR second, instead of the other way around. This change reflects a growing awareness that CC-CPR is largely ineffective and that a patient's best chance for recovery is early defibrillation and associated definitive therapy.

This may seem an extreme statement, particularly to those who have not witnessed the all too common tableaux played out in intensive care units around the world of the brain dead or vegetative cardiac arrest victim consuming tens of thousands of dollars in medical resources.

The staggering cost of CC-CPR in teaching, healthcare, and patient/family emotional and financial resources when weighed against the dubious benefit suggests that society might have been better served if the CC-CPR program had never been implemented. The conclusion seems inescapable that CC-CPR is most effective at producing individuals who either are brain dead, or in a persistent vegetative state.

The problem with CC-CPR (or any in-field resuscitation technique) is cerebral ischemia. While mechanical or other device-oriented means of optimizing CC-CPR may well be developed, and the first-response use of defibrillators may become more commonplace, the fundamental problem of ischemic time before restoration of adequate circulation remains.

For many of the 325,000 persons in the United States who will experience sudden cardiac death (SCD) in the coming year, there will be little or no possibility of rescue. Cardiac arrest will occur without warning, often in situations not conducive to activation of the EMS. However, for many of those patients, there will have been a warning that they are at increased risk of SCD. A prior myocardial infarct (MI), familial history of arrhythmic disease, or iatrogenic risk such as CABG or angioplasty, will often provide ample warning that SCD could occur. In MI alone the incidence of SCD within the first year following infarct is 14% [14]. The development of more sophisticated markers for SCD in post MI patients, such as increased R-R interval regularity, is also making it possible to identify with increasing accuracy those who are at risk of SCD [15].

What can be done to improve the disappointing overall success rate of CPR? Does increasing the ability to identify patients at risk for SCD offer the possibility of therapeutic interventions such as anti-arrhythmic drugs and implantable defibrillators? Is there some way to pre-medicate or pre-treat patients who are at risk to increase their chances of surviving an ischemic episode with intact mentation?

A review of the literature in experimental cerebral resuscitation and the pathophysiology of cerebral ischemia (CI) suggests a number of approaches using both pre- and post-insult medication which may provide protection against cerebral ischemia for those at risk for SCD and which have acceptable costs and risk-to-benefit ratios.

While a wide range of post-insult interventions are currently being investigated in animal and clinical trials, relatively little attention has been paid to the possibility of pre-medication of the at risk population combined with post-insult therapy. Additionally, despite almost universal agreement that CI is a multifactorial insult, there has been little or no research aimed at developing a multimodal method of managing the multiple insults and compromises to brain metabolism that are known to occur.

Before suggestions are put forth for prevention and/or amelioration of ischemic injury, it is desirable to briefly review the requirements for adequate cerebral perfusion and the basic mechanisms of cerebral ischemic injury as they are currently understood.

Requirements For Adequate Cerebral Perfusion

Normal cerebral blood flow (CBF) in man is typically in the range of 45-50 ml/min/100g between a mean arterial pressure (MAP) of 60 and 130 mmHg [20]. When CBF falls below 20 to 30 ml/min/100g, marked disturbances in brain metabolism begin to occur, such as water and electrolyte shifts and regional areas of the cerebral cortex experience failed perfusion [21]. At blood flow rates below 10 ml/min/100g, sudden depolarization of the neurons occurs with rapid loss of intracellular potassium to the extracellular space [22].

The Mean Arterial Pressure (MAP) necessary for cerebral viability following extended resuscitation efforts in dogs has been found to be above 40 mm Hg [23]. It has been speculated that a minimum MAP of 45 to 50 mm Hg is required to preserve cerebral viability in man [24].

Unfortunately, as is now well documented, conventional CC-CPR is generally incapable of consistently delivering MAPs much above 30 mm Hg in man [25,26]. A clinical evaluation of manual and mechanical CPR (using a pneumatically driven chest compressor and ventilator) demonstrated that only 3 of 15 acute cardiac arrest patients presenting for emergency room resuscitation had MAPs above 40 mm Hg [27].

It should be emphasized that these studies evaluated a highly selected patient population, where the underlying cause of cardiac arrest was primary cardiac failure without other organ system failure, dehydration, sepsis, or pulmonary hypoxia as an underlying cause.

Quite often, the patient presenting for cryopreservation suffers from a variety of pathologies which can be expected to further reduce the ability of closed chest CPR to deliver adequate MAP or adequate arterial blood oxygenation (paO2). Pneumonia, pulmonary and systemic edema, hemorrhage, sepsis, liver failure, space-occupying lesions of the lungs, and a host of other pathologies can all compromise gas exchange and reduce vascular tone and circulating blood volume. Even in the patient experiencing optimum machine-delivered CPR, lung compliance and blood gases tend to deteriorate rapidly during CPR, perhaps as a result of pulmonary edema secondary to high intrathoracic venous pressures [28].

As the foregoing analysis makes clear, many, if not most, cryopreservation patients will suffer significant periods of cerebral anoxia, ischemia, or hypoperfusion before they receive more effective cardiopulmonary support such as OC-CPR [29], extracorporeal circulation utilizing a membrane or bubble oxygenator [30], or high impulse CPR [31,32].

Mechanisms of Ischemic Injury

Early observations on the mechanisms of ischemic injury focused on relatively simple biochemical and physiological changes which were known to result from interruption of circulation. Examples of these changes are: loss of high-energy compounds [16], acidosis due to anaerobic generation of lactate [17], and no reflow due to swelling of astrocytes with compression of brain capillaries [18]. Subsequent research has shown the problem to be far more complex than was previously thought, involving the action and interaction of many factors [19].

Biochemical Events

Within 20 seconds of interruption of blood flow to the mammalian brain under conditions of normothermia, the EEG disappears, probably as a result of the failure of high-energy metabolism. Within 5 minutes, high-energy phosphate levels have virtually disappeared (ATP depletion) [33] and profound disturbances in cell electrolyte balance start to occur: potassium begins to leak rapidly from the intracellular compartment and sodium and calcium begin to enter the cells [34]. Sodium influx results in a marked increase in cellular water content, particularly in the astrocytes [35].


Normally, calcium is present in the extracellular milieu at a concentration 10,000 times greater than the intracellular concentration. This 10,000:1 differential is maintained by at least the following four mechanisms: (1) active extrusion of calcium from the cell by an ATP-driven membrane pump [36]; (2) exchange of calcium for sodium at the cell membrane driven by the intracellular to extracellular differential in the concentration of Na+ as a result of the cell membrane's Na+ -- K+ pump [37], (3) sequestration of intracellular calcium in the endoplasmic reticulum by an ATP-driven process [38], and (4) accumulation of intracellular calcium by oxidation-dependent calcium sequestration inside the mitochondria [39].

The loss of cellular high-energy compounds during ischemia causing the loss of the Na+ -- K+ gradient, virtually eliminates three of the four mechanisms of cellular calcium homeostasis. This, in turn, causes a massive and rapid influx of calcium into the cell [40]. Mitochondrial sequestration, the remaining mechanism, causes overloading of the mitochondria with calcium and diminished capacity for oxidative phosphorylation. Elevated intracellular Ca++ activates membrane phospholipases and protein kinases. A consequence of phospholipase activation is the production of free fatty acids (FFA's) including the potent prostaglandin inducer, arachidonic acid (AA). The degradation of the membrane by phospholipases almost certainly damages membrane integrity, further reducing the efficiency of calcium pumping and leading to further calcium overload and a failure to regulate intracellular calcium levels following the ischemic episode [41]. Additionally, FFAs almost certainly have other degradative effects on cell membranes [42].

The production of AA as a result of FFA release causes a biochemical cascade ending with the production of thromboxane and leukotrienes. Both these compounds are profound tissue irritants which can cause platelet aggregation, clotting, vasospasm, and edema [42,43,44], with resultant further compromise to restoration of adequate cerebral perfusion upon restoration of blood flow.

Free Radicals

During ischemia, the hydrolysis of ATP via AMP leads to an accumulation of hypoxanthine [45]. Increased intracellular calcium enhances the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). Upon reperfusion and reintroduction of oxygen, XO may produce superoxide and xanthine from hypoxanthine and oxygen [46,47]. Even more damaging free radicals could conceivably be produced by the metal catalyzed Haber-Weiss reaction as follows [48-51]:

O2- + H2O ----Fe3 ------> O2 + OH-+ OH-

Iron, the transition metal needed to drive this reaction, is present in abundant quantities in bound form in living systems in the form of cytochromes, transferrin, hemoglobin and others. Anaerobic conditions have long been known to release such normally bound iron [52,53,54]. Indirect experimental confirmation of the role of free iron in generating free-radical injury has come from a number of studies which have confirmed the presence of free-radical breakdown products such as conjugated dienes [55,56] and low molecular weight species of iron [57].

During reperfusion and re-oxygenation, significantly increased levels of several free-radical species that degrade cell and capillary membranes have been postulated: 1) O2-, OH-, and free lipid radicals (FLRs). O2- may be formed by the previously described actions of XO and/or by release from neutrophils which have been activated by leukotrienes (see discussion below of the role of leukocytes in ischemia-reperfusion injury).

Re-oxygenation also restores ATP levels, and this may in turn allow active uptake of calcium by the mitochondria, resulting in massive calcium overload and destruction of the mitochondria [58].

Mitochondrial Dysfunction

Calcium loading and free-radical generation are no doubt major contributors to the mitochondrial ultrastructural changes which are known to occur following cerebral ischemia [59]. In addition to the structural alterations observed, there are biochemical derangements such as a marked decrease in adenine nucleotide translocase and oxidative phosphorylation. There is also an accumulation of FFAs, long-chain acyl-CoA, and long-chain carnitines. Of these alterations, the accumulation of long-chain acyl-CoA is perhaps most significant, since intramitochondrial accumulation of long-chain acyl-CoA is known to be deleterious to many different mitochondrial enzyme systems [60].

Lactic Acidosis

While it is clearly not the sole or even the major source of injury in ischemia, lactic acidosis does apparently contribute to the pathophysiology of ischemia [64,65]. It has been shown, for instance, that lactate levels above a threshold of 18 - 25 micromol/g result in currently irreversible neuronal injury [66,67,68].

Decrease in pH as a consequence of lactic acidosis has been shown to injure and inactivate mitochondria. Lactic acid degradation of NADH (which is needed for ATP synthesis) may also interfere with adequate recovery of ATP levels post ischemically [69]. Lactic acid can also increase iron decompartmentalization, thus increasing the amount of free-radical mediated injury [70].


A rapidly growing body of evidence indicates that excitatory neurotransmitters, which are released during ischemia, play an important role in the etiology of neuronal ischemic injury [71,72,73]. Those areas of the brain which show the most "selective vulnerability" to ischemia, such as the neocortex and hippocampus, are richly endowed with excitatory AMPA (alpha-amino-hydroxy-5-methyl-4-isoxazole proprionic acid) and NMDA (N-methyl-d-aspartate receptors) [74].

Initially there was much optimism that blockade of the NMDA receptor would provide protection against delayed neuronal death following global cerebral ischemia [75,76,77]. The use of NMDA receptor blocking drugs has shown significant promise in ameliorating focal cerebral ischemic injury; a number of studies have demonstrated marked reduction in the severity of ischemic injury to focal areas (particularly the poorly perfused "penumbra" surrounding the no-flow area) as a result of treatment with glutamate-blocking drugs such a dextrorophan [78] or the experimental anticonvulsant MK-801 [79]. In vitro studies with cultured neurons have demonstrated that excitatory neurotransmitters cause neuronal injury and death even in the absence of hypoxic or ischemic injury [80]. In vivo studies have confirmed a massive release of glutamate and aspartate during both regional and global cerebral ischemia [81].

In regional or focal cerebral ischemic injury, the NMDA receptor remains activated for a long period due to the prolonged interval of poor perfusion in the area at the edges of the infarct (the "penumbra"). However, in complete or global ischemia there is good resumption of blood flow following restoration of circulation with prompt uptake of glutamate and aspartate and resultant relatively rapid inactivation of the NMDA receptors [82]. Another factor limiting the role of the NMDA receptor in mediating injury in global cerebral ischemia may be the rapid and pronounced drop in pH which occurs in global as opposed to focal ischemia, since low pH is known to inactivate the NMDA receptor. These reasons are probably why NMDA receptor inhibitors have not proved effective in preventing global cerebral ischemic injury [83,84]. Recently, attention has turned to non-NMDA antagonists such as inhibitors of the kainate and AMPA receptors [85].

The mechanisms by which excitotoxins cause cell injury is not yet fully understood. It is known that they facilitate calcium entry into neurons [86]. However, these agents are neurotoxic even in cell culture where the medium is calcium free [87]. In the case of kainate and AMPA receptor activation, the likely mode of injury is sensitization of the CA1 pyramidal cells during ischemia such that when normal signaling is restored at the end of the ischemic insult, and normal intensity input from the Schaffer collaterals is resumed, lethal cell injury results, perhaps from abnormal calcium regulation in the CA1 cells or other metabolic derangements not yet understood.

Neutrophil Activation

Since the late 1960s, polymorphonuclear leukocytes (PMNLs) and monocytes/macrophages have been implicated as significant causes of pathology in cerebral ischemia. During the last decade there has been a veritable explosion of research documenting the role of PMNLs in reperfusion injury. Most of the initial work done in this area focused on PMNL-mediated reperfusion injury to the myocardium, establishing that PMNL activation and subsequent plugging and degranulation (resulting in release of oxidizing compounds) is responsible for the no-reflow phenomenon following myocardial ischemia [88,89,90]. In particular, the work of Engler has demonstrated that PMNL activation is responsible for plugging at least 27% of myocardial capillaries and is further responsible for the development of edema and arrhythmias upon reperfusion [91].

To what extent leukocyte plugging occurs in the brain following global cerebral ischemia remains controversial [92]. Anderson, et al. have examined the question of how rapidly leukocyte plugging occurs following cerebral ischemia using a bilateral carotid artery plus hypotension model in the dog. They noted no leukocyte plugging after 3 hours of reperfusion following a 40-minute ischemic episode [93].

However, it is clear from a growing body of work that neutrophils are a major mediator of ischemic injury in a variety of organ systems and that their acute activation is responsible for many of the effects of ischemia observed in the brain and other body tissues, including the loss of capillary integrity and the degradation of ultrastructure upon reperfusion [94].

When PMNLs are activated they generate large amounts of hydrogen peroxide. A large fraction of the hydrogen peroxide, aided by myeloperoxide (also released by activated PMNLs), reacts with the halides Cl-, Br-, or I- to produce their corresponding hypohalous acids (HOX) [95]. Because the concentration of Cl- is more than a thousand times greater than the other halides, the hydrogen peroxide-myeloperoxidase system probably generates Cl- most often in the form of HOCl. HOCl is more commonly known as household bleach and is capable of damaging a wide range of organic molecules including most of those that make up the structure of the cells and proteinaceous extracellular matrix [96]. As Klebanoff has pointed out, the amounts of HOCl generated by the neutrophil are awesome: 106 neutrophils can generate 2 x 107 mol of HOCl – enough to destroy 150 million E. Coli in a matter of milliseconds [97].

However, the direct destructive effects of HOCl are probably limited in vivo by a variety of mechanisms [98]. Most probably the hypohalous acids act to inflict the lion's share of injury by interacting with PMNL, collagenase, elastase, gelatinase, and other proteinases. As is shown in the diagram below, it is now believed that the oxidants released from the neutrophil create a halo of oxidized alpha-1-proteinase inhibitor that allows released elastase (and probably others of the 20 or so known neutrophil-secreted proteolytic enzymes [99]) to begin degrading the extracellular matrix, thus destroying capillary integrity and interfering with tissue metabolism and anabolism.

In complete circulatory arrest, it is clear that neutrophil activation with accompanying release of HOCl and activation of elastase is a key factor in initiating the systemic cascade of inflammation/immune response which terminates in delayed multisystem organ failure [100]. The extent to which this pathway is a factor in acute global cerebral ischemic injury in cardiac arrest is not yet clear.

Hypoperfusion Following Reperfusion

An apparently significant contributor to reperfusion injury is hypoperfusion after restoration of spontaneous circulation. The work of Hossman, et al [101], and Sterz, et al [102], has demonstrated the critical importance of providing adequate circulatory support following global cerebral ischemia. Loss of autonomic regulation, depressed myocardial function secondary to ischemic insult of the myocardium, and autonomic dysfunction all serve to depress MAP and cerebral perfusion following restoration of circulation. Both Hossman's and Sterz's work has demonstrated significant improvements in neurological outcome if circulation is supported both extracorporeally and/or with pressors during reperfusion.

Histological Ultrastructural Change

Ischemic changes in cell architecture begin almost as rapidly as ischemic changes in biochemistry. Within seconds of the onset of cerebral ischemia, brain interstitial space almost completely disappears. Loss of interstitial space is a consequence of cell swelling secondary to sodium influx and failure of membrane ionic regulation. There have been several studies of the ultrastructural alterations associated with prolonged global cerebral ischemia. Notable is the work of Kalimo et al in the cat [103], as well as Karlsson and Schultz [104], and Van Nimwegen, et al [105] in the rat. These investigators describe the following changes in common in these animals' brain ultrastructure after varying periods of global cerebral ischemia (GCI):

1) Changes At 10 Minutes

After 10 minutes of GCI, a significant number of cells (but not all) show clumping of nuclear chromatin and a modest increase in electron lucency (probably due to dilution of the cytosol by extracellular fluid). After 30 minutes, further changes include increased cytoplasmic swelling (particularly in the astrocytes), swelling and shape change of the mitochondria, and some loss of mitochondrial matrix density. Microtubules disappear and there is detachment of the ribosomes from the cisternae of the endoplasmic reticulum. There is also disassociation of the polyribosomes, and single ribosomes lose their compact structure with associated failure of protein synthesis. Of note is the stability of the lysosomes over this time course [106].

2) Changes At 60 Minutes

After 60 minutes of GCI, the above changes have become more pronounced with more conspicuous swelling of the ER cisternae. The mitochondria begin to show slight inner matrix swelling and occasional flocculent densities (probably due to accumulated calcium).

3) Changes At 120 Minutes

After 120 minutes of GCI, the changes discussed above are more pronounced and a larger number of mitochondria exhibit the presence of flocculent densities evidencing calcium overload which is currently considered irreversible. Published electron micrographs reveal intact lysosomes and seem to confirm other studies which indicate that lysosomal rupture and subsequent catastrophic autolysis is not a feature of early (1 - 4 hours) ischemic injury [107].

From a cryonics (i.e., information-theoretic perspective), it is important to point out that throughout even a 120-minute-period of normothermic cerebral ischemia, the appearance of the plasma membrane layers, including synapses and myelin sheaths, is only altered modestly. Indeed, the first ultrastructural changes associated with what is currently considered lethal cell injury are to the mitochondria and ribosomes, and these do not usually appear until after 30 minutes of GCI.

At least one study of post-mortem ultrastructural degradation has been conducted on a small number of human subjects [108]. The histological and ultrastructural changes experienced in patients with 25 to 85 minutes of GCI, and without extensive pre-mortem brain trauma or pre-mortem cerebral no-reflow of prolonged duration, closely parallel those observed in animal models of GCI: astrocytic edema, clumping of nuclear chromatin, disassociation of the polyribosomes, detachment of the ribosomes from the ER cisternae, and swelling of the mitochondria with the presence of flocculent densities. Stability of the lysosomes and conservation of the structure of the neuropil over this time-course are well documented.

Opportunities For Intervention

With the understanding of the mechanisms of the pathophysiology of cerebral ischemia having evolved to the point outlined above, many possible interventions suggest themselves. Indeed, the literature of cerebral resuscitation is a vast one and is growing rapidly with the release of papers exploring a variety of monomodal approaches to treating cerebral injury secondary to both global and regional ischemic insults.

However, despite the widely held belief that cerebral ischemic injury is multifactorial in nature, there has been almost no work done examining multimodal methods of treatment. There is also almost a complete absence of studies which address the potential of pre-treatment in ameliorating cerebral ischemic injury, particularly pretreatment with nonproprietary agents such as antioxidant nutrients. This kind of approach is of particular importance to the cryonics community where a significant number of patients present for cryopreservation in a slow failure mode that allows for active intervention.

The approach to protecting cryopreservation patients against cerebral ischemic injury outlined in this text is a multimodal approach which address the following known sources of cerebral ischemic injury:

1) Numerous studies have suggested a cerebroprotective effect for a variety of calcium channel blockers administered post-insult [109,110,111].

2) Free radical damage: Free radicals have long been understood to be a major source of cerebral ischemic pathology. Similarly, there have been a number of studies which suggest that free radical associated ischemic injury can be reduced greatly or eliminated by pre- or post-insult treatment with nutritional antioxidants such as vitamin E [112,113,114], selenium [115], vitamin C [116], and beta carotene [117]. Theoretical considerations also suggest other possible therapeutic agents such as those known to elevate neuronal (intracellular) glutathione levels for protection from cerebral ischemic injury [118,119].

3) Phospholipase activation has been implicated as a significant source of injury in both cold and warm ischemia. The phospholipase inhibitor quinacrine has reduced cold ischemic injury in an organ preservation model [120] as well as myocardial reperfusion injury [121]. Quinacrine may be effective in attenuating normothermic cerebral ischemic injury as well.

4) The importance of mitochondrial dysfunction in preventing recovery following global cerebral ischemia has been demonstrated in a recent study by Rosenthal, et al. They demonstrated the effectiveness of acetyl-l-carnitine in improving both neurological function and normalizing brain high energy metabolism in the dog following 10 minutes of normothermic cardiac arrest [122].

5) Protection against the deleterious effects of excitotoxicity has been addressed in a number of ways, including the use of both NMDA and kainate receptor inhibiting drugs. As has been previously discussed, excitotoxicity is clearly a significant source of reperfusion injury and must be addressed in any multimodal therapeutic approach to cerebral ischemia. The best compound(s) to use to achieve this effect has not been determined by the author as of this writing.

6) As was previously noted, extracorporeal perfusion to support MAP, facilitate reperfusion through initial hypertension, insure adequacy of cerebral perfusion, and allow for induction of mild hypothermia have been shown to be beneficial in achieving a favorable outcome following 10 to 12 minute periods of global cerebral ischemia.

7) Inhibition of the inflammatory cascade and the adhesion and degranulation of polymorphonuclear lymphocytes by both drug treatment and by their removal via filtration have been shown to lessen reperfusion injury in the lungs and heart. As a consequence, they presumably lessen the likelihood of development of the post resuscitation syndrome, at least in extracerebral tissues [123].


As the foregoing has hopefully made clear, neuronal ischemic changes occur rapidly with significant structural changes being observed over a time-course of minutes rather than hours. The significance of these changes in terms of damage to identity-critical structures (i.e., those encoding memory and personality) is not currently known since we do not yet understand how memory is encoded, or more generally, which brain structures (gross or ultrastructural) are critical to mentation.

As a consequence of our ignorance about what structures need to be preserved, it is the opinion of this author that a very conservative approach to cryopreservation patient transport should be followed. In practice, what this means is that every reasonable effort should be made to minimize cerebral ischemic injury. Achieving a reasonable cost versus benefit tradeoff in actual practice will naturally be a matter of some debate. An attempt has been made in the development of this protocol to strike a reasonable balance between cost and complexity and anticipated benefit to the patient. A fairly conservative approach has been used in the application of new technologies without a proven track record of clinical success in cerebral resuscitation.

The author has been active in the fields of cerebral resuscitation and cryonics long enough to have observed a number of "fads" and "hot new techniques" come and go. An attempt has been made here to apply only those research modalities which have shown promise in a number of researchers' hands, and whenever possible, to have in-house verification of the effectiveness of these modalities.


1) American Heart Association and National Research Council, Standards for Cardiopulmonary Resuscitation (CPR) and emergency cardiac care (ECC). J Amer Med Assoc (Suppl.) 1974;227:833-68

2) Ibid.

3) Weale FE, Rothwell-Jackson RL. The efficacy of cardiac massage. The Lancet 1960;1:990-96

4) Eisenberg MS, Harwood BT, Cummins RO, Reynolds-Haertle R, Hearne TR. Cardiac arrest and resuscitation: A tale of 29 cities. Ann of Emer Med 1990;19:179-86.

5) Kentsch M, Stendel M, Berkel H. Early prediction of prognosis in out-of-hospital cardiac arrest. Intensive Care Med 1990;16:378-83.

6) Troiano P, Masaryk J, Stueven HA, et al. The effect of bystander CPR on neurologic outcome in survivors of prehospital cardiac arrests. Resuscitation 1989;17:91-98.

7) Bossaert L, Van Hoeyweghen R. The Cerebral Resuscitation Study Group. Resuscitation 1989;17m Suppl.:S55-S69.

8) Del Guercio LRM, Feins NR, Cohn JD, et al. A comparison of blood flow during external and internal cardiac massage in man. Circulation 1965;Suppl. 1:171-80.

9) Troiano P, Masaryk J, Stueven HA, et al. The effect of bystander CPR on neurologic outcome in survivors of prehospital cardiac arrests. Resuscitation 1989;17:91-98.

10) Bengtsson M, et al. A psychiatric-psychological investigation of patients who had survived circulatory arrest. Acta Psychiat Scan 1969;45:327.

11) Lucas BGB. Cerebral anoxia and neurologic sequelae after cardiac arrest. In Stephenson HE, ed. Cardiac Arrest and Resuscitation, 4th Ed. St Louis:The CV Mosby Co. 1974: 681-707.

12) Myerburg RJ, Conde CA, Sung RJ. Clinical, electrophysiologic, and hemodynamic profiles of patients resuscitated from prehospital cardiac arrest. Amer J Med 1980;68:568.

13) Del Guercio LRM. Open chest cardiac massage: An overview. Resuscitation 1987;15:9-11.

14) Luria MH, Knoke JD, Margolis RM, et al. Acute myocardial infarction: prognosis after recovery. Ann Inter Med 1976;85:561-63.

15) Odemuyiwa O, Farrell TB, Malik M, Bashir Y, et al. Comparison of the predictive characteristics of heart rate variability index and left ventricular ejection fraction for all-cause mortality arrhythmic events after acute myocardial infarction. Amer J Cardio 1991;8:434-39.

16) Reichelt KL. The chemical basis for the intolerance of the brain to anoxia. Acta Anesthesiol Scand 1978; Suppl. 29:35-46.

17) Rhenchrona S. Brain acidosis. Ann Emerg Med 1985;14:770-76.

18) Ames A III, et al. Cerebral ischemia II. The no-reflow phenomenon. Amer J Pathol 1968;52:437-53.

19) Kaplan J, Dimlich RVW, Biros MH, Hesges J. Mechanisms of Ischemic cerebral injury. Resuscitation 1987;15:149-169.

20) Dearden NM. Ischaemic brain. The Lancet 1985: August 3: 255.

21) Ibid.

22) Hertz L. Features of astrocyte function apparently involved in the response of central nervous tissue to ischemia-hypoxia. J Cereb Blood Flow Metab 1981;1:143-53.

23) McDonald JL. Systolic and mean arterial pressure during manual and mechanical CPR in humans. Annal Emerg Med 1982;11:292-295.

24) Ibid.

25) Tatsura A, Kentara D, Tsukahara I, et al. Cerebral blood flow during conventional, new and open chest cardiopulmonary resuscitation in dogs. Resuscitation 1984;12:147-154.

26) Del Guercio LRM, Feins NR, Cohn JD, et al. A comparison of blood flow during external and internal cardiac massage in man. Circulation 1965;Suppl 1:171-80.

27) McDonald JL. Systolic and mean arterial pressure during manual and mechanical CPR in humans. Annal Emerg Med 1982;11:292-295.

28) Ornato JP, Bryson BL, Donovan PJ, et al. Measurement of ventilation during cardiopulmonary resuscitation. Crit Care Med 1983;11:79-82.

29) Yashon D, Wagner FC, Massopust LC, et al. Electrocortigraphic limits of cerebral viability during cardiac arrest and resuscitation. Am J of Surg 1971;121:728-31.

30) Carden DL, Martin GB, Nowak RM, et al. The effect of cardiopulmonary bypass resuscitation on cardiac arrest induced lactic acidosis in dogs. Resuscitation 1989;17:153-161.

31) Ornato JP, Levine Rl, Young DS, et al. The effect of applied chest compression force on systemic arterial pressure and end tidal carbon dioxide concentration during CPR in human beings. Ann of Emerg Med 1989;18:732-737

32) Maier GW, Tyson GS, Olsen CO, et al. The physiology of external cardiac massage: high impulse cardiopulmonary resuscitation. Circulation 1984;70: 86-101.

33) Siesjo BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metab 1981;1:155-85.

34) Heuser D, Guggenberger H. Ionic changes in brain ischemia and alterations produced by drugs. Br J Anesth 1985;57:23.

35) Hertz L. Features of astrocyte function apparently involved in the response of central nervous tissue to ischemia-hypoxia. J Cereb Blood Flow Metab 1981;1:143-53.

36) Carafoli E, Crompton M. Curr Topics Memb Transport 1978;10:151-216.

37) Carafoli, ibid.

38) Blaustein MP, Ratzlaff R, Kendrick N. The regulation of intracellular calcium in presynaptic nerve terminals. Proc NY Acad Sci 1978;307:195-212.

39) Mitchell P, Moyle J. Chemiosmotic hypothesis of oxidative phosphorylation. Nature 1967;213:137-139.

40) White BC, Wiegenstein JG, Winegar CD. Brain ischemia and anoxia: Mechanisms of injury. J Amer Med Assoc 1984;251:1586-90.

41) Farber JL, Chien KR, Mittnacht S. The pathogenesis of irreversible cell injury in ischemia. Amer J Pathol 1981;102:271-81.

42) Wolfe LS. Eicosanoids: prostaglandins, thromboxanes, leukotrienes and other derivatives of carbon-20 unsaturated fatty acids. J Neurochem 1982;38:1-14.

43) Raichle ME. The pathophysiology of brain ischemia. Ann Neurol 1983;13:2-10.

44) Mullane KM, Salmon JA, Kraemer R. Leukocyte derived metabolites of arachidonic acid in ischemia-induced myocardial injury. Fed Proc 1987;46:2422-33.

45) Tien M, Aust SD. Comparative aspects of several models of lipid peroxidation systems. In Lipid Peroxides in Biology and Medicine. K. Yagi, ed. New York:Academic Press. 1982:23-39.

46) McCord JM. Oxygen derived free radicals in postischemic tissue injury. N Eng J Med 1985;312:159-163.

47) Kleihues K, Kobayashi K, Hossman KA. Purine nucleotide metabolism in the cat brain after one hour of complete ischemia. J Neurochem 1974;23:417-25.

48) Rhenchrona S. Brain acidosis. Ann Emerg Med 1985;14:770-76.

49) Fridovich I. Superoxide radical: An endogenous toxicant. Annul Rev Pharmacol Toxicol 1983;23:239-57.

50) McCord JM. The superoxide free radical: Its biochemistry and pathophysiology. Surgery 1983;94:412-14.

51) Tien M, Svingen BA, Aust SD. An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation. Arch Biochem Biophys 1982; 216:142-51.

52) Komara KS, Nayini NR, Bialick HA. Brain iron delocalization and lipid peroxidation following cardiac arrest. Ann Emer Med 1986;15:384-88.

53) Babbs CF. Role of iron ions in the genesis of reperfusion injury following successful cardiopulmonary resuscitation: Preliminary data and a biochemical hypothesis. Ann Emerg Med 1985;14:777-83.

54) White BC, Krause GS, Aust SD. Postischemic tissue injury by iron-mediated free radical lipid peroxidation. Ann Emerg Med 1985;14:804-09.

55) Nayni NR, White BC, Aust SD, et al. Post resuscitation iron delocalization and malondialdehyde production in the brain following prolonged cardiac arrest. J Free Radic Biol Med 1985;1:111-16.

56) Bromont C, Marie C, Bralet J. Increased lipid peroxidation in vulnerable brain regions after transient forebrain ischemia in rats. Stroke 1989;20:918-24.

57) Babbs CF. Role of iron ions in the genesis of reperfusion injury following successful cardiopulmonary resuscitation: Preliminary data and a biochemical hypothesis. Ann Emerg Med 1985;14:777-83.

58) Safar P. Cerebral resuscitation after cardiac arrest. A review. Circulation 1986;74 (Suppl IV):138.

59) Carnitine Biosynthesis, Metabolism and Functions, Frenkel RA, McGarry JD, eds. New York:Academic Press. 1980:321-340.

60) Karmazyn M. The 1990 Merck Frost Award: Ischemic and reperfusion injury in the heart: Cellular mechanisms and pharmacological interventions. Can J Physiol Pharmacol 1991; 69:719-730.

61) Siesjo BK, Folbergrova J, MacMillan V. The effect of hypercapnia on the intracellular pH in the brain , evaluated by bicarbonate-carbonic acid method and from the creatine phosphokinase equilibrium. J Neurochem 1972;19:2483-95.

62) Folbergrova J, MacMillan V, Siesjo BK. The effect of moderate and marked hypercapnia upon the energy state and upon the cytoplasmic NADH/NAD+ ratio of the rat brain. J Neurochem 1972;19:2497-2505.

63) Paljarvi L, Soderfeldt B, Kalimo H. The brain in extreme respiratory acidosis: A light and electron microscopic study in the rat. Acta Neuropathol 1982;58:87-94.

64) Siesjo BK. Cell damage in the brain: A speculative synthesis. J Cerebr Blood Flow Metab 1981;1:155-85.

65) Biros MH, Dimlich RW, Barsan WG. Post-insult treatment of ischemia-induced cerebral lactic acidosis in the rat. Ann Emerg Med 1985;15:397-404.

66) Rhenchrona S, Rosen I, Siesjo B. Brain lactic acidosis and ischemic cell damage: I. Biochemistry and neurophysiology. J Cereb Blood Flow Metab 1981;1:297-311.

67) Kalimo H, Rhencrona S, Soderfeldt, et al. Brain lactic acidosis and ischemic cell damage: Histopathology. J Cereb Blood Flow Metab 1981;1:313-27.

68) Rhencrona S, Rosen I, Smith ML. Effect of different degrees of brain ischemia and tissue lactic acidosis on the short-term recovery of neurophysiologic and metabolic variables. Exp Neurol 1985:87:458-73.

69) Lowry OH, Passonneau JV, Rock MK. The stability of pyridine nucleotides. J Bio Chem 1961;236:2756-59.

70) Siesjo BK, Bendek G, Koide T, et al. Influence of acidosis on lipid peroxidation of brain tissues in vitro. J Cereb Blood Flow Metab 1985;5:253-58.

71) Jorgensen MB, Diemer NH. Selective neuron loss after cerebral ischemia in the rat: Possible role of transmitter glutamate. Acta Neurol Scand 1982;66:536-46.

72) Rothman S. Synaptic release of excitatory amino acid neurotransmitters mediates anoxic cell death. J Neurosci 1984;4:1884-91.

73) Diemer NH, Johansen FF, Benveniste H, et al. Ischemia as an excitotoxic lesion: protection against hippocampal neuron loss by denervation. Acta Neurochir Suppl 1993;57:94-101.

74) Monaghan DT, Holets RV, Toy DW, Cotman CW. Anatomical distributions of four pharmacologically distinct 3H-glutamate binding sites. Nature 1983;306:176-179.

75) Barnes DM. NMDA Receptors trigger excitement. Science 1987;239:254-56.

76) Benveniste H, Jorgensen MB, Diemer NH, Hansen AJ. Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia. Acta Neurol Scand 1988;78:529-36.

77) Ito U, Spatz M, Walker JT, Klatzo I. Experimental cerebral ischemia in mongolian gerbil: Light microscopic observations. Acta Neuropathol 1975; 32:209-33.

78) Steinberg GK, Saleh J, DeLaPaz R, et al. Pretreatment with the NMDA antagonist dextrophan reduces cerebral injury following transient focal ischemia in rabbits. Brain Res 1989;18:382-86.

79) Ozyurt E, Graham DI, Woodruff GN, McCulloch J. Protective effect of the glutamate antagonist, MK-801 in focal cerebral ischemia in the cat. J Cerebr Blood Flow Metab 1988;8:138-43.

80) Rothman S. Synaptic release of excitatory amino acid neurotransmitters mediates anoxic cell death. J Neurosci 1984;4:1884-91.

81) Hagberg H, Lehmann A, Sandberg M, et al. Ischemia-induced shift pf inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 1985;5:413-19.

82) Diemer NH, Johansen FF, Jorgensen MB. N-methyl-d-aspartate and non n-methyl-d-aspartate antagonists in global cerebral ischemia. Supplement III: Stroke 1990;21:39-41.

83) Stertz F, Yuval L, Safar P, Radovsky A, et al. Effect of excitatory amino acid receptor blocker MK-801 on overall, neurologic, and morphological outcome after prolonged cardiac arrest in dogs. Anesth 1989;71:907-918.

84) Lanier WL, Perkins WJ, Karlsson BR, et al. The effects of dizoclipine maleate (MK-801) an antagonist of the N-methyl-d-aspartate receptor, on neurologic recovery and histopathology following complete cerebral ischemia in primates. J Cerebr Blood Flow Metab 1990;10:252-61.

85) Sheardown MJ, Nielsen EO, Hansen AJ, et al. 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline: A neuroprotectant for cerebral ischemia. Science 1990;247:571-74.

86) Berdichevsky E, Riveros N, Sanchez-Aimess S, Orrego F. Kainate, n-methyl aspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro. Neurosci Lett 1983;36:75-80.

87) Rothman S. Synaptic release of excitatory amino acid neurotransmitters mediates anoxic cell death. J Neurosci 1984;4:1884-91.

88) Engler RL, Dahlgren MD, Morris DD, et al. Role of leukocytes in response to acute myocardial ischemia and reflow in dogs. Am J Physiol 1986;251:H314-H322.

89) Schmid-Schobein GW. Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation. Federation Proc 1987;46:2397-401.

90) Engler R. Consequences of activation and adenosine mediated inhibition of granulocytes during myocardial ischemia. Federation Proc 1987;46:2407-412.

91) Mullane KM, Salmon JA, Kraemer R. Leukocyte derived metabolites of arachidonic acid in ischemia-induced myocardial injury. Federation Proc 1987;46:2422-2433.

92) Kochanek PM, Hallenbeck JM. Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 1992;23:1367-1379.

93) Anderson ML, Smith DS, Nikoa S, et al. Experimental brain ischemia: Assessment of injury by magnetic resonance spectroscopy and histology. Neurol Res 1990;12:195-204.

94) Halliwell B, ed. Oxygen Radicals and tissue injury: Proceedings of a Brook Lodge Symposium. Augusta, MI. USA, 27-29 April, 1987. Bethesda, MD:Federation of American Societies for Experimental Biology. 1988:1-143.

95) Klebanoff SJ. Phagocytic cells: Products of oxygen metabolism. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press. 1988:391-444.

96) Test ST, Weiss SJ. The generation and utilization of chlorinated oxidants by human neutrophils. Adv Free Radical Biol Med 1986;2:91-116.

97) Klebanoff SJ. Phagocytic cells: Products of oxygen metabolism. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: basic principles and clinical correlates. New York: Raven Press. 1988:391-444.

98) Weiss SJ. Tissue destruction by neutrophils. In: Epstein FH, ed. Mechanisms of disease. New Eng J Med 1989;320:365-76.

99) Henson PM, Henson JE, Fitlschen C, et al. Phagocytic cells: Degranulation and secretion. In: Gallin JI, Goldstein IM, Snyderman R, eds. Inflammation: Basic Principles and Clinical Correlates. New York:Raven Press. 1988:363-80.

100) Bersten A, Sibbald WJ. Acute lung injury in septic shock. Crit Care Clin 1989;5:49-80.

101) Hossmann KA. Resuscitation after prolonged global cerebral ischemia in cats. Crit Care Med 1988;16:964-71.

102) Sterz F, Leonov Y, Safar P, et al. Hypertension with or without hemodilution after cardiac arrest in dogs. Stroke 1990;20:1178-84

103) Kalimo H, Garcia JH, Kamijyo Y, et al. The ultrastructure of brain death II. Electron microscopy of feline cortex after complete ischemia. Virchow's Arch B Cell Path 1977;25:207-220.

104) Karlsson U, Schultz RL. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. III. Structural changes after exsanguination and delayed perfusion. J Ultrastruc Res 1966;14:57-63.

105) Van Nimwegen D, Sheldon, H. Early postmortem changes in cerebellar neurons of the rat. J Ultrastruc Res 1966;14:36-46.

106) Ibid.

107) Hawkins HK, Ericsson JL. Lysosome and phagasome stability in lethal cell injury. Amer J Path 1972;68:255-78.

108) Klimo H, Garcia, JH, Kamijyo Y, et al. Cellular and subcellular alterations of human CNS. Arc Pathol 1974;97:352-59.

109) White BC, Gadzinski DS, Hoehner PJ, et al. Cerebral cortical perfusion during and following resuscitation from cardiac arrest in dogs. Am J Emerg Med 1983;1:128-34.

110) Winegar CP, Henderson O, White BC, et al. Early amelioration of neurologic deficit by lidoflazine after 15 minutes of cardiopulmonary arrest in dogs. Ann Emer Med 1983;12:471-77.

111) Vaagenes P, Rinaldo C, Safart P. Amelioration of brain damage by lidoflazine after prolonged ventricular fibrillation cardiac arrest in dogs. Crit Care Med 1984;12:846-55.

112) Yoshida S. Brain injury after ischemia and trauma, the role of vitamin E. Ann NY Acad Sci 1989;570:219-36.

113) Uenohara H, Imaizumi S, Suzuki J, Yoshimoto T. The protective effect of mannitol, vitamin E, and glucocorticoid in experimental cerebral ischemia - influence on lipid peroxidation, energy metabolism and brain edema. No Shinkei Geka 1987;6:613-22.

114) Kinuta Y, Kikuchi H, Ishikawa M, et al. Lipid peroxidation in focal cerebral ischemia. J Neuro Surg 1989;71:421-9.

115) Poltronieri R, Cevese A, Sbarbati A. Protective effect of selenium in cardiac ischemia and reperfusion. Cardioscience 1992;3:155-60.

116) Kinuta Y, Kikuchi H, Ishikawa M, et al. Lipid peroxidation in focal cerebral ischemia. J Neuro Surg 1989;71:421-9.

117) Uenohara H, Imaizumi S, Suzuki J, Yoshimoto T. The protective effect of mannitol, vitamin E, and glucocorticoid in experimental cerebral ischemia - influence on lipid peroxidation, energy metabolism and brain edema. No Shinkei Geka 1987;6:613-22.

118) Menasche EP, Grousset C, Gaudel Y, et al. Maintenance of the myocardial thiol pool by N-acetylcysteine. An effective means of improving cardioplegic protection. J Thorac Cardiovasc 1992;103:936-44.

119) Aberola A, Such L, Gil F, et al. Protective effect of n-acetylcysteine on ischaemia-induced myocardial damage in canine heart. Nauyn Schmiedebergs Arch Pharmacol 1991;343:505-10.

120) Belzer FO, Hoffman RM, Miller DT, et al. A new perfusate for kidney preservation. Transplant Proc 1984;16:3241-42.

121) Otani H, Engelman RM, Breyer RH, et al. Mepacrine, a phospholipase inhibitor. A potential tool for modifying myocardial reperfusion injury. J Thorac Cardiovasc Surg 1986;92:247-54.

122) Rosenthal RE, Williams R, Yolanda E, et al. Prevention of postischemic canine neurological injury through potentiation of brain energy metabolism by acetyl-l-carnitine. Stroke 1992;23:1312-18.

123) Huddleston VB. Multisystem organ failure: Background and etiology. In: Multisystem Organ Failure: Pathophysiology and Clinical Implications. Huddleston VB, ed. St Louis:The Mosby Co. 1992:3-14.