Message: #2633 - BPI Tech Brief #4
Date: 08 Mar 94 01:09:45 EST
From: Mike Darwin <>


      It is a core objective of BPI TECH BRIEFS to not only 
report significant in-house progress in both cryonics research 
and technology but also to educate the technologically  
sophisticated public about various aspects of the science which 
underlies cryonics.  Other than cryobiology and the neurobiology 
of learning and memory no area of investigation impacts cryonics 
more than the pathophysiology of cerebral ischemia.  The nature 
of ischemic lesions and the rapidity of their development is 
likely to be of critical importance to the workability of 
cryonics.  Clearly, if a period of ischemic injury destroys those 
structures responsible for human mentation and memory then no 
technology of preservation or recovery (given our current 
understanding of physics) will be of any use to a patient so 
injured.  Unfortunately, our understanding of the workings of 
memory and identity are still in their infancy.  

      Our understanding of the nature of ischemic injury is more 
advanced, but still far from complete.  The following article is 
adapted from the first Chapter of the HUMAN CRYOPRESERVATION 
LEVEL 1 TRANSPORT PROTOCOL, Seventh Edition (Biopreservation, 

by Michael G. Darwin, President
Biopreservation, Inc.

        In 1960 Kouwenhoven, Jude, and Knickerbocker reported 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 

        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 hospital3,4 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 what CC-CPR is most 
effective at is 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 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 SCD15.

      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- 
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 review briefly 
the requirements for adequate cerebral perfusion and the basic 
mechanisms of cerebral ischemic injury as they are currently 

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 mmHg20.  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 perfusion21.  At blood flow rates below 
10 ml/min/100g, sudden depolarization of the neurons occurs with 
rapid loss of intracellular potassium to the extracellular 

      The Mean Arterial Pressure (MAP) necessary for cerebral 
viability following extended resuscitation efforts in dogs has 
been found to be above 40 mm Hg23.  It has been speculated that a 
minimum MAP of 45 to 50 mm Hg is required to preserve cerebral 
viability in man24.

      Unfortunately, as is now well documented, conventional CC-
CPR is generally incapable of consistently delivering MAPs much 
above 30 mm Hg in man25,26.  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 Hg27.

      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 cryonic suspension 
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 (pa02).  
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 

      As the foregoing analysis makes clear, many, if not most, 
cryonic suspension patients will suffer significant periods of 
cerebral anoxia, ischemia, or hypoperfusion before they receive 
more effective cardiopulmonary support such as OC-CPR29, 
extracorporeal circulation utilizing a membrane or bubble 
oxygenator30, or high impulse CPR31,32.

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 
compounds16, acidosis due to anaerobic generation of lactate17, 
and no reflow due to swelling of astrocytes with compression of 
brain capillaries18.  Subsequent research has shown the problem 
to be far more complex than was previously thought, involving the 
action and interaction of many factors19.

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 cells34.  Sodium influx results in a 
marked increase in cellular water content, particularly in the 


      Normally, calcium is present in the extracellular milieu at 
a concentration 10,000 times greater than it is present 
intracellularly.  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 pump36, 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+ pump37, 3) 
sequestration of intracellular calcium in the endoplasmic 
reticulum by an ATP-driven process38, and 4) accumulation of 
intracellular calcium by oxidation-dependent calcium 
sequestration inside the mitochondria39.

      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 cell40.  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 episode41.  
Additionally, FFAs almost certainly have other degradative 
effects on cell membranes42.

      The production of AA as a result of FFA release causes a 
biochemical cascade ending with the production of throxboxane and 
leukotrienes.  Both these compounds are profound tissue irritants 
which can cause platelet aggregation, clotting, vasospasm, and 
edema42,43,44, 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 hypoxanthine45.  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 oxygen46,47.  Even more damaging free radicals could 
conceivably be produced by the metal catalyzed Haber-Weiss 
reaction as follows48-51:

    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 iron52,53,54.  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 dienes55,56 
and low molecular weight species of iron57.

      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 

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 ischemia59.  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 systems60.

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 ischemia64,65.  It has been shown, for 
instance, that lactate levels above a threshold of 18 - 25 
micromol/g result in currently irreversible neuronal 

      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 
ischemically69.  Lactic acid can also increase iron 
decompartmentalization, thus increasing the amount of free-
radical mediated injury70.


      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 
injury71,72,73.  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 ischemia75,76,77.  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 dextrorophan78 or the 
experimental anticonvulsant MK-80179.  In vitro studies with 
cultured neurons have demonstrated that excitatory 
neurotransmitters cause neuronal injury and death even in the 
absence of hypoxic or ischemic injury80.  In vivo studies have 
confirmed a massive release of glutamate and aspartate during 
both regional and global cerebral ischemia81.

      In regional or focal cerebral ischemic injury, the NMDA 
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 receptors82.  
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 
injury83,84.  Recently, attention has turned to non-NMDA 
antagonists such as inhibitors of the kainate and AMPA 

      The mechanisms by which excitotoxins cause cell injury is 
not yet fully understood.  It is known that they facilitate 
calcium entry into neurons86.  However, these agents are 
neurotoxic even in cell culture where the medium is calcium 
free87.  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 ischemia88,89,90.  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 reperfusion91.

      To what extent leukocyte plugging occurs in the brain 
following global cerebral ischemia remains controversial92.  
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 episode93.

      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 reperfusion94.

      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 matrix96.  
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 milliseconds97.

      However, the direct destructive effects of HOCl are 
probably limited in  vivo by a variety of mechanisms98.  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 enzymes99) to begin 
degrading the extracellular matrix, thus destroying capillary 
integrity and interfering with tissue metabolism and anabolism.


Figure 1:  The role of PMNL in mediating ischemic injury (from 
Weiss, S.J., New Eng J Med 1989;320:365-76).

      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 failure100.  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 al101, and Sterz, et al102, 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 

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 cat103, as well as Karlsson and Schultz104, 
and Van Nimwegen, et al105 in the rat.  These investigators 
describe the following changes in common in these animals' brain 
ultrastructure after varying periods of global cerebral ischemia 

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 course106.

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 

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 injury107.

      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 
subjects108.  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 cryonic suspension in a slow failure mode 
that allows for active intervention.

      The approach to protecting cryonic suspension 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-

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 E112,113,114, selenium115, vitamin 
C116, and beta carotene117.  Theoretical considerations also 
suggest other possible therapeutic agents such as those known to 
elevate neuronal (intracellular) glutathione levels for 
protection from cerebral ischemic injury118,119.

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 model120 as well as myocardial 
reperfusion injury121.  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 arrest122.


Figure 2:  The Pathophysiology of Cerebral Ischemia.

Schematic summary of the hypothesized mechanics of tissue injury 
in cerebral ischemia during both the circulatory arrest (left) 
and reperfusion (right) intervals.  During normal conditions 
intracellular calcium (Ca++) levels are maintained at 
approximately 100 nM.  Ca++ regulation is achieved by the plasma 
membrane Ca/Mg-ATPase and the ATP dependent uptake of Ca++ into 
the endoplasmic reticulum (ER) and mitochondria.  The release of 
bound Ca++ from the ER store is believed to be triggered by 
inositol-1,4,5 triphosphate (IP3) and/or by free arachidonic acid 
(AA).  Release of Ca++ bound in the mitochondria is not thought 
to occur until the ER stores are depleted.  The initial response 
of many different cell types to stimulation--i.e., ligand-
receptor interaction, hormone receptor binding, chemotactic 
peptide binding to polymorphonuclear leukocytes, or presynaptic 
or post-synaptic neurotransmitter binding , in an increase in 
Ca++ due to release of intracellular ER-bound Ca++, an influx of 
extracellular Ca++, or both.  Changes in many intracellular 
enzyme activities, including phospholipases and protein kinases, 
the polymerization of g-actin to f-actin, and that of tubulin to 
microtubules, all occur at different "set points" of Ca++.  
Therefore much of the control of intracellular processes is 
related to the level of Ca++. During ischemia (left), in all 
cells (including neurons) the level of ATP decreases rapidly to 
near zero.  This causes an increase in free calcium, even without 
an increase in IP3.  The addition of 2-deoxyglucose to cells, 
which acts as an ATP sink, causes a rapid increase in Ca++.  
Increases in Ca++ activate phospholipase A2 (p.lase), which 
breaks down membrane phospholipids (PL) into free fatty acids 
(FFA), particularly AA.  The AA causes increased activity of the 
cyclooxygenase pathway to produce prostaglandins (PG), including 
thromboxane (TX) A2, the lipoxygenase pathway to produce 
leukotrienes (LT), or both.  Furthermore, during ischemia the 
hydrolysis of ATP via AMP leads to accumulation of hypoxanthine 
(HX).  Increased Ca++ enhances the conversion of xanthine 
dehydrogenase (XD) to xanthine oxidase (XO), priming the neuron 
for the production of the oxygen free radical O2- 
intracellularly, once O2 is reintroduced.  During reoxygenation 
(right), significantly increased levels of at least three free 
radical species (in oblique boxes) that result in direct and 
indirect damage to cell membranes and the extracellular matrix 
(and thus lead to edema and microcirculatory failure) may be 
formed: O2-, OHo, and free lipid radicals (FLR).  O2- may be 
formed from two sources: 1) the previously described XO system 
and 2) activation of neutrophils in the microvasculature due to 
increased LT production by the neurons or simply by absent blood 
flow and consequent margination and diapedesis of neutrophils 
from the microvasculature.  Increased O2- production leads to 
increased H2O2 production as a result of the intracellular action 
of SOD. H2O2 is controlled by intracellular catalase.  Increased 
O2- production leads to increased OHo, due to the Fenton reaction 
(Fe++ +H2O2--->Fe+++.+OH+OHo) with iron liberated from ferritin, 
and the Haber-Weiss reaction (O2-+H2O2--->OH-+ OHo).. Each or all 
of these oxidants can result in lipid peroxidation and the 
production of LFRs.  All free radicals can cause leaky membranes 
and currently irreversible cell damage.  Furthermore, 
reoxygenation restores ATP via oxidative phosphorylation, which 
may result in massive uptake of Ca++ into mitochondria.  Thus, 
increased Ca++ as a result of ischemia and reoxygenation, by 
itself, and by triggering free radical reactions, may well be the 
principal cause of neuronal necrosis during reperfusion.

The above text and figure are reproduced with some changes from 
Safar, P., and Bircher, N.G., Cardiopulmonary Cerebral 
Resuscitation. 1988; W.B. Saunders Company, Ltd., London, UK. pp. 
236-37.Figure 2. The Pathophysiology of Cerebral Ischemic Injury

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 


     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 
signifance 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 suspension 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 

      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 

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-

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-

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-

28) Ornato JP, Bryson BL, Donovan PJ, et al. Measurement of 
ventilation during cardiopulmonary resuscitation. Crit Care Med 

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 

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-

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 

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 

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 

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

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 

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 biochemicalhypothesis. Ann Emerg Med 

68) 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 Frosst Award: Ischemic and 
reperfusion injury in the heart: Cellular mechanisms and 
pharmacological interventions. Can J Physiol Pharmacol 1991; 

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 

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. Biocheminstry 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 

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 axidosis 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 

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 

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 
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 

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-

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 

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 

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 
metyabolites 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 destrucion 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 

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 

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 

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 

122) Rosenthal RE, Williams R, Yolanda E, et al. Prevention of 
postischemic canine neurological injury through potentiation of 
brain energy metabolism by acetyl-l-lcarnitine. Stroke 

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. 
        Copyright 1993 by Mike Darwin