X-Message-Number: 2633
Date: 08 Mar 94 01:09:45 EST
From: Mike Darwin <>
Subject: SCI.CRYONICS BPI TECH BRIEF #4
BPI TECH BRIEF #4
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,
1994).
THE PATHOPHYSIOLOGY OF CEREBRAL ISCHEMIA
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
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 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
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 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
space22.
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
pressures28.
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
astrocytes35.
Calcium
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
mitochondria58.
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
injury66,67,68.
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
ischemically69. Lactic acid can also increase iron
decompartmentalization, thus increasing the amount of free-
radical mediated injury70.
Excitotoxins
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
receptors85.
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.
***ILLUSTRATION NOT INCLUDED
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
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 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
(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 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
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 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-
insult109,110,111.
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 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.
***ILLUSTRATION NOT INCLUDED
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
tissues123.
Summary
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
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.
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Copyright 1993 by Mike Darwin
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