X-Message-Number: 16165
From: 
Date: Tue, 1 May 2001 01:30:27 EDT
Subject: INTRODUCTION TO ISCHEMIA, Part II

GLOBAL ISCHEMIA: A COMPREHENSIVE INTRODUCTION, PART II

By Mike Darwin, CEO Kryos, Inc.

Introduction  

As noted in above, for 325,000 persons in the United States who will 
experience sudden cardiac death (SCD) in the coming year, there will be no 
possibility of rescue.  In such cases, cardiac arrest occurs without warning, 
frequently in situations not conducive to activation of the Emergency Medical 
System (EMS). In post-MI patients the incidence of SCD within the first year 
following infarct is 14% [39]. 

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? 

A review of the literature in experimental cerebral resuscitation and the 
pathophysiology of cerebral ischemia (CI) suggests a combination of 
therapeutic interventions may prove invaluable.

While a wide range of post-insult interventions are currently being 
investigated in animal and clinical trials, and 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.

Normal cerebral blood flow (CBF) in the human is typically in the range of 
45-50 ml/min/100g of brain tissue as long as mean arterial pressure (MAP) is 
in the range of 60-130 mm Hg [40]. When CBF falls below 20 to 30 
ml/min/100g/brain tissue, marked disturbances in brain metabolism begin to 
occur, such as water and electrolyte shifts, with regional areas of the 
cerebral cortex experiencing failed perfusion [40]. At blood flow rates below 
10 ml/min/100g/brain tissue, sudden depolarization of the neurons occurs with 
rapid loss of cellular ionic homeostasis [41].

The Mean Arterial Pressure (MAP) necessary for cerebral viability following 
extended resuscitation efforts in dogs has been found to be above 40 mm Hg. 
It has been speculated that a minimum MAP of 45 to 50 mm Hg is required to 
preserve cerebral viability in man [42].

Unfortunately, as is now well documented, conventional closed-chest CPR is 
generally incapable of consistently delivering MAPs >30 mm Hg in man [43]. 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 [42]. 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 [44].

As the foregoing analysis makes clear most SCD patients will suffer 
significant periods of cerebral anoxia, ischemia, or hypoperfusion resulting 
in currently irreversible brain damage. This effectively excludes these 
patients from receiving more effective cardiopulmonary support (such as open 
chest CPR [OCCCPR] [45], or the use of extracorporeal circulation utilizing a 
membrane oxygenator [46]) as a bridge to definitive repair of their defective 
coronary circulation.

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 [47], acidosis due to anaerobic generation of lactate 
[48] , and no reflow due to swelling of astrocytes with compression of brain 
capillaries [49]. Subsequent research has shown the problem to be far more 
complex than was previously thought, involving the action and interaction of 
many factors [50].

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 phosphates 
have been exhausted (ATP depletion) [51] and profound disturbances in cell 
electrolyte balance start to occur: potassium begins to leak rapidly from the 
neurons and glial cells, and sodium and calcium begin to enter the cells 
[52]. Sodium influx, particularly in the astrocytes results in a marked 
increase in cellular water content, [41]. Concurrent with the rapid shift in 
ions there is marked leakage of ascorbate from the neurons and glial cells. 
Ascorbate, in the presence of free iron (see discussion under Free Radicals 
below) is one of the most potent pro-oxidants known.

Calcium

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 
[53], 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 [53], 3) sequestration of 
intracellular calcium in the endoplasmic reticulum by an ATP-driven process 
[54], and 4) accumulation of intracellular calcium by oxidation-dependent 
calcium sequestration inside the mitochondria [55-57].

The loss of cellular high-energy compounds during ischemia causes the loss of 
the Na+/K+ gradient which 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 [58]. Mitochondrial sequestration, the 
remaining mechanism, causes overloading of the mitochondria with calcium and 
diminishes its capacity for oxidative phosphorylation. Elevated intracellular 
Ca++ activates membrane phospholipases and protein kinases. A consequence of 
phospholipase activation is the release 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. 
During the reperfusion phase after cerebral ischemia, calcium accumulates in 
mitochondria, and a burst of free radical formation occurs, conditions that 
favor the activation of the mitochondrial permeability transition pore. As a 
result of this injury to the mitochondria cytochrome C (cyto C) is released 
resulting in activation of at least three caspases. The caspases are one of 
the proteases in the cyto-c pathway for triggering apoptosis. In vivo models 
of global and regional cerebral ischemia have demonstrated release of large 
amounts of caspase 3 which is a known apoptosis-inducing protease. More 
recently, in vitro studies have demonstrated that other caspases are also 
being released. Attention has focused on caspase-9 which is released from 
isolated mitochondria on treatment with calcium. 

Similarly, in neuronal cell culture models, apoptosis-inducing agents trigger 
translocation of caspase-9 from mitochondria to the nucleus. Of greater 
relevance has been the very recent demonstration in vivo, in a model of 
transient global cerebral ischemia, that caspase-9 is released from the 
mitochondria. This study demonstrated accumulation of caspase-9 in the 
neuronal nuclei of hippocampal and other vulnerable neurons exhibiting early 
post-ischemic changes preceding apoptosis. Loss of mitochondrial barrier 
function during neuronal damage from ischemia and subsequent calcium influx, 
may therefore, play an important role in liberating certain caspases [59-61]. 

    The production of AA as a result of FFA release causes a biochemical 
cascade ending with the production of thromboxane and leukotrienes [62, 
63],and the release of platelet activating factor [64]. These compounds are 
profound tissue irritants [65], which can cause platelet aggregation [66], 
clotting, vasospasm, and edema with resultant further compromise to 
restoration of adequate cerebral perfusion upon restoration of blood flow.

Free Radicals

    During ischemia, the hydrolysis of ATP to AMP leads to an accumulation of 
hypoxanthine [67]. 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 [68, 69]. Even more damaging free radicals could 
conceivably be produced by the metal catalyzed Haber-Weiss reaction as 
follows [67, 68, 70-73]:

    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 [74], [75]. 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 [76] and low 
molecular weight species of iron [77]. Paradoxically, the damaging effects 
resulting from the release of free iron from cytochromes, hemoglobin and 
possibly other iron-containing proteins during ischemia (iron delocalization) 
is likely greatly exacerbated by the endogenous antioxidant and free radical 
scavenger ascorbic acid (vitamin C). Ascorbate (reduced vitamin C) is an 
important enzyme cofactor, neuromodulator, and antioxidant that is stored at 
millimolar concentrations in the cytosol of cerebral astrocytes. 
Approximately 80% of the body stores of ascorbate are in the brain, and 
ascorbate is released in large quantities during ischemia [78]. Despite its 
status as an antioxidant, in the presence of free iron, ascorbic acid is one 
of the most potent facilitators of the Fenton-Haber-Weiss reaction known, and 
its solo administration during reperfusion following ischemia is associated 
with increased neuroinjury [79], [80].

During reperfusion and re-oxygenation, significantly increased levels of 
multiple free-radical species that degrade cellular and capillary membranes 
have been postulated: O2-, OH-, and free lipid radicals (FLRs).  O2- may be 
formed by the previously described actions of XO and/or by release from 
neutrophils that have been activated by leukotrienes (see discussion below of 
the role of leukocytes in ischemia-reperfusion injury). Below is a table 
listing the free radicals currently known or suspected to be implicated in 
cerebral and systemic ischemia-reperfusion injury.

MULTIPLE REACTIVE OXYGEN SPECIES

Species       Symbol      Source        Target
o Nitric Oxide    l NO   NOS             Receptor, l O2-
o Superoxide    l O2-        PMN, Mito.   Ferritin, SOD
o Hydroxyl R     l OH-        H202/Fe2+         DNA, organics
o O2 Triplet        l OO l       Air                 Organic l R     
o O2 Singlet       :OO         Mitochon.     Organic R=R
o Peroxynitrite  ONOO-     NOl +l O2-       DNA 
o H. Peroxide    HO:OH     SOD              GSH, Fe2+ 

Figure 1: Multiple free radical species known or suspected to mediate 
ischemia reperfusion injury. (PLEASE NOTE: THE FORMATTING OF THE ABOVE TABLE 
MAY NOT SURVIVE E-MAIL TRANSMISSION/)

PARP and NFkB

Recently, free radicals have been demonstrated in vivo and in vitro to cause 
damage to both mitochondrial and nuclear DNA resulting in activation of 
poly-(ADP-ribose)-polymerase (PARP) which is part of the cell's DNA repair 
system [81-83]. Activation of PARP results in mitochondrial "futile cycles" 
with consequent consumption of up to 30% to 60% of the cell's available 
metabolic capacity via massive depletion of NAD and ATP resulting in direct 
cell death from failure of high energy metabolism [84].

PARP activation triggers apoptosis in experimental models of sepsis and 
ischemia [59], [85] and increases mitochondrial metabolism during injury 
resulting in amplified oxidative stress and generation of additional damaging 
free radicals and cytotoxic wastes. Free radicals with or without PARP 
activation may also be primary initiator of nuclear factor kappa B (NFk B) 
production (Gilad, Wong et al. 1998; Hickenbottom, Grotta et al. 1999; Szabo 
and Billiar 1999).

NFk B activates genes for production of TNFa, resulting in increased 
collagenase activity with resulting capillary leak. NFk B is also thought to 
activate lipoprotein lipase, induce fever, increase tissue catabolism, 
inducible nitric oxide synthase (iNOS) and consequently (NO), and cause 
peroxynitrite formation. Other deleterious effects of NFk B activation are 
production of cyclooxygenase II (COX-2) with concurrent production of 
inflammatory prostaglandins, increase [I-CAM] and accompanying up-regulation 
of neutrophil production, adhesion and degranulation, and increased renal 
tissue factor expression with resultant activation of the plasma enzyme 
coagulation system

Re-oxygenation also restores ATP levels, which may in turn allow active 
uptake of calcium by the mitochondria, resulting in massive calcium overload 
and destruction of the mitochondria [86].

Mitochondrial Dysfunction

    Calcium loading and free-radical generation are no doubt major 
contributors to the mitochondrial ultrastructural changes that are known to 
occur following cerebral ischemia.  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 [87, 
88].  Of these alterations, the accumulation of long-chain acyl-CoA is 
perhaps most significant, since intra-mitochondrial accumulation of 
long-chain acyl-CoA is known to be deleterious to many different 
mitochondrial enzyme systems [89].

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 [51], [90].  It has been shown, for instance, that lactate levels 
above a threshold of 18 - 25  M/g result in irreversible neuronal injury [70, 
91, 92].

    Decrease in pH as a consequence of lactic acidosis has been shown to 
injure and inactivate mitochondria.  Lactic acid consumption of NADH (which 
is needed for ATP synthesis) may also interfere with adequate recovery of ATP 
levels post ischemically [93]. Lactic acid can also increase iron 
decompartmentalization, thus increasing the amount of free-radical mediated 
injury [94].

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 injury [95-97]. 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) glutamate receptors [98].

    Initially there was much optimism that blockade of the NMDA receptor 
would provide protection against delayed neuronal death following global 
cerebral ischemia [99], [100], [101]. The use of NMDA receptor blocking drugs 
has shown significant promise in ameliorating focal cerebral ischemic injury. 
A number of studies have demonstrated a marked reduction in the severity of 
ischemic injury in focal areas (particularly the poorly perfused "penumbra" 
surrounding the no-flow area) as a result of treatment with 
glutamate-blocking drugs such a dextrorophan [102] or the experimental 
anticonvulsant MK-801 [101].  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 [

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