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 [ Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=16165