X-Message-Number: 16178 From: Date: Thu, 3 May 2001 00:21:27 EDT Subject: INTRODUCTION TO ISCHEMIA, Part III GLOBAL ISCHEMIA: A COMPREHENSIVE INTRODUCTION, PART III By Mike Darwin, CEO Kryos, Inc. Part of the last installment was not transmitted to Cryonet. Therefore the entire section on excitotoxicity is being repeated at this time. 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 [95]. In vivo studies have also confirmed a massive release of glutamate and aspartate during both regional and global cerebral ischemia [100]. In regional or focal cerebral ischemic injury, the NMDA receptor remains activated for a long period due to the prolonged interval of poor perfusion in the area at the edges of the infarct (the "penumbra"). However, in complete or global ischemia there is good resumption of blood flow following restoration of circulation with prompt uptake of glutamate and aspartate and rapid inactivation of the NMDA receptors [103]. Another factor limiting the role of the NMDA receptor in mediating injury in global cerebral ischemia may be the rapid and pronounced drop in pH which occurs in global (as opposed to focal) ischemia, since low pH is known to inactivate the NMDA receptor. These reasons are probably why NMDA receptor inhibitors have not proved effective in preventing global cerebral ischemic injury [104]. Recently, attention has turned to non-NMDA receptor antagonists such as inhibitors of the kainate and AMPA receptors [105], [106]. The mechanisms by which excitotoxins cause cell injury is not yet fully understood. It is known that they facilitate calcium entry into neurons [107-109]. However, the excitatory amino acids released during cerebral ischemia are neurotoxic, even in cell culture where the medium is calcium free [96]. In the case of kainate and AMPA receptor activation, the likely mode of injury is sensitization of the CA1 pyramidal cells during ischemia such that when normal signaling is restored at the end of the ischemic insult, and normal intensity input from the Schaffer collaterals is resumed, lethal cell injury results, perhaps from abnormal calcium regulation in the CA1 cells or other metabolic derangements not yet understood. Neutrophil Activation Since the late 1960s, polymorphonuclear leukocytes (PMNLs) and monocytes/macrophages have been implicated as significant causes of pathology in cerebral ischemia. During the last decade there has been a veritable explosion of research documenting the role of PMNLs in reperfusion injury. Most of the initial work done in this area focused on PMNL-mediated reperfusion injury to the myocardium, establishing that PMNL activation and subsequent plugging and degranulation (resulting in release of oxidizing compounds) is responsible for the no-reflow phenomenon following myocardial ischemia [110]. In particular, the work of Engler demonstrated that PMNL activation is responsible for plugging at least 27% of myocardial capillaries and is further responsible for the development of edema and arrhythmias upon reperfusion [111]. To what extent leukocyte plugging occurs in the brain following global cerebral ischemia remains controversial [112-114]. Anderson, et al. have examined the question of how rapidly leukocyte plugging occurs following cerebral ischemia using a bilateral carotid artery plus hypotension model in the dog. They noted no leukocyte plugging after 3 hours of reperfusion following a 40-minute ischemic episode [115]. However, it is clear from a growing body of work that neutrophils are a major mediator of ischemic injury in a variety of organ systems, and that their acute activation is responsible for many of the effects of ischemia observed in the brain and other body tissues, including the loss of capillary integrity and the degradation of ultrastructure upon reperfusion [116]. 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) [117]. Because the concentration of Cl- is more than a thousand times greater than the other halides, the hydrogen peroxide-myeloperoxidase system probably generates Cl- most often in the form of HOCl. HOCl is more commonly known as household bleach, and is capable of damaging a wide range of organic molecules including most of those that make up the structure of the cells and proteinaceous extracellular matrix [118], [119]. As Klebanoff has pointed out, the amounts of HOCl generated by the neutrophil are awesome: 106 neutrophils can generate 2 x 107 mole of HOCl - enough to destroy 150 million E. Coli in a matter of milliseconds [117]. However, the direct destructive effects of HOCl are probably limited in vivo by a variety of mechanisms [120]. Most probably the hypohalous acids act to inflict the majority of injury by interacting with collagenase, elastase, gelatinase, and other proteinases. As is shown in the diagram below, it is now believed that the oxidants released from the neutrophil create a halo of oxidized alpha-1-proteinase inhibitor that allows released elastase (and probably others of the 20 or so known neutrophil-secreted proteolytic enzymes) to begin degrading the extracellular matrix, thus destroying capillary integrity and interfering with tissue metabolism and anabolism [121]. In complete circulatory arrest, it is clear that neutrophil activation with accompanying release of HOCl and activation of elastase is a key factor in initiating the systemic cascade of inflammation/immune response which terminates in delayed multisystem organ failure [122]. The extent to which this pathway is a factor in acute global cerebral ischemic injury in cardiac arrest is not yet clear. Hypoperfusion Following Reperfusion An apparently significant contributor to reperfusion injury is hypoperfusion after restoration of spontaneous circulation. The work of Hossman, et al., [123] and Sterz, et al., [124] 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. Sedimentation A heretofore unappreciated source of reperfusion injury is the sedimentation of the formed elements of the blood in both large and small blood vessels under the influence of gravity. Sedimented red blood cells and leukocytes, particularly in the presence of acidosis, become sticky and adhere to each other. Thus, after 15 minutes of global ischemia the dependent portion of all areas of the vasculature become filled with a hyperviscous sludge of blood cells which is refractory to resuspension when perfusion is reinitiated. Research at Critical Care Research (CCR) in Rancho Cucamonga, CA has shown that this hyperviscous cell sludge is forced into the small caliber dependent vessels when the circulatory system is repressurized at the start of reperfusion. This is one of the reasons why achieving blood washout is so difficult in human cryopatients despite perfusion with in excess of 100 liters of perfusate. When flow is first restarted during an attempt to achieve blood washout in cryopatients after a prolonged ischemic episode the plasma column layering the top of the larger caliber vessels is the path of least resistance and is quickly replaced with perfusate, while the sludge of formed blood elements remains in place. As pressures and flows are increased, packed cells are forced into capillaries. These capillaries are already decreased in diameter (which typically less than that of red blood cell; 7.7 microns) from endothelial and parenchymal cell swelling and do not allow flow of the packed cell sludge. This results in uneven, patchy perfusion and prolonged or permanent sequestration of tissues supplied by these plugged vessels from restoration of flow. This interferes with adequate cryoprotectant agent (CPA) distribution and can lead to severe osmotic damage if vessels are eventually opened to flow from dehydration of adjacent tissue by CPA. Limits To Reversibility: Histological Ultrastructural Change? Understanding the time course and nature of the biochemical, ultrastructural and gross changes which occur as a result of systemic normothermic ischemia, with special attention to the brain (the most vulnerable organ) is essential to determining the realistic limits to resuscitation given known and foreseeable technology. Ischemic changes in cell architecture begin almost as rapidly as ischemic changes in biochemistry. Within seconds of the onset of cerebral ischemia, brain interstitial space almost completely disappears. Loss of interstitial space is a consequence of cell swelling secondary to sodium influx and failure of membrane ionic regulation. There have been several studies of the ultrastructural alterations associated with prolonged global cerebral ischemia. Notable is the work of Kalimo et al., in the cat, [125] as well as Karlsson and Schultz, [126] and Van Nimwegen, et al., [127] in the rat. These investigators describe the following changes in common in these animals' brain ultrastructure after varying periods of global cerebral ischemia (GCI): 1) Changes At 10 Minutes After 10 minutes of GCI, a significant number of cells (but not all) show clumping of nuclear chromatin and a modest increase in electron lucency (probably due to dilution of the cytosol by extracellular fluid). After 30 minutes, further changes include increased cytoplasmic swelling (particularly in the astrocytes), swelling and shape change of the mitochondria, and some loss of mitochondrial matrix density. Microtubules disappear and there is detachment of the ribosomes from the cisternae of the endoplasmic reticulum. There is also disassociation of the polyribosomes, and single ribosomes lose their compact structure with associated failure of protein synthesis. Of note is the stability of the lysosomes over this time course [127]. 2) Changes At 60 Minutes After 60 minutes of GCI, the above changes have become more pronounced with more conspicuous swelling of the ER cisternae. The mitochondria begin to show slight inner matrix swelling and occasional flocculent densities (probably due to accumulated calcium). 3) Changes At 120 Minutes After 120 minutes of GCI, the changes discussed above are more pronounced and a larger number of mitochondria exhibit the presence of flocculent densities evidencing calcium overload, which is currently considered irreversible. Published electron micrographs reveal intact lysosomes and seem to confirm other studies which indicate that lysosomal rupture and subsequent catastrophic autolysis is not a feature of early (1-4 hours) ischemic injury [128]. From a feasibility of reversal standpoint (i.e., likely contemporary periods of normothermic cardiac arrest after which successful resuscitation can be achieved) 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. The extent to which these changes are irreversible remains unknown. However, by 120 minutes of noromothermic circulatory arrest it is likely that micro and macrovascular clotting may be underway effectively preventing restoration of circulation to the organism without very technologically demanding and dramatic interventions such as low flow CPB with asanguineous solutions containing thrombolytics, sub-micron PFC-emulsion-based oxygen delivery capability, and complex mixtures of drugs and metabolites to restore homeostasis upon thrombolysis. Also required would be reversal of endothelial and parenchymal edema, and restoration of normothermic metabolism. For the foreseeable future, in the absence of pre-arrest intervention, a period of ~30 minutes of normothermic cardiac arrest would seem the outer limit achievable with current technology [86]. At least one study of post-mortem ultrastructural degradation has been conducted on a small number of human subjects [129]. The histological and ultrastructural changes observed in humans with 25 to 85 minutes of GCI, without extensive pre-mortem brain trauma or pre-mortem cerebral no-reflow of prolonged duration, closely parallel those observed in animal models of GCI. The changes typically seen are 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. End of Part III [References Available Upon Request] Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=16178