Injury During Ischemia

In the ischemic phase of I/R injury, the predominant mechanisms of injury result from tissue hypoxia or anoxia and stasis in the microcirculation (Fig. 7-1). The degree to which tissue from various organs is capable of tolerating ischemia varies widely and depends on each tissue’s baseline metabolic demand. Ischemia of human skeletal muscle under normothermic conditions is tolerated for more than 2 hours, whereas histologic evidence of ischemic injury develops in the jejunum after only 30 minutes of ischemia.³

During ischemia, mitochondria deprived of O₂ can no longer produce adenosine triphospate (ATP) by oxidative phosphorylation; consequently, cellular (ATP) falls rapidly with a concomitant increase in adenosine diphosphate (ADP), adenosine mononphosphate (AMP), and Pi. Glycolysis is stimulated, but is unable to produce sufficient ATP to meet the needs of the cell. The intracellular pH decreases with accumulation of lactic acid, progressively inhibiting glycolysis and activating the Na⁺/H⁺ antiporter that pumps H⁺ out of the cell. Because of ATP deficiency, the entering Na⁺ ions are not pumped out of the cell by Na⁺/K⁺ ATPase. High intracellular Na⁺ inhibits the Na⁺/Ca²⁺ antiporter, producing high intracellular Ca²⁺, which, in turn, activates degradative enzymes, including phospholipase A2, and proteases, such as calpains. In addition, ATP deficiency impairs ATP-dependent repair processes.⁴ Consequently, prolonged ischemia will lead to cell membrane damage and necrotic cell death.

As ischemia progresses, reactive oxygen species (ROS) are produced by and accumulate within the mitochondria.^4,5 Increased mitochondrial matrix ROS together with increased cytosolic Ca²⁺, elevated Pi, and depletion of adenine nucleotides are most of the conditions needed for opening of the mitochondrial membrane permeability transition pore (MPTP), which causes necrotic cell death.^6,7 The data indicate that the increase of mitochondrial ROS during ischemia primes the mitochondrial MPTP for opening, and that the magnitude of ROS damage to the mitochondria is the most important factor that determines the duration of opening of the MPTP, and therefore, the extent of tissue damage.⁴ Pore opening, however, is inhibited by the low intracellular pH during ischemia, but occurs during reperfusion when the pH increases, as is discussed in “Injury During Reperfusion” section in this chapter.

Tissue hypoxia leads to mobilization of neutrophils into the interstitium, where they have both beneficial and deleterious effects on tissue during reperfusion.⁸ Migration of neutrophils and macrophages to sites of inflammation is dependent on hypoxia-adaptive pathways.^9,10 Activated neutrophils release the soluble mediators glutamate¹¹ and adenine nucleotides (in the form of ATP or AMP)^12,13 during ischemia that are converted to adenosine at the vascular endothelial surface. Adenosine protects the function of the microvascular endothelial barrier by re-establishing endothelial cell-cell contact after neutrophil transmigration. Transcellular metabolism (neutrophils provide ATP as a substrate for enzymes located on the endothelial membrane) and signaling are enhanced by hypoxia-induced transcriptional increases in functional endothelial surface apyrase (CD39), 5′-ectonucleotidase (CD73), and adenosine receptors (AdoRA_2B).^13,14 Polymorphonuclear neutrophils have a deleterious effect on local tissue by releasing factors that can disrupt the endothelial barrier. Activation of neutrophils by β₂ integrins sti­mulates neutrophils to release soluble factors that induce endothelial cytoskeletal rearrangement, gap formation, and increased permeability. One neutrophil-derived permeabilizing factor is heparin-binding protein (HBP), also known as azurocidin or CAP37. HBP induces Ca²⁺-dependent cytoskeletal changes in endothelial cells and triggers macromolecular leakage in vivo.¹⁵

Injury During Reperfusion

Reperfusion injury represents the complex response to tissue injury when blood flow is restored after ischemia (Fig. 7-2). There are metabolic, thrombotic, and inflammatory components of reperfusion injury. The degree to which reperfusion either restores tissue integrity or exacerbates ischemic injury depends primarily on the duration of the ischemia. It is paradoxical that moderate ischemia followed by reperfusion may cause more fulminant postischemic tissue injury than seen with ischemia alone. However, without reperfusion, the loss of function in the brain, gut, heart, or limb may have a more catastrophic outcome than if perfusion is not restored.

Reperfusion is characterized by a burst of ROS production (primarily by the mitochondrial electron transport chain), increased Ca²⁺ uptake into the mitochondrial matrix, and a shift of the intracellular pH from acidic toward neutral.⁶ Mitochondrial oxidative damage leading to programmed cell death is the major cause of tissue loss produced by reperfusion.¹⁶ The two forms of programmed cell death during reperfusion are apoptosis and necrosis.

Increased ROS generated during both ischemia and reperfusion can initiate apoptosis and necrosis, cell death programs mediated through the mitochondria. The BCL-2 family of proteins is central to regulating mitochondrial apoptosis. Antiapoptotic members, including BCL-2 and BCL-XL, act by blocking proapoptotic members, including BAX and BAK. The latter initiate apoptosis by increasing the permeability of the outer mitochondrial membrane, causing the release of proapoptotic proteins, such as cytochrome c.¹⁷ BH3-only proteins, proapoptotic BCL-2 family members, can initiate apoptosis by directly activating BAX and BAK or by blocking BCL-2 and BCL-XL.¹⁷ ROS initiate mitochondrial apoptosis by activating BH3-only proteins or by triggering opening of the mitochondrial MPTP, causing release of cytochrome c.⁷ Alternatively, apoptosis may occur via ROS-dependent lipid peroxidation of cardiolipin, a mitochondria-specific phospholipid of the inner membrane, which binds strongly to cytochrome c. The latter dissociates from peroxidized cardiolipin and is released from the inner membrane.¹⁸ The evidence, however, suggests that ROS-dependent necrosis, rather than apoptosis, is the main mechanism of cell death caused by I/R injury.^6,7,19

Programmed necrotic cell death may be mediated by signaling via a tumor necrosis factor (TNF) death receptor^20–22 or by direct opening of the mitochondrial MPTP.^16,17 Both processes require calcium and oxidative stress.^16,17 TNF receptor 1 (TNFR1) signaling for necrotic cell death begins with a conformational change caused by binding of TNF to the receptor. This conformational change promotes binding of cytosolic TNF receptor-associated death domain (TRADD) to a cytosolic domain of the receptor, initiating formation of a complex consisting of receptor-interacting protein 1 (RIP1) kinase, TNF receptor-associated factor 2/5 (TRAF2/5), cellular inhibitor of apoptosis 1 (cIAP1) and cellular inhibitor of apoptosis 2 (cIAP2). The TNFR1 complex is internalized and produces secondary cytosolic complexes, including the necrosome. The latter comprises Fas-associated death domain (FADD) protein, inactivated caspase 8, RIP1, and receptor-interacting protein 3 (RIP3). Activation of necrotic signaling requires phosphorylation of RIP1 and RIP3 and is critically dependent on ROS production by the mitochondrial electron transport system.²¹ The ultimate effector in this pathway is not yet established; however, the present data suggest that signaling leads to opening of the mitochondrial MPTP.²⁰

Direct opening of the mitochondrial MPTP is central to tissue injury caused by ischemia and reperfusion.^16,17,19 The mitochondrial MPTP is a nonselective protein channel spanning the inner and outer mitochondrial membranes at points of contact. When open, the pore permits free passage of water and solutes less than 1.5 kDa, causing osmotic damage to the mitochondria and uncoupling mitochondrial oxidative phosphorylation, the major source of cellular ATP, by dissipation of the pH gradient and the electrical potential across the inner mitochondrial membrane.^4,6 Mitochondrial damage and collapse of oxidative phosphorylation promote disruption of metabolism and ionic homeostasis initiated before pore opening during ischemia, and thereby, promote and expand necrotic cell death. The composition of mitochondrial MPTP is not yet solved, but the voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and mitochondrial phosphate carrier (PiC) appear to be constituents.⁶ Cyclophilin D (CyP-D), a prolyl isomerase in the mitochondrial matrix, is an essential component of the mitochondrial MPTP and the central regulator of pore opening.¹⁶ Pore opening depends on mitochondrial calcium concentration (Ca²⁺ m) but is relatively insensitive to Ca²⁺ m per se, occurring at very high Ca²⁺ m. The sensitivity of pore opening to Ca²⁺ m is greatly enhanced by oxidative stress caused by increased mitochondrial ROS production during ischemia and a surge of mitochondrial ROS production during reperfusion.⁶ Oxidative stress promotes binding of CyP-D to an ANT/PiC dimer, facilitating a Ca²⁺-dependent conformational change in these proteins and opening of the mitochondrial MPTP. The evidence supports activation of CyP-D via binding of p53, which accumulates in the mitochondrial matrix in response to oxidative stress.¹⁶ Low intracellular pH that occurs during ischemia inhibits pore opening; however, during reperfusion, when blood flow is restored, the intracellular pH increases, and the mitochondrial MPTP opens.⁶

Reperfusion of ischemic tissue produces a burst of ROS production by the mitochondrial ETC and by parenchymal and endothelial cell reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and xanthine oxidase, neutrophils and macrophages, and necrotic cell death with opening of the MPTP.²³ These processes lead to release of normally sequestered cellular proteins and protein fragments in addition to extracellular and cell surface accumulation of oxidatively modified proteins and lipids. Some of these structures have molecular configurations recognized by Toll-like receptors (TLR) of the innate immune system as “danger-associated molecular patterns” (DAMPS),²⁴ and a growing body of work supports TLR-mediated inflammation as central to I/R injury.²⁵ Reperfusion-associated DAMPS include heat shock protein 60 (HSP60), a ligand for TLR4; high-mobility group protein B1 (HMGB1), a ligand for TLR2, 4, and 9; low-molecular-weight hyaluronic acid, a ligand for TLR2 and 4; the extra domain A of fibronectin (EDA), a ligand for TLR2 and TLR4; and cardiac myosin, a ligand for TLR2 and 8.²⁶ Ligand binding to TLRs present on parenchymal cells, endothelial cells, and circulating leukocytes activates TLR signaling pathways that lead to apoptosis or to nuclear factor kappa B (NF-κB) signaling.²⁶ Inflammation induced by TLR-mediated NF-κB signaling is a significant contributor to I/R injury.²⁵ NF-κB is a transcription factor present in the cytosol in an inactive complex with inhibitor of NF-κB (I-κB). Signaling via TLRs leads to removal and proteolysis of I-κB, permitting NF-κB to translocate to the nucleus, where it initiates transcription of proinflammatory genes, including those that code for TNF-α, interleukin-1β (IL-1β), and IL-6, as well as intercellular adhesion molecule-1 (ICAM-1), all of which recruit neutrophils and macrophages to the damaged tissue.^23,25,27 In addition, NF-κB signaling may be initiated by binding of TNF to the TNFR1 receptor.²¹

Activation of innate immunity in response to DAMPS, during reperfusion, is not limited to their recognition by TLRs, but also occurs in response to their recognition by natural antibodies.²⁷ These antibodies belong predominantly to the immunoglobulin-M (IgM) class, are secreted by B-1 cells (a specialized subset of B-cells) and are germline encoded.²⁴ Many of these antibodies recognize epitopes formed by oxidative modification of lipids and proteins (e.g., during reperfusion). Some recognize normally sequestered self-antigen, for example, non-muscle myosin heavy chain II released as a consequence of cell injury during both ischemia and reperfusion.²⁸ Reperfusion elicits an inflammatory response dependent on activation of complement by natural IgM antibodies.²⁸ Binding of IgM natural antibodies to target molecules on the cell surface or in the extracellular environment activates the complement system, causing production of cleavage products, including complement component C3b, which tags cells and other targets for removal by macrophages, and C5a, a fragment chemotactic for neutrophils.²⁷ Alternatively, the complement system may be activated in the absence of antibody by mannose-binding lectin (MBL) bound to cell surface carbohydrates.²⁹ Complement activation, detected as tissue-bound C3b and MBL, occurs during ischemia and increases during reperfusion.³⁰

A variety of inflammatory mediators are delivered to reperfused tissue by blood-borne cells (macrophages, lymphocytes, neutrophils, mast cells, platelets).^31,32 Noncellular elements, such as the complement system, ROS, nitric oxide (NO), and proinflammatory and anti-inflammatory cytokines are also believed to modulate the complex scenario of reperfusion injury. The presence of O₂, washout of lactatic acid, development of tissue edema, and influx of numerous cell types during reperfusion produce a complex environment in which numerous mediators contribute to inflammation and tissue injury. The cellular and molecular components of the I/R response will be discussed individually.

Mitochondrial Injury

The mitochondria are a dynamically growing and shrinking network of double-membrane organelles that comprise the primary energy-generating system of eukaryotic cells. Energy is generated by coupling ATP production with oxidative transfer of electrons from reduced substrates to molecular O₂ (i.e., oxidative phosphorylation).⁵⁵ This process of energy conversion is mediated by the mitochondrial electron transport chain that consists of five multiunit protein complexes tightly configured within the mitochondrial inner membrane.⁵⁶ The largest of these is Complex I, which consists of 46 protein subunits and 9 redox centers, 1 of which is flavin mononucleotide (FMN) located at the terminus of the hydrophilic arm extending into the mitochondrial matrix. High-energy electrons from reduced nicotinamide adenine dinucleotide (NADH) enter the ETC by reducing the FMN of Complex I. The electrons pass through Complex I to its coenzyme Q (CoQ) reductase site located in the inner membrane and where CoQ is reduced. The latter shuttles the electrons to Complex III, which couples oxidation of reduced CoQ with reduction of (passage of the electrons to) cytochrome c located in the intermembrane space. Finally, the electrons are passed to Complex IV, which couples oxidation of cytochrome c with reduction of molecular O₂ to water. Complex II can also shuttle electrons to CoQ by coupling conversion of succinate to fumarate with reduction of flavin adenine dinucleotide (FAD), which is present at the mitochondrial matrix side of the complex. Importantly, passage of two electrons through Complexes I, III, and IV is coupled with transfer of protons from the mitochondrial matrix across the inner membrane to the intermembrane space (i.e., a total of eight protons per electron pair).⁵⁶ This process generates a pH gradient and a membrane potential used by Complex V to generate ATP from ADP and Pi.

Approximately 90% of inhaled O₂ is consumed by mitochondria and reduced to water to produce energy. Under optimal physiologic conditions, 2% to 4% of the O₂ consumed by mitochondria can capture single electrons that leak along the complexes of the electron transport chain generating the ROS superoxide anion (O^•−).⁵⁷ Oxidative stress caused by excess accumulation of ROS is held in check by mitochondrial superoxide dismutase, which converts it to H₂O₂ (another ROS) and catalase, which converts the latter to H₂O and O₂.⁵⁷ During tissue ischemia, when O₂ tension in the mitochondria is reduced (hypoxia), ROS generation paradoxically is increased, overwhelming oxidative defense enzymes and causing an excessive accumulation of ROS (oxidative stress).⁵ This happens because under tissue hypoxia with reduced availability of O₂ as the final electron acceptor of the ETC, the NADH/NAD+ ratio in the mitochondrial matrix increases, thereby saturating the FMN of Complex I with electrons. Saturated FMN transfers electrons to available O₂ in the matrix, producing O^•−.⁵⁷ This FMN-dependent process is amplified by (1) the accumulation of reduced CoQ generated by both Complex I and II under the condition of low electron flow, and (2) the presence of a pH gradient across the inner membrane. Under these conditions, present during ischemia, electrons back up through Complex I to its FMN, where electrons are passed to O₂, generating O^•−.⁵⁷ With the onset of reperfusion, O₂ returns to meet the conditions of increased NADH/NAD+, accumulated reduced CoQ, electron-saturated FMN, and an incapacitated electron transport chain. The surge of O₂ thus provides a sink for flow of electrons from reduced FMN leading to a burst of O^•−. The duration of this burst, and therefore, the extent of acute reperfusion damage are dependent on the duration and extent of ROS accumulation in the preceding ischemic phase of I/R. The evidence supports Complex I as the main source of this reperfusion burst of ROS generation.⁴

Complement

The complement system has long been recognized as an important mediator in innate immune defense and inflam­mation.⁵⁸ Complement can be activated by any of three pathways—the antibody-dependent classical pathway, the alternative pathway, or the MBL-associated serine protease pathway. The classical pathway is initiated when IgM or IgG antigen-antibody complexes bind to C1, the first component of complement. Activation of the alternative pathway is triggered by microbial surfaces and complex polysaccharides, and results in the generation of C3. The lectin pathway is initiated by binding of the MBL protein to mannose and glucosamine residues on bacterial cell walls. Activation of these proteins results in cleavage of complement factors 4 and 2, followed by subsequent activation of C3.

There is considerable evidence to support a role of the complement system in hind limb I/R injury. In a model of rodent hind limb I/R, there was direct correlation between increased complement deposition, tissue injury, and increased glycolysis.⁵⁹ Complement receptor 2–deficient (CR2^−/−) mice exhibit deficient IgM secretion and are protected against I/R injury by defective B cells.⁶⁰ IgM-secreting B-cells clones were studied from CR2 knockout mice and wild-type mice. Only one clone was found to restore injury to previously protected mice, thus suggesting that a single clone of self-reactive IgM can initiate complement-dependent I/R injury.⁶¹ In a follow-up study, peptides that bound the IgM clone were found to block injury in wild-type mice, a novel observation providing a potential therapeutic venue.⁶² Complement inhibition has been demonstrated to improve skeletal muscle contractility after I/R.⁶³ C5-deficient mice have been shown to exhibit decreased lung vascular permeability, lung myeloperoxidase levels, and serum alanine aminotransferase levels, along with less skeletal muscle injury after reperfusion.⁶⁴ Because the complement system is well described, and numerous inhibitors are available for each pathway, a beneficial role for these inhibitors in the brain, intestine, liver, and myocardium has been described.⁵⁸

Nitric Oxide

NO is known to regulate vascular tone and to have potent anti-inflammatory and antithrombotic properties at the endothelial surface. It is also known to have concentration-dependent toxic effects on tissue and organ systems.^65,66 The primary physiologic effect of NO results from stimulation of the activity of guanylate cyclase, accumulation of intracellular cyclic guanosine monophosphate, and a consequent decrease in intracellular calcium that results in relaxation of smooth muscle, decreased cardiac contractility, and reduced platelet and inflammatory cell activation. NO is produced from L-arginine by oxidation of one or two equivalent guanido nitrogens by NO synthase (NOS) in a reaction that requires O₂, and the cofactors NADPH and tetrahydrobiopterin. There are at least three distinct genes for NOS; neuronal NOS (nNOS or NOS-1) and endothelial NOS (eNOS or NOS-3) are dependent on micromolar concentrations of calcium for activity and are constitutively expressed. The inducible isoform, iNOS (or NOS-2), is calcium independent and widely expressed in a variety of cells after exposure to specific stimuli. iNOS is often referred to as the high-output source of NO because it can be strongly induced by proinflammatory stimuli. However, the enzyme does not produce NO at a rate substantially greater than does eNOS or nNOS; rather, more of the protein can be transiently induced and activated at normal levels of calcium.^67,68

One of the first observations implicating NO as a marker of ischemic injury was made by Beckman.⁶⁵ His laboratory went on to propose that the production of NO and O₂ radicals by neurons when the ischemic or hypoxic brain is reperfused might contribute to cerebral injury. Ischemia is believed to depolarize neuronal membranes and cause synaptic discharge of the excitatory neurotransmitter glutamate, which, in turn, opens the voltage-dependent, N-methyl-d-aspartate–specific glutamate receptor/ionophore and allows calcium to accumulate in the neuron. Calcium then activates an O₂-dependent nNOS that oxidizes arginine to produce NO when O₂ is readmitted to the brain by reperfusion. NO reacts with the O₂ radical superoxide (O^•−), also produced by reperfusion, to form peroxynitrite (ONOO⁻). ONOO⁻ has been shown to interact directly with proteins (i.e., the mitochondrial electron transport chain, prostaglandin synthase) and amino acids (oxidizes critical cysteine residues, leading to deactivation of proteins), and triggers membrane lipid peroxidation, thereby altering membrane permeability.⁶⁹ ONOO⁻ is a powerful oxidant capable of nitrating phenolic moieties, such as tyrosine or tyrosine residues in proteins, and increases after traumatic brain injury.⁷⁰ This powerful molecule also activates mitochondrial-dependent apoptosis, poly (ADP-ribose) polymerase (PARP),⁶⁹ and NF-κB.⁷¹ Alternatively, some in vitro studies have shown that ONOO⁻ may inhibit the generation of NF-κB.^72,73 ONOO⁻ is believed to diffuse for several micrometers before decomposing to form the powerful and cytotoxic oxidants hydroxyl radical and nitrogen dioxide. Beckman’s hypothesis is consistent with available evidence on the protective action of glutamate antagonists and O₂ radical scavengers in limiting cerebral infarction after focal ischemia.⁶⁶ Furthermore, ONOO⁻ decomposition catalysts are known to limit reperfusion injury in a variety of organ systems.⁷⁴

There are also anti-inflammatory effects of NO that deserve mention. NO donors have been shown to ameliorate tissue injury in in vivo and ex vivo models of intestinal, neuronal, and hepatic I/R.^75–77 NO is known to inhibit activation of NF-κB,^78,79 which regulates a wide variety of proinflammatory cytokines and adhesion molecules. NO alone is an important antiatherosclerotic autacoid with antiaggregatory effects on platelets and with antioxidant, anti-inflammatory, and antiproliferative effects on the vasculature.⁸⁰ NO inhibits vascular permeability in the postischemic intestinal microcirculation, probably by preventing endothelial cell contraction and gap formation.⁸¹ Endogenous NO also inhibits neutrophil adhesion to endothelium.⁸² Finally, NO regulates the activation of platelets by decreasing aggregation and adhesion responses.⁸³ The duration of ischemia, magnitude of the injury, and specific organ bed will determine whether NO has a protective or deleterious effect. In contrast to the deleterious effects of NO when synthesized in large amounts by inflammatory cells via iNOS, the protective effects of NO appear to be related primarily to endogenous sources of constitutively synthesized NO generated by eNOS or nNOS or exogenous pharmacologic doses of NO donors.⁸⁴

Endothelin

Endothelin (ET), a 21-amino acid peptide with potent vasoconstrictor properties,⁸⁵ is produced mainly in endothelial cells, yet it is present throughout the entire cardiovascular system. Shear stress, growth factors, vasoactive hormones, and I/R are major stimuli for its release. Three distinct human ET-related genes have been cloned: “classic” ET-1, ET-2, and ET-3.⁸⁶ ET-1 is the most predominant isoform in blood vessels and is synthesized by endothelial cells. ET-1 is produced as a precursor peptide that is cleaved by a converting enzyme into its active form. ET plays a major role in the regulation of vascular tone through its potent vasoconstrictive actions. ET acts on two different types of G-protein–coupled receptors: ET-A and ET-B. ET-1, the most potent vasoconstrictor, mediates its effects mainly through ET-A receptors present on vascular smooth muscle cells.⁸⁷ ET-B receptors mediate both vasodilatory effects of ET through prostacyclin and NO (ET-B₁) and vasoconstrictive effects on vascular smooth muscle (ET-B₂).^88,89

Increased production, upregulation of its receptors, enhanced sensitivity, and potentiation of the action of serotonin and norepinephrine all potentiate ET’s actions during I/R.^90,91 Bosentan, a mixed ET-A/ET-B endothelin receptor antagonist, was shown to exert a marked tissue protective effect, as assessed by histologic evaluation in a rat model of myocardial I/R. This effect was probably the result of antioxidant protection derived from preservation of levels of the antioxidant enzymes superoxide dismutase and catalase in the treated animals.⁹² McMurdo et al⁹³ showed that the vasoconstrictive effects of ET-1 in the coronary vasculature of the rabbit were primarily mediated by the ET-A receptor. However, FR139317, an ET-A receptor antagonist, failed to decrease infarct size after the induction of ischemia and reperfusion in that same model.⁹³ A more contemporary assessment of the action of BQ-123, a selective ET-A receptor antagonist, revealed decreased myocardial levels of lipid peroxidation; an increase in NO, glutathione, catalase, and superoxide dismutase activity; and a decrease in the ratio of infarcted area to area at risk compared with I/R alone.⁹⁴ Further evidence has proved that ET-A antagonism is useful in I/R. The ET-A antagonists ABT-627, S-0139, BSF208075, and LU135252 all show significant tissue protection in models of I/R.^95–98

Adenosine

Adenosine is an endogenous nucleoside produced in part by degradation of ATP. Formation of adenosine follows the metabolism of ATP into its by-products (ADP, AMP, and cyclic AMP [cAMP]). These by-products increase during ischemia partly as a result of the inability of mitochondria to rephosphorylate ATP.⁹⁹ Depletion of the adenosine pool occurs during reperfusion because of washout and conversion of adenosine first to inosine and then to xanthine through the action of xanthine oxidase.¹⁰⁰ Adenosine acts as a hormone and mediates its effects through the actions of four G-protein–coupled receptors (A₁, A_2A, A_2B, A₃). These receptors are expressed widely and differentially in most cells and vascular beds of the body, including cells involved in the immune response. Specific agonists and antagonists and knockout mice have elucidated the role of each receptor in the cardiovascular system.¹⁰¹ The majority of data on the beneficial effects of adenosine treatment of I/R have involved the myocardium.

Adenosine has a multitude of physiologic effects, which makes it an ideal treatment of I/R. Adenosine promotes preservation of vascular flow both through endogenous vasodilator properties and by opposing the action of vasoconstrictor substances.^102,103 It can inhibit multiple cell types involved in immunity, including neutrophils,¹⁰¹ and can enhance recovery of the depleted intracellular nucleotide pool.¹⁰⁴ Finally, adenosine can reduce the production of O₂-derived free radicals and reduce intracellular calcium through the opening of receptor-activated K_ATP channels.^105,106 Multiple animal experiments and human clinical trials have established adenosine as a viable therapy for myocardial I/R. We refer the reader to two excellent reviews on this subject^100,107 and to a discussion of the two most important human studies documenting the effectiveness of adenosine in reducing infarct size in patients presenting with anterior wall myocardial infarction (MI)^108,109 presented in the Clinical Trials section of this chapter.

The protective effects of adenosine have been demonstrated in other organ systems. In a mouse model of skeletal muscle I/R, the role of adenosine receptors was elucidated by using specific agonists. The results showed that the A₃ receptor could induce potent cytoprotection of skeletal muscle through phospholipase C receptor signaling.¹¹⁰ A recent study on the role of ATL-146e, an adenosine A₂ receptor agonist, showed improved hind limb function at 48 hours after reperfusion in a rabbit model of spinal I/R injury.¹¹¹ Activation of the adenosine A_2A receptor with the selective agonist DWH-146e also resulted in protection in a model of renal I/R injury, most likely by reducing neutrophil infiltration and suppressing the molecules involved in neutrophil rolling and adhesion (P-selectin and ICAM-1).¹¹² Finally, a large amount of data is also available to show the beneficial effects of aden­osine in models of hepatic I/R.¹¹³

Adrenomedullin

Adrenomedullin (AM) is a peptide with powerful vasodilatory properties that was originally isolated from human pheochromocytoma.¹¹⁴ AM binds to a specific AM-binding protein-1 (AMBP-1) receptor in human plasma, which has been identified as complement factor H.¹¹⁵ Besides its vasodilatory effects, mediated by increasing both cAMP and endothelium-derived NO, AM inhibits the alternative complement pathway through AMBP-1 binding and can modulate neutrophil and monocyte function.^116–118 During gut I/R, AM levels increase significantly after reperfusion, but not with ischemia alone, whereas levels of AMBP-1 decrease with ischemia and remain depressed during reperfusion. Administration of AM/AMBP-1 after I/R dramatically downregulates inflammatory cytokines (TNF-α, IL-1β, IL-6), attenuates tissue injury (as manifested by decreased levels of alanine and aspartate aminotransferase and lactate), and improves survival.¹¹⁹ Studies in AM knockout mice have delineated the protective effects of AM in renal I/R. Taken together, results from these studies show that eNOS, which is stimulated by AM, may play a protective role, but the role of iNOS remains to be delineated in renal I/R.¹²⁰ Similar findings (attenuation of inflammatory cytokines and improved NO) have also been found to be responsible for attenuating injury in a hepatic model of I/R.¹²¹

Heme Oxygenase System and Carbon Monoxide

In humans, heme oxygenase (HO) opens up the heme ring and releases equimolar quantities of three biologically active molecules: biliverdin IXα, reduced iron, and carbon monoxide (CO).¹²² Three different isoforms of HO catalyze the degradation of heme. HO-1 is also known as HSP32. HO-1 is the only isoform that is inducible. Maines et al¹²³ first described induction of HO-1 during ischemia and reperfusion in a model of kidney I/R, the activity of which goes from half that of HO-2 at baseline to 10-fold more than its counterpart after induction. HO-1 expression is increased in various models of hypoxia and exogenous stress mediated by a variety of oxidant-sensitive regulatory molecules.¹²⁴

The protective actions of HO-1 induction are manifold. O₂-derived free radical damage is known to be a major component of the pathophysiology of I/R injury. Heme production has been shown to accumulate in excess of its needs on resumption of the circulation in I/R.¹²⁵ First, the heme molecule is an effective catalyst for the production of powerful oxidants (O^•−), and H₂O₂).¹²⁶ Induction of HO-1 decreases the levels of heme itself, hence protecting against free radical damage–mediated cellular injury and the induction of further inflammatory signals. Second, metabolism of the heme molecule generates biliverdin, and subsequently, bilirubin, both of which have been shown to exhibit strong antioxidant activity.¹²⁷ The third product of heme metabolism is CO, which provides protection against I/R at many cellular levels. Like NO, CO has been recognized as a vasodilator substance through its activation of soluble guanylate cyclase.¹²⁸ In a model of lung ischemia and reperfusion injury, CO has been shown to inhibit apoptosis and protect cultured cells through a p38 mitogen-activated protein kinase (MAPK) and caspase-3–mediated mechanism.¹²⁹ In a model of transplant-induced I/R injury, CO provided anti-inflammatory protection by suppressing the early expression of TNF-α and IL-6. Possible mechanisms for this protection include downregulation of the MEK/ERK1/2 signaling pathway.¹³⁰ Furthermore, working with a mouse hind limb I/R injury model, Patel et al have demonstrated that inhaled CO can protect skeletal muscle from structural injury and energy depletion.¹³¹ Finally, through its regulatory role on platelet–vessel wall interactions, CO has also been shown to decrease platelet aggregation.¹³² Although the idea of using CO as a therapeutic agent might be complicated by its well-known potential toxic effects, the ability to reliably deliver it and also to measure its levels in humans makes it an attractive agent for the treatment of I/R-mediated injury.

Anticoagulants and Ischemia-Reperfusion

Following restitution of vascular flow by either open surgical or endovascular means, anticoagulants have been the mainstay of clinical therapy after acute vascular occlusion. In clinical practice, unfractionated heparin, and recently, low-molecular-weight heparin are the agents most widely used to prevent extension of arterial thrombus, and hence, improve vascular flow in the affected organ. Experimental studies in animal models have indicated that unfractionated heparin may modulate endothelial cell permeability and pH.¹³³ Recent experimental studies of the effect of enoxaparin administered soon after the onset of hind limb ischemia revealed a significant decrease in markers of local thrombosis but no significant salvage of skeletal muscle.¹³⁴ In contrast to the findings noted in ischemic skeletal muscle, some data suggest that enoxaparin administered 30 hours after the onset of traumatic brain injury in a rodent model decreases tissue edema and infarct size.¹³⁵ This finding may indicate that ischemic brain tissue may benefit from treatment with low-molecular-weight heparin when administered after an acute injury. More recently, attention has focused on new antithrombotic agents, tested previously in models of sepsis and shock, such as activated protein C (APC)¹³⁶ and antithrombin III,¹³⁷ for their potential role in the treatment of I/R injury. APC’s mode of action depends on its initial binding to thrombomodulin, whereupon activated thrombomodulin then decreases the activity of thrombin. In experimental models of renal reperfusion injury, APC prevented histologic changes and the inflammation associated with I/R injury.¹³⁸ In rodents subjected to bilateral lower extremity I/R, antithrombin III decreased remote lung and local skeletal muscle injury, but did not alter the biochemical profile of tissue inflammation.¹³⁹

Hypothermia

Hypothermia is known to reduce cellular metabolism and thus decrease demand for O₂ and nutrients during times of stress. Thus, hypothermia has been shown to decrease skeletal muscle injury in isolated gracilis muscle¹⁴⁰ and spinal cord dysfunction^141,142 in response to I/R injury. In vascular and cardiovascular surgery, moderate hypothermia must be used cautiously to protect tissue yet avoid devastating coagulopathy. To this end, localized spinal cord cooling has been used by Black et al,¹⁴³ Cambria et al,¹⁴⁴ Davison et al,¹⁴⁵ and El-Wahsh¹⁴⁶ to ameliorate spinal cord injury. In clinical applications of organ transplantation, explanted organs are routinely stored under hypothermic conditions to decrease basal metabolic rates.¹⁴⁶ Total body hypothermia combined with total circulatory arrest has been useful in replacement of the great vessels.^147–149 New technology has now made systemic hypothermia feasible in patients with acute ischemic events. Current endovascular systems can provide rapid and safe systemic cooling and have been evaluated in patients with acute MI in the COOL MI (Hypothermia as an Adjunctive Therapy to PCI in Patients with Acute Myocardial Infarction) and ICE-IT (Intravascular Cooling Adjunctive to Percutaneous Coronary Intervention) trials. Results from both of these trials have been presented in scientific meetings, but have not been published yet in peer-reviewed journals. Initial conclusions from the available data include that mild systemic hypothermia is safe and well tolerated during coronary intervention in patients presenting with acute MI, and that although neither of the two trials met its primary end point, which was a reduction of infarct size, patients with anterior infarction, who were successfully cooled to the target temperature of ≤35° C, appear to have a significant reduction in infarct size.

Neurogenic Inflammation

Recent studies have shown that vagal nerve stimulation inhibits cytokine synthesis induced by endotoxin by activating the cholinergic anti-inflammatory pathway.^150–156 Vagal stimulation has also been shown to decrease myocardial I/R injury by decreasing tissue injury and inflammation.^153,154 In a rodent model of suprarenal aortic occlusion, vagal nerve stimulation has been found to decrease cytokine synthesis in serum and pulmonary/visceral tissue and shock during reperfusion.¹⁵⁰ Further studies have shown that the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) is an essential component in the vagus nerve–based “cholinergic anti-inflammatory pathway” that regulates levels of TNF, high-mobility group protein B1 (HMGB1), and other cytokines during inflammation. Examination of the effects of cholinergic stimulation with the agonists nicotine or GTS-21 given before or after bilateral renal I/R injury in rats revealed that pretreatment with either agonist significantly attenuated the renal dysfunction and tubular necrosis induced by renal ischemia. Similarly, TNF-α expression and leukocyte infiltration of the kidney were markedly reduced after treatment with cholinergic agonists.¹⁵⁷

Compartment Syndrome

Acute limb compartment syndrome is often associated with acute penetrating arterial or blunt/crush injury, followed by reperfusion.¹⁵⁸ Isolated arterial and venous injuries can give rise to lower extremity compartment syndrome.¹⁵⁹ In the setting of combined injury, the combination of edema and ischemia may increase the need to perform fasciotomy. Iliofemoral thrombosis causing phlegmasia cerulea dolens can produce compartment syndrome. Embolectomy, thrombolysis, and bypass surgery can cause compartment syndrome at an incidence of up to 21%. In this setting, the condition is known as postischemic compartment syndrome and is caused by revascularization of the leg after a prolonged period of ischemia. The pathophysiology of compartment syndrome is believed to be due to a reduction in the arteriovenous pressure gradient. Specifically, as edema and intracompartmental pressure increase, the veins are compressed, and venous pressure increases. The increase in venous pressure decreases the arteriovenous gradient, thereby resulting in diminished or absent local perfusion. Reduced venous drainage as a result of increased interstitial pressure exacerbates the development of edema, such that arteriolar compression develops and leads to muscle and nerve ischemia. Sensory changes in the nerves, manifested by clinical complaints of paresthesias and hyperesthesia, may develop after only 30 minutes of ischemia. Irreversible nerve damage begins after 12 to 24 hours of total ischemia. Failure to decompress all fascial compartments usually leads to muscle ischemia, rhabdomyolysis, and renal insufficiency. Treatment is directed toward improving tissue perfusion pressure, usually by expediently performing fasciotomy.^160,161 A high index of suspicion is required to make this diagnosis, particularly in sedated or unconscious patients.

No–Reflow Phenomenon

One of the most formidable advanced consequences of I/R injury is the “no-reflow phenomenon,” whereby, despite restoration of blood flow to the muscular arteries, occlusion of the microvasculature results in tissue ischemia and anoxic injury. In 1968, Ames et al¹⁶² described the no-reflow phenomenon in the brain, and May et al¹⁶³ reported the same phenomenon 10 years later in skin flaps. In the no-reflow state, the flow in the reperfused tissue is disturbed because of downstream microembolization of platelets, neutrophils, thrombus, and atheroma particles, as well as endothelial cell dysfunction and severe microvascular damage. Clinically, this syndrome is associated with tissue hypoperfusion in the absence of a significant obstruction in the named arteries, and in its advanced forms, it can result in loss of the affected organ or limb.

Myocardial Stunning/Arrhythmias

Myocardial stunning is defined as reversible, transient myocardial dysfunction persisting after reperfusion despite the absence of irreversible damage.^164,165 This kind of injury may require inotropic support. A multitude of conditions contribute to myocardial stunning, including decreased ATP resynthesis, coronary microvascular spasm/plugging, and cytotoxic injury secondary to reactive O₂ metabolites or increased intracellular calcium release and uptake.^166,167 Reperfusion arrhythmias may cause sudden death after surgical revascularization or coronary thrombolysis. These arrhythmias may be due in part to rapid and sudden changes in ion concentrations within ischemic tissue on reperfusion.^168–170 Staged gradual reflow or transient acid reperfusion may reduce the incidence of malignant arrhythmias.¹⁷¹

Central Nervous System

Reperfusion injury of the central nervous system (CNS) may contribute to the morbidity and mortality of stroke, head trauma, cerebrovascular surgery, and deep hypothermic circulatory arrest. Disruption of the blood-brain barrier leading to increased vascular permeability results in cerebral edema, increased intracranial pressure, and leukocyte migration into brain tissue.¹¹ As mentioned earlier in this chapter, leukocytes release proteases, ROS, and lipid mediators that result in irreversible tissue damage. The clinical manifestations of CNS I/R injury usually coincide with severe changes in sensory, motor, or cognitive function, and often lead to death.

Gastrointestinal System

Gastrointestinal I/R injury is characterized by a decrease in intestinal barrier function. Under normal physiologic conditions, the gastrointestinal mucosa separates the bacteria of the bowel lumen from the vascular space and the interstitium of the bowel wall. After I/R, there is an increase in intestinal permeability that allows translocation of bacteria from the bowel lumen into the portal and systemic circulation.^87,172,173 Activation of complement and circulating leukocytes by translocated bacteria may eventually lead to development of the systemic inflammatory response after gastrointestinal I/R injury. The relatively unpredictable nature of gastrointestinal I/R injury has led to widespread adoption of a policy to consider a second-look operation within 24 hours of acute mesenteric or celiac revascularization.^174–176

Multiorgan Dysfunction Syndrome

Remote organ injury after I/R is a leading cause of death, with mortality often linked to the number of failed organ systems. The pulmonary system is the most frequently injured organ in patients with multiple-organ dysfunction syndrome,¹⁷⁷ and onset of the syndrome is commonly preceded by the development of acute respiratory insufficiency 24 to 72 hours after the initiating ischemic event.^178,179 Respiratory failure is often followed by hepatic, renal, gastrointestinal, myocardial, and CNS dysfunction. In its terminal stages, multiorgan dysfunction is characterized by dysfunction of the coagulation and immune systems, which leads to thrombosis, disseminated intravascular coagulation, and immunocompromise.

Coronary Interventions

Two of the most encouraging trials are the AMISTAD-I¹⁰⁸ (Acute Myocardial Infarction Study of Adenosine Trial) and AMISTAD-II studies,¹⁰⁹ which have provided evidence that adenosine reduces infarct size in patients presenting with anterior MI when given in conjunction with thrombolysis or primary angioplasty therapy. Specifically, a 3-hour infusion of adenosine at 70 μg/kg/min produced a significantly lower infarct size (11% vs 27% in controls as measured by technetium-99m sestamibi tomography; P = .023), and this finding correlated with fewer adverse clinical events. As previously discussed, adenosine has direct protective effects on organ parenchymal cells; vasodilatation of native arteries, but also anti-inflammatory effects that may inhibit neutrophil adhesion and migration into the myocardium; cytokine release from mononuclear cells; release of O₂ radicals; and antiplatelet effects that may have a role in maintaining infarct artery patency.

Several studies of experimental animal models of acute MI have also documented the effectiveness of ischemic conditioning (applications of brief periods of ischemia) in reducing infarct size. When ischemic conditioning is applied before the I/R episode, it is called ischemic preconditioning and is applicable mostly during operations for the performance of either a heart¹⁸⁰ or leg bypass procedure.¹⁸¹ Ischemic conditioning can also be applied at the time of reperfusion (ischemic postconditioning), making it applicable for patients who are being treated for an acute ischemic event. Postconditioning can be applied either as inflation and deflation of the angioplasty balloon after reopening the occluded (usually coronary) artery or as inflation and deflation of a blood pressure cuff around an unaffected arm or leg (remote conditioning). Several small clinical studies have demonstrated that postconditioning by coronary angioplasty protects the human heart during acute MI.¹⁸² The initial promise presented by these studies has lead to postconditioning being currently evaluated in nine ongoing clinical studies, including the DANAMI-3 (DANish Study of Optimal Acute Treatment of Patients With ST-elevation Myocardial Infarction) with 2000 anticipated patient enrollments. Clinicians hope that these ongoing studies either alone or in combination will provide data that will have adequate statistical power to detect the cardioprotective properties of postconditioning.¹⁸³

Other treatments targeting the biochemical pathways that are activated during IR, including the production of ROS, cytosolic and mitochondrial Ca^2+-overload, complement and neutrophil activation, endothelial dysfunction, and shift in metabolism and substrate use, have been investigated for patients with acute MI with mostly disappointing results.^184,185 The most encouraging exception among these works is a pilot study evaluating the cardioprotective effects of cyclosporine, which in addition to its well-known immunosuppressive properties, is a potent inhibitor of MPTP.¹⁸⁶ In a randomized trial of 58 patients, the investigators reported that administration of the medication immediately before primary coronary intervention reduced the size of the infarct (measured using surrogate biomarkers and cardiac magnetic resonance imaging) by as much as 40%. This study has provided the preliminary data for a larger European trial, does Cyclosporine ImpRove Clinical oUtcome in ST elevation (CIRCUS), which aims to confirm the beneficial effects of cyclosporine in a multicenter, randomized, placebo-controlled, double-blinded fashion.

Transplantation Interventions

In the field of organ transplantation, the strategies used to improve organ preservation and minimize I/R injury have not changed very much in the last 10 years, with cold preservation being the mainstay procedure. More recently, additives to the cold storage solution, including the p38 MAPK inhibitor FR 167653 (with anti-inflammatory properties) and the colloid polyethylene glycol, which appear to reduce ATP depletion and calcium accumulation within the cells, have been successfully used to reduce I/R damage after transplantation.¹⁸⁷ In transplantation, the benefit of ischemic preconditioning of the donor is still under evaluation, with most studies not showing a clinical benefit with respect to graft or graft-recipient survival.¹⁸⁸ In the direction of treatments applied to the donor before harvesting of the organs, a study of pretreatment of donors with low-dose dopamine demonstrated a reduction of the need for dialysis after kidney transplantation.¹⁸⁹ The investigators believe that the mechanism of protection from dopamine involves direct interaction with the cellular membrane (related to the dihydroxy-phenolic ring structure of the dopamine molecule), which is capable of protecting endothelial cells from oxidative stress during cold storage reperfusion.

Lower Extremity Interventions

Therapeutic intervention for acute lower extremity arterial occlusion has centered on open surgical intervention or thrombolysis. Several prospective, randomized trials were designed to compare thrombolytic therapy with direct operative intervention^190–193 in a post hoc scenario (i.e., after the onset of ischemia). The STILE (Surgery vs. Thrombolysis for Ischemia of the Lower Extremity) trial showed that patients with peripheral artery occlusion and ischemia of less than 14 days’ duration who were treated with catheter-directed urokinase or alteplase had significantly lower amputation rates, shorter hospital stays, and fewer subsequent surgical interventions than those initially treated with surgery did.¹⁹⁰ These benefits were achieved at the cost of a greater risk of bleeding complications than in patients treated by surgery alone. In contrast, for patients with ischemia lasting longer than 14 days, surgery resulted in significantly less ongoing or recurrent ischemia than fibrinolysis did. In the TOPAS trial (Thrombolysis or Peripheral Arterial Surgery), intra-arterial infusion of urokinase reduced the need for open surgical procedures and yielded similar rates of amputation and death despite a higher frequency of hemorrhagic complications.¹⁹³ Even with these major milestones in the management of acute arterial occlusion, high rates of limb loss and mortality continue to be documented in this complex patient population.¹⁹¹

A cell-free extracorporeal perfusion system used to minimize both local and systemic injury has been evaluated in patients with severe limb ischemia (sensorimotor loss, rest pain/gangrene).¹⁹⁴ In this post hoc treatment study, reperfusion of the ischemic limb was achieved with hypertonic, hyperoncotic perfusate containing antioxidants delivered through the arterial tree with initial venous drainage. Rapid lactate washout and decreased neutrophil activation, serum levels of F₂-isoprostanes (marker of free radical–mediated systemic lipid peroxidation), and mortality were observed in patients treated with the study perfusion method versus controls. In this small study, modification of limb perfusion in patients with severe limb ischemia via a simple and rapid (15-20 minutes) method provided beneficial systemic effects.

Another post hoc therapeutic intervention, controlled reperfusion using a simple blood bag perfusion system, has been found to reduce reperfusion injury and facilitate return of normal function.¹⁹⁵ Patients with severe, acute lower limb ischemia were allocated to treatment with either embolectomy alone or surgical embolectomy plus controlled reperfusion via a simplified perfusion system. Although the duration of ischemia was greater in the patients who underwent controlled reperfusion, there were no deaths or amputations in this treatment group. In contrast, in patients who had shorter periods of ischemia treated by embolectomy alone, there were three in-hospital deaths and two major amputations. This study strongly suggests that the results of conventional embolectomy for acute, severe lower limb ischemia can be improved by controlled reperfusion.

The state of our knowledge for the treatment/prevention of I/R injury in patients undergoing vascular operations is quite limited. Almost all of the clinical work thus far has focused on prevention of I/R after the performance of major vascular operations, mostly aortic repairs for aneurysmal and occlusive disease. The two main interventions evaluated in small pilot studies are ischemic preconditioning and administration of antioxidants. A recent review comparing the outcomes from vascular and endovascular surgical procedures with and without the use of remote ischemic preconditioning demonstrated that perioperative mortality was not clearly different between the two groups, whereas MIs were reduced in the remote ischemic preconditioning group, and unplanned critical care admissions tended to increase in the remote ischemic preconditioning group. The authors concluded that based on current evidence from small pilot trials, there are too few data to be able to say whether remote ischemic preconditioning has any beneficial or harmful effects and that the safety of this technique needs to be confirmed in adequately powered randomized trials.¹⁸¹

A similar review compared the effects of antioxidant treatment on a variety of ROS- and inflammation-related markers in patients who underwent vascular and endovascular surgical procedures. The authors demonstrated that antioxidant supplementation might reduce oxidative stress and activation of inflammatory pathways. The correlation of this finding to clinical outcomes in these patients was evaluated in only one study, which demonstrated an association between plasma malondialdehyde (one of the most frequently used biomarkers of lipid peroxidation and oxidative stress) levels and the need for transfer to the intensive care unit during vascular operations. The authors concluded that their data pointed to a potential therapeutic advantage associated with anti­oxidant supplementation in patients undergoing vascular operations.¹⁹⁶