Ischemia-Reperfusion Injury (IRI) Department of Pathophysiology Shanghai Jiao-Tong University School of Medicine

What is ischemia-reperfusion injury? In majority situations, blood reperfusion can reduce ischemia-induced tissue and organ injury, resulting in the structural and functional recovery. In some circumstances, however, blood reperfusion may induce or aggravate the further reversible even irreversible cell damage and tissue or organ injury, especially for a prolonged ischemia. This phenomenon has been termed IRI. In majority situations, blood reperfusion can reduce ischemia-induced tissue and organ injury, resulting in the structural and functional recovery. In some circumstances, however, blood reperfusion may induce or aggravate the further reversible even irreversible cell damage and tissue or organ injury, especially for a prolonged ischemia. This phenomenon has been termed IRI.

CHAPTER 1 Etiology and Factors Influencingfactors Influencing factors Etiology

Etiology Recovery of blood supplementation following ischemia Recovery of blood supplementation following ischemia Microcirculation revascularization during shock treatment Coronary artery convulsion relief Application of new medical technologies Application of new medical technologies PTCA, Thrombolytic therapy Cardiac bypass surgery Cardiac bypass surgery Cardiopulmonary resuscitationin sudden arrest of heart beat Cardiopulmonary resuscitation in sudden arrest of heart beat Others: organ transplantation Others: organ transplantation

Factors Duration of ischemia Collateral circulation formation Dependency on oxygen supply Condition of reperfusion The speed of reperfusion The components of reperfusion solution

Experimental study showed Calcium paradox Oxygen paradox PH paradox

CHAPTER 2 The pathogenesis of IRI Intracellularcalcium overload Intracellular calcium overload The increase of free radicals Neutrophil activation

■ ■ Conception and properties Free radicals Free radicals are a highly reactive group of atoms, molecules or radicals, which carry unpaired electron in out orbital. The properties of these radicals are as following: short life time and powerful oxidative ability. Free radicals are a highly reactive group of atoms, molecules or radicals, which carry unpaired electron in out orbital. The properties of these radicals are as following: short life time and powerful oxidative ability.

Oxygen free radicals (OFR) Nitrogen free radicals (NFR) Lipid free radicals (LFR) Others: chlorine radicals (Cl. ), methyl radicals (CH 3. ) ■ ■ Classification

▲ ▲ Reactive oxygen species (ROS) ROS are composed of oxygen-derived free radicals (OFR), and non free radical substances such as hydrogen peroxide and singlet oxygen. H2O2、 1O2H2O2、 1O2 Non free radical ROS Free radical ROS ROSOFR （ O 2 、 OH ）. ● Oxygen free radicals (OFR) OFR is referred to oxygen-derived free radicals.

(NFR) Nitrogen free radicals (NFR) NFR is defined as nitrogen-derived free radicals (also identified as reactive nitrogen species, RNS). NO NO ONOO, NO 2 RNS Lipid free radicals (LFR) Lipid free radicals (LFR) Lipid free radicals are referred to middle metabolic products resulting from the chain reaction of lipid peroxidation, which is produced by interaction of OFR and non-saturated fatty acid.LLOLOO LFR

■ ■ Metabolism of the free radicals O2O2 O2O2 ˉ · e O 2 OH· + H 2 O 3e 3H + 2e O 2 H 2 O 2 2H + 4e O 2 2H 2 O 4H + Production of oxygen free radical ● Production of oxygen free radical

OH Sources of OH▲ Fe 2+ H 2 O 2 + H 2 O 2 O 2 + OH + OH + · O2O2O2O2 H 2 O OH + H ； H 2 O H + + OH ‒ heterolysis homolysis ▲ · O2O2O2O2  Sources of autoxidation Enzyme oxidation poison O2O2 e-e- Mitochondria O2O2 · ionic irradiation

Scavenge the free radical Scavenge the free radical Enzymes superoxide dismutase (SOD) catalase (CAT) gluthione peroxidase (GSH-Px)▲ 2O 2 + 2H + H 2 O 2 + O 2. - SOD 2H 2 O 2 2H 2 O + O 2 CAT H 2 O 2 + 2GSH 2H 2 O + O 2 GSH-Px vitamin-E, -A, and -C; cysteine; glutathione; albumin; allopurinol; ceruloplasmin Non-enzyme substances▲

■ ■ Mechanisms of free radical increase The increase of xanthine oxidase (XO) formation in VEC The increase of xanthine oxidase (XO) formation in VEC ischemia ATP↓ calcium overload calcium-dependent proteases ↑

Neutrophil respiratory burst Neutrophil respiratory burst IRI induces the activation of complement and endothelial cells and the increase of chemokine such as, which further attract and activate neutrophils. This activation of neutrophils then intakes large amounts of molecular oxygen and produce OFR by respiratory burst. IRI induces the activation of complement and endothelial cells and the increase of chemokine such as C3a and leukotriene, which further attract and activate neutrophils. This activation of neutrophils then intakes large amounts of molecular oxygen and produce OFR by respiratory burst.

The increase of one electron deoxidization in mitochondria The increase of one electron deoxidization in mitochondria 95%O 2 Cytochrome C oxidases O 2 H 2 O 【 Normal 】 O 2 H 2 O 【 Normal 】 4e - O 2  OFR 【 ischemia 】 2%O 2 respiratory chain enzyme↓ 4e - 95%O 2 respiratory chain enzyme↓ O 2 H 2 O↓ 【 reperfusion 】 e-e-e-e- ↓ Transferring the electrons↓ ╳

The increase of catecholamine and its oxidazition The increase of catecholamine and its oxidazition sympathetic-adrenal medulla (  ) vanilmandelic acid (normal) monoamine oxidase adrenochrome ischemia and hypoxia CA release OFR

■ ■ Mechanisms of free radical-induced IRI The increase of membrane lipid peroxidation (MIP) The increase of membrane lipid peroxidation (MIP) OFR interacts with non-saturated fatty acids from membrane lipids and further induce lipid peroxidation reaction, which results in the structural alteration and dysfunction of membrane. ROS induces oxidation of lips, proteins and nucleic acid. OFR are extremely reactive to interact with lipids, proteins and nucleic acids.

Membrane structural damage MIP leads to the abnormal state of membrane non- saturation, which results in the decreases of membrane fluidity and permeability.▲ Membrane protein function inhibition MIP induces the deactivation and malfunction of membrane receptors and ionic pumps, which produces the signal pathway blockage.▲ Mitochondrial function damage MIP induces mitochondrial dysfunction and further decreases ATP generation.▲

Protein denaturalization and decreased enzyme activity Protein denaturalization and decreased enzyme activity Nucleic acid damage and chromosome aberration Nucleic acid damage and chromosome aberration Others: Others: Mediation of a series of reaction important for IRI, such as releasing inflammatory factors; decreasing nitric oxide; promoting the expression of adhesion molecules and the adherence between neutrophils and vessels. Mediation of a series of reaction important for IRI, such as releasing inflammatory factors; decreasing nitric oxide; promoting the expression of adhesion molecules and the adherence between neutrophils and vessels. Intracellular calcium overload Intracellular calcium overload

Calcium overload The phenomenon of cellular structure damage and dysfunction caused by intracelluar calcium increasing abnormally is termed “calcium overload”. ■ ■ Mechanisms of IRI-induced calcium overload Disorder of Na + /Ca 2+ exchange Disorder of Na + /Ca 2+ exchange Na + /Ca 2+ exchange protein ▲ Na + /H + exchange protein ▲ Protein kinase C (PKC) ▲

Membrane permeability damage Membrane permeability damage The integrity and permeability of membrane is impaired during ischemia-reperfusion. These damages do not occur only in sarcoplasmic reticulum (SR) but also in mitochondria, lysosomes and other cellular membranes. Therefore, Ca 2+ can flow into the cytoplasm through damaged membrane according to the gradient. Mitochondrial injury Mitochondrial injury CA increase CA increase

■ ■ Mechanisms of calcium overload-induced IRI Promotion of OFR generation Promotion of OFR generation Aggravation of acidosis Aggravation of acidosis Damage of cellular membrane Damage of cellular membrane Mitochondrial dysfunctions Mitochondrial dysfunctions Activation of other enzymes (proteinases, nucleases) Activation of other enzymes (proteinases, nucleases)

Neutrophil activation It has been manifested that the capillary damage and dysfunction which mediated by neutrophil activation play an important role in IRI.

Cell adhesion molecule (CAM) increase Activated neutrophils can increase the expression of CAMs, which including the selectins, integrins (CD11/CD18) and immunoglobulin superfamily (ICAM- 1, VCAM-1). ■ ■ Leukecyte accumulation induced by IRI Inflammatory factors or chemokines increase Inflammatory factors or chemokines increase Activated neutrophils can adhere to endothelial cells or blood cells, then to release the inflammatory factors after margination and aggregation, such as TXA2, leukotrienes, prostaglandin and so on, to increase the permeability of endothelial cell monolayer.

■ ■ Mechanism of leukecyte-mediated IRI Microvascular damage Microvascular damage The ischemia region could not be reperfused sufficiently after relieving the occlusion to recover the blood flow. -No-reflow phenomenon microvessel hemorheological alteration Abnormal regulation of inflammatory reactions Abnormal regulation of inflammatory reactions Machinery blockage action

CHAPTER 3 The alterations of function and metabolism induced by IRI The alteration of IRI in brain The alteration of IRI in heart The alteration of IRI in other organs

Myocardial IRI The major IRI in heart includes arrhythmia, reversible contractile dysfunction, alterations of myocardium untrastructure and metabolism. ■ Lower myocardial diastolic & contractility function Myocardium stunning ● Myocardium stunning ▲ ▲ A form of IRI to reversible loss of myocardial contractility ▲ ▲ Its mechanism is associated with OFR and Ca 2+ overload Myocardium stunning is termed that cardiac contractile function is impaired temporarily but reversibly for a period of hours to days after ischemia-reperfusion.

■ Reperfusion arrhythmia Ventricular tachycardia and fibrillation are the major manifestation of reperfusion arrhythmias. The occurrence of reperfusion arrhythmia ● The occurrence of reperfusion arrhythmia ▲ ▲ Ischemia period before reperfusion ▲ ▲ Ischemia degree ▲ ▲ The cardiac myocytes with recoverable capability existed ▲ ▲ The speed of reperfusion blood The mechanism of reperfusion arrhythmia ● The mechanism of reperfusion arrhythmia ▲ ▲ OFR and calcium disturbance ▲ ▲ Sodium and potassium homeostasis ▲ ▲ The ununiformity of action potential duration (APD)

■ Alterations of myocardium metabolism Decreased generation of ATP and CP ● Decreased generation of ATP and CP Mitochondrial functional loss ● Mitochondrial functional loss ■ Alterations of myocardium ultrastructure Cellular membrane damage ● Cellular membrane damage Myofibrils break down and contractile bands occur ● Myofibrils break down and contractile bands occur Mitochondria swelling ● Mitochondria swelling Cristae fragmentation and solution Cristae fragmentation and solution

Cerebral IRI Cerebral IRI includes cytotoxic edema and apoptosis or death of brain, which causes the manifestation of intracranial hypertension such as vomiting and coma. ■ The alteration of cerebral energy metabolism Enhancing lipid peroxidation reaction ● Enhancing lipid peroxidation reaction During IRI, the accumulation of free fatty acids such as arachidonic acid and stearic acid as substrates produces OFR and peroxidative lipids by lipid peroxidation, due to the increased degradation of cerebral phospholipid.

■ The alteration of cerebral amino acid metabolism Stimulant amino acid decrease ● Stimulant amino acid decrease Repressive amino acid increase ● Repressive amino acid increase ▲ ▲ Glutamic acid ▲ ▲ Aspartic acid ▲ ▲ Alanine ▲ ▲  -Aminobutyric acid ▲ ▲ Taurine ▲ ▲ Glycine ■ The alteration of cerebral histology ▲ ▲ Cerebral edema ▲ ▲ Cerebral cellular necrosis

IRI in other organs The IRI also can occur in other organs besides heart and brain. For example, liver and kidney are the organs which studied extensively in IRI. It has been implicated in the pathogenesis of a variety of clinical conditions including trauma, hypovolemic and endotoxic shock, transplantation, etc.

CHAPTER 4 Pathophysiological basis of prevention and treatment for IRI Scavenge the free radicals Control the reperfusion conditions Relieve calcium overload Improve the metabolism

Control the reperfusion conditions ■ Shorten the ischemia period Lower pressure ● Lower pressure ■ Improve the reperfusion conditions Lower flow speed ● Lower flow speed Lower temperature ● Lower temperature Lower pH ● Lower pH Lower concentration of calcium and sodium ● Lower concentration of calcium and sodium

Scavenge the free radicals ■ Enzymes ■ Non-enzyme substances Relieve calcium overload ■ Calcium antagonists ■ Calcium channel blockers Improve the metabolism ■ Energy supplementation ■ Cell protector application

Ischemic preconditioning (IPC) IPC is defined as short period-ischemic stress can significantly led to the protection of tissue and organs with subsequent longer IRI, which is also an adaptive mechanism.