Ischemia-Reperfusion Injury (IRI)

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

2. 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.

3. CHAPTER 1 Etiology and Factors • Etiology • Influencingfactors

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

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

6. Experimental study showed • Calcium paradox • Oxygen paradox • PH paradox

7. CHAPTER 2 The pathogenesis of IRI •The increase of free radicals •Intracellularcalcium overload •Neutrophil activation

8. Free radicals ■Conception and properties 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.

9. ■Classification • Oxygen free radicals (OFR) • Nitrogen free radicals(NFR) • Lipid free radicals (LFR) • Others: chlorine radicals (Cl.), methyl radicals (CH3.)

10. ●Oxygen free radicals (OFR) OFR is referred to oxygen-derived free radicals. ▲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. OFR （O2、OH•） . ROS Free radical ROS Non free radical ROS H2O2、 1O2

11. NFR is defined as nitrogen-derived free radicals (also identified as reactive nitrogen species, RNS). NO• ONOO , NO2 RNS 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. L• LO• LOO• LFR • Nitrogen free radicals (NFR) • Lipid free radicals (LFR)

12. ● Production of oxygen free radical e ˉ · O2 O2 2e O2 H2O2 2H+ 4e O2 2H2O 4H+ 3e O2 OH· + H2O 3H+ ■Metabolism of the free radicals

13.  Sources of ▲ · O2 · O2 Sources of OH• ▲ e- · Fe2+ O2 + H2O2 O2 O2 + OH• + OH+ Mitochondria H2O OH• + H• ； H2O H+ + OH‒ homolysis heterolysis ionic irradiation autoxidation Enzyme oxidation poison

14. Enzymes superoxide dismutase (SOD) catalase (CAT) gluthione peroxidase (GSH-Px) ▲ SOD 2O2 + 2H+ H2O2 + O2 CAT 2H2O2 2H2O + O2 GSH-Px H2O2 + 2GSH 2H2O + O2 Non-enzyme substances ▲ - vitamin-E, -A, and -C; cysteine; glutathione; albumin; allopurinol; ceruloplasmin . • Scavenge the free radical

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

16. Neutrophil 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.

17. 95%O2 Cytochrome C oxidases O2 H2O 【Normal】 4e- 2%O2 respiratory chain enzyme↓ O2OFR 【ischemia】 Transferring the electrons↓ 95%O2 respiratory chain enzyme↓ ╳ O2 H2O↓ 【reperfusion】 4e- e- • The increase of one electron deoxidization in mitochondria

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

19. 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. ■ Mechanisms of free radical-induced IRI OFR are extremely reactive to interact with lipids, proteins and nucleic acids.

20. 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. ▲

21. 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. • Intracellular calcium overload • Protein denaturalization and decreased enzyme activity • Nucleic acid damage and chromosome aberration

22. 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+/Ca2+ exchange Na+/Ca2+ exchange protein ▲ Na+/H+ exchange protein ▲ Protein kinase C (PKC) ▲

23. 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, Ca2+ can flow into the cytoplasm through damaged membrane according to the gradient. • Mitochondrial injury • CA increase

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

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

26. 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. • 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

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

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

29. ■Lower myocardial diastolic & contractility function ●Myocardium stunning Myocardium stunning is termed that cardiac contractile function is impaired temporarily but reversibly for a period of hours to days after ischemia-reperfusion. ▲A form of IRI to reversible loss of myocardial contractility ▲Its mechanism is associated with OFR and Ca2+ overload Myocardial IRI The major IRI in heart includes arrhythmia, reversible contractile dysfunction, alterations of myocardium untrastructure and metabolism.

30. ■ Reperfusion arrhythmia Ventricular tachycardia and fibrillation are the major manifestation of reperfusion arrhythmias. ● 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 ▲OFR and calcium disturbance ▲Sodium and potassium homeostasis ▲The ununiformity of action potential duration (APD)

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

32. ■The alteration of cerebral energy metabolism ●Enhancing lipid peroxidation reaction During IRI, the accumulation of free fatty acidssuch as arachidonic acid and stearic acid as substrates produces OFR and peroxidative lipids by lipid peroxidation, due to the increased degradation of cerebral phospholipid. 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.

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

34. 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.

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

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

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

38. 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.

39. Thank You