Chapter 11 Ischemia- reperfusion injury Zhao Mingyao BMC.ZZU

1955, Sewell ligated coronary artery of dog, restore blood flow after deligation. What happened ? Brief history Simple phenomenon

Clinical ： Shock, DIC Bypass surgery Fibrinolytic therapy Cardiopulmonary operation Organ transplantation

Concept of Ischemia-Reperfusion Injury The restoration of blood flow after transient ischemia may induce further reversible or irreversible cellular injury

Features of IRI ： 1. reversible  irreversible 2. Massive in organs 3.participating factors oxygen paradox calcium paradox pH paradox

Without O 2 Normal O2 supply Without Ca 2+ with Ca 2+ Deteriorate injury Acidosis Correcting acidosis effect Perfusion fluid pH paradox Ca 2+ paradox O 2 paradox

Section 1 Etiology of IRI 1. Duration of ischemia 2. Dependency on O 2 supply 3. The condition of reperfusion: reperfusion pressure, speed, T, Na +, Ca 2+, K +, Mg 2+

Effect of Ischemic time on perfusion arrhythmia of rat Ischemic time incidence rate

Section 2 mechanism of IRI

Part 1. Injury of free radicals

Concept and Types of FR Free radicals are atoms or molecules with unpaired electrons in their outer orbital 1.Non-lipid free radicals 2.Lipid free radicals

(1) Oxygen free radical （ OFR) ---Induced by O2 Rective Oxygen Species (ROS) OFR H 2 O 2 Types O· - 2 OH· 1 O 2 Classification peroxynitrite

(2) Lipid radicals types ： L· LO· Alkoxyl LOO· (3)Cl· 、 CH 3 · (Methane ) 、 NO ·

1. Generation of free radical 1) Initiation 2) Propagation 3) Degradation

(1)Production and scavenging of OFR 1) Origin of O· - 2 ： ① Mt ② Natural oxidation of some substances ③ Enzyme catalysis ④ Toxin acting on cell

2 ） Production process of OFR Single electron reduction O 2 + e O 2  O 2 + 2e + 2H + H2O2 H2O2 H2O2H2O2 O e + 3H + HO  + H 2 O O e + 4H + 2 H 2 O Cytaa 3 SOD nse Single electron reduction of O 2

Haber-Weiss reaction (without Fe  2 ) O 2 － + H 2 O 2 O 2 + OH  +OH   SLOW

Fenton type of Haber-Weiss reaction ( with Fe  3 ) O 2 － + H 2 O 2 O 2 + OH  +OH  Fast  Fe  2 What significance ？？？

3 ） Scavenging of OFR ① Low molecule scavenger ② Enzymatic scavenger Water-soluble Lipid-soluble

① low molecule scavenger *hydrofacies of intra- or extracell: Cysteine 、 Vit C 、 Glutathione *Cellular lipid ： Vit E 、 Vit A Cytosol ： NADPH

② Enzymatic scavenger Catalase (CAT) Glutathione peroxidase (GSH-Px) Superoxide dismutase (SOD)

Dismutation reaction Single electron reduction of O 2 2O 2  + 2H + H 2 O 2 + O 2 H2O2H2O2 SOD nse ?

GSH-Px : containing selenium scavenging large biological molecule peroxide LOOH + 2GSH GSSG + LOH + H 2 O GSSG + NADPH + H + 2GSH + NADP + GSH-Px GSH reductase

(2) Mechanism of OFR ↑during IRI

1) Mitochondria pathway Ca 2+ enter Mt  Single electron reduction of O2 ↑ O - 2 · ↑ hypoxia  MnSOD 

Xanthine oxidase (XO )10% xanthine dehydrogenase(XD) 90% Ca 2 + sensitive enzyme 2) Xanthine oxidase(XO) pathway↑

Ischemia: ATP degradation Hypoxathine↑↑ Reperfusion: (1)Ca 2+ →protease XO （ 2 ） restore O 2 xanthine + O· H 2 O 2 O· H 2 O 2 + uric acid O2O2 O2O2 XD OH · XO role in formation of OFR

3)Neutrophil pathway NADH(I) NADPH(II) + O 2 NADPH oxidase H + + O - 2 ·+H 2 O 2 NADH oxidase C 3,LTB 4 Activates NP hexose bypass activation Respiratory burst

4) Catecholamine autooxidation pathway Adr Methyl transferase monoamine oxidase Vanillylmandelic acid (VMA) Renal excretion 80% during stress adrenochrome O 2 - · 

(3) The detrimental effects of OFR to tissue 1 ） Lipid membrane 2 ） Protein: channel, pump, 3 ） Enzyme 4 ） Nuclear acid : DNA

Membrane lipid peroxidation

Protein break Protein ~ -S-S- CH 3 -S- Lipid-lipid ~ O Two sulfur ~ Lipid –pro ~ Amino acid oxidation OH HO fatty acid oxidation OH HO MDA released by oxidated fatty acid Biomacromolecle crosslinkage Malondialdehyde (MDA)

DNA disruption and chromosome aberration induced by OH  about 80% damage OH (hydroxyl)

Part 2 Calcium overload

1. Ca 2+ transportation and distribution Na + - Ca 2+ cotransportor Ca 2+ pump Ca 2+ binding Pr Mt SR Ca 2+ Ca 2+ Channel

2. Mechanism of ~ ① Na + - Ca 2+ exchange↑: H + -Na + ↑; Na + - Ca 2+ ↑(forward mode reverse mode); PKC triggers ② ATP ↓: mitochondria damage, energy precursor ↓ ③ Membrane permeability ↑ ④ catecholamine ↑

α1α1 NE GqPLC P1P1 IP 3 Ca 2+ SR DG PKC H+H+ Na + Ca 2+ filament PKC activating Na + /Ca 2+ exchanger indirectly

3. The detrimental effects of Ca 2+ overload to tissue (1) Activating Ca 2+ -activated protease (2) Defects in membrane permeability activating phospholipase A 2 OFR (3) Hypercontracture and reperfusion arrhythmia cellular electrical action (4)Mitochondria damage

Part 3. The endothelial injury and neutrophil activation

1.The role of neutrophil activated ① Swelling ② Adhesion ③ Infiltration ④ Release: arachidonic acid, PAF, lysosomal enzyme ⑤ Respiratory burst ⑥ Cell adhesion molecules(CAM): selectins, integrins, immunoglobulin superfamily

2. Mechanism of no-reflow phenomenon Vaso-endothelial damage Vaso-endothelial edema Occlusion of microvascular luman

Rulo: 肉膜

3.NO and ONOO - production NO in VEC(eNOS), little, physiological NO in inflammatory cell(iNOS), rich, cytotoxic (Mt respiration, aconitase activity, DNA synthesis) and OONO - peroxynitrite

Free radicals with a nitrogen center ① Nitric oxide(NO) NADP + O2O2 NADPH L-arginine L-citrulline + NO NOS

② Peroxynitrite, ONOO - NO+O 2.- ONOO - H2OH2O NO 2. + OH. + H + acidic Killing bacterial & tumor

Protein rupture Pro-pro linkage -S-S- CH 3 -S- O disulfide linkage Lipid-pro linkage Oxidation of AA Inhibit function of protein: lipid-protein- collagen linkage

OFR Ca 2+ VEC -NP ？ Ca 2+ overload results in cellular death Brief summary Change of metabolism & energy

Section 3 Body change during IRI

1. Heart （ 1 ） Reperfusion arrhythmia ATP-sensitive K + channel open: hyperpolarization long chain acylcarnitines & lysophospholipids released reduced conduction velocity

AP shortening + conduction slowing = re-entrant arrhythmia Generation of ectopic beats

（ stunning （ 2 ） Myocardial stunning Myocardial contractile function is temporarily but reversibly impaired for a period of hours to days 5 min ischemia, reperfusion, 40min later restoring 1 hr ischemia, reperfusion, a month later restoring

stunning Mechanism of myocardial stunning OFR Ca 2+ overload No-reflow ATP↓ + contractile protein sensitivity ↓ for Ca 2+

（ 3 ） Myocardial metabolism ATP depletion ATP substrate catabolized, rushed out

Energy SourcesEnergy PoolEnergy Use Fatty Acids Lactate Pyruvate O2O2 Glycolysis (Anaerobic) Glucose Glucose-1-PO 4 Glycogen TCA cycle (Aerobic) ATP ADPPC C Myokinase Ca ++ ATPase Ca + + CA ++ T T ATP-M + A MA + ADP CONTRACTIONCONTRACTION CPK Processes Involving Energy Production and Utilization by the Myocardium Ca ++ tricarboxylic acid cycle phosphocreatine

（ 4 ） Myocardial ultrastructure Histopathologic features in the myocardium following reperfusion. Contraction band necrosis Interstitial haemorrhage Neutrophilic plugging Distal platelets–fibrin microembolisation

2. Cerebral, Hepatic, Pulmonary, Renal ischemia-reperfusion injury Structure Metabolism function change

Section 4 Section 4 Principle of prevent & treatment 1. Controlling reperfusion condition 2. Antioxidant and OFR scavenging agents 3. Inhibition of neutrophil activation 4. Ca 2+ antagonists or Ca 2+ channel blocker

Zhao Mingyao