Ischemia-Reperfusion Injury and Free Radicals Jianzhong Sheng MD PhD

History In 1955，Sewell et al., reported that ventricular fibrillation occurred in dogs when sudden return of blood flow to the coronary artery that was ligated

The injury of ischemic cardiac muscles was Myocardial reperfusion injury was first postulated in 1960 by Jennings et al. in their description of the histologic features of reperfused ischemic canine myocardium. The injury of ischemic cardiac muscles was more severe after reperfusion than before. 证实再灌注会引起心肌超微结构不可逆性坏死，包括爆发性水肿，组织结构崩解，收缩带形成和线粒体内磷酸钙颗粒形成。

1967，Bulkley and Hutchins found that reflow of blood induced necrosis of cardiac myocytes after coronary artery bypass graft surgery. 1981, Greenberg confirmed that reperfusion induced severe damage of the intestinal mucosal cells of cat after 3 hours of ischemia. 通过几十年的观察研究，人们逐渐认识了这种缺血、恢复血流灌注后反常的有害现象，对其进行了较为深入的研究。

Clinic: Shock, DIC microcirculation reperfusion Coronary reperfusion、artery bypass graft Restoration of blood supply in reimplantation of limb, transplantation of organs.

What is ischemia-reperfusion injury? The restoration of blood flow after transient ischemia may be associated with further reversible or irreversible cell damage, which is called ischemia-reperfusion injury or reperfusion injury.

3 Key points are: In the definition of ischemia-reperfusion injury Ischemia for a long time Reestablishment of blood flow More severe injury

Characteristics of ischemia-reperfusion injury: 1. Reversible  Irreversible injury 2. Having reported in heart, kidney, liver, lung, brain, intestine, skeletal muscles Calcium paradox Oxygen paradox pH paradox

Etiology of ischemia-reperfusion injury Cause Ischemia followed by reperfusion

Which factors are involved in reperfusion injury Generally speaking, Duration of ischemia the longer period of ischemia, the more severe injury Severity of ischemia the more grievous ischemia, the more severe injury Speed of reperfusion the faster reperfusion of blood, the more severe injury Ischemia preconditioning increasing tolerance to reperfusion injury

Why are more severe injury induced by reestablishment of blood flow after ischemia? MICROVASCULAR DAMAGE—NO-Reflow Phenomenon Vaso-endothelial edema ATP depletion  decreased Na+-K+ pump function  Na+ and water  entering cell  endorthelial edema Vaso-endothelial damage WBC adherence  OFR increase and NO decrease Microvascular obstruction Squeeze of the coronary arteries induced by ischemic myocardium and by interstitial edema of myocardium Adherence, Aggregation and Activation of WBC

Pathogenesis of ischemia-reperfusion injury

1. Injury of free radicals

(1) Free radical Free radical——atoms, molecules or ions with unpaired electrons on an otherwise open shell configuration. These unpaired electrons are usually highly reactive, so radicals are likely to take part in chemical reactions. Oxygen free radical Lipid radical

(2) Oxygen free radical, OFR Types： the superoxide anion (O2-) the hydroxyl radical (OH ·) singlet oxygen (1O2 ) hydrogen peroxide (H2O2)

(3) Lipid free radicals： The interaction of oxygen free radicals with polyunsaturated fatty acids in the phospholipids of cell membrane leads to the formation of lipid free radicals. Types： Fatty acid radical (L·) Lipid peroxide（LOO·） (4) Others: Cl·, CH3·, NO· 氧化还原过程中产生的具有高活性的一系列中间产物

(5) Generation and elimination of oxygen free radicals 1) Origin of O·-2： a. Mitochondria b. Oxidation of some chemicals in body. c. Catalysis by enzymes d. Stimulation of cells with toxins

O2  SOD (6) Generation of OFR O2 + e Cytaa3 O2 + 3 e + 3H+ 2 H2O HO + H2O H2O2 2 H2O O2 + 4 e + 4H+ SOD, Superoxide dismutase

Haber-Weiss reaction (without Fe3 ) O2－ + H2O2 O2 + OH +OH  SLOW hydroxyl radical; ferrum

Fenton-Haber-Weiss reaction O2－ + H2O2 O2 + OH +OH  FAST

Cysteine, Vit C, glutathione (6) Elimination of oxygen free radicals 1）Small MW scavenging agents Dihydrocoenzyme II Cysteine, Vit C, glutathione Vit E、 Vit A

2）Enzymatic scavenging agents Catalase (CAT) Superoxide dismutase MnSOD CuZnSOD Peroxydase （H2O2）

(7) The mechanisms of increased generation of oxygen free radicals during ischemia-reperfusion

1) Mitochondria pathway Ca2+entering MT O2+e↑ Mn− SOD + O-2 Mn+ − SOD + O2 O-2↑ · Hypoxia Mn-SOD  Superoxide dismutase Pm O2 呼吸链受抑 NADH蓄积供电子

2) Xanthine oxidase pathway Xanthine oxidase (XO) 10% Xanthine dehydrogenase (XD) 90% Ca+2

Effect of XO on formation of OFR Ischemia: ATP comsumption↑ Hypoxanthine ↑ (1) Ca2+ overload→ activating protein kinase O2 Reperfusion: xanthine + O·-2+ H2O2 XD XO (2) Restoration of O2 supply O2 O·-2+ H2O2 +Uric acid Effect of XO on formation of OFR OH ·

H+ + O-2·+H2O2 3) Neutrophil pathway NADH(I) NADPH(II) + O2 Activating C3, LTB4 (Complement C3 Leukotriene B4 ) Hexose shunt activity↑ cellular respiration ↑ NADH oxidase NADH(I) NADPH(II) H+ + O-2·+H2O2  + O2 NADPH oxidase

4) Catecholamines  Adr Remove O-2· adrenochrome Methyl transferase vanillylmandelic acid (normal) monoamine oxidase Stress 80% O2 Remove O-2· adrenochrome

(8) Alterations induced by OFR 1) lipid peroxidation Alteration of membrane lipid Function inhibition of membrane proteins Enhance of arachidonic acid metabolism Blockage of ATP production in mitochondria membrane 4造成生物膜损害

2) Injury of chromosome and nuclear acid 80% induced by OH

Destructive effects of OFR: Attacking membrane structure such as mitochondria membrane  interfering with energy metabolism Attacking DNA  changing genetic information  cell death Initiating lipid peroxidation  increasing permeability of membrane and inducing destruction of membrane  cell death Destroying proteins  decreased enzyme activity  metabolic disorder

How to maintain Intracellular calcium Calcium Overload Intracellular calcium concentration abnormally increases and leads to cell and tissue damages How to maintain Intracellular calcium at normal level? Ca2+ Pump on cell membrance Na+ - Ca2+ exchage pump Ca2+ Pump on mitochondrial membrane Ca2+ Pump on endoplasmic reticulum

2. Calcium overload Ca2+ Binding proteins Mt SR Ca2+pump Ca2+ channel Na + -Ca 2+ exchanger Ca2+ Binding proteins Mt SR Ca 2+ Ca2+pump Ca2+ channel 1 钠-钙交换过程： 钙跨膜转运机制顺着某些离子的浓度梯度弥散。 2 影响交换的因素： 决定因素：细胞外Na+浓度 影响细胞外Na+浓度的因素： Na+-K+交换 “钠-钙交换过程是可逆的。 1 钙泵（calcium pump) （耗能） 2 钠-钙交换(Na+-Ca2+ exchange) （间接耗能） 3 钙通道（calcium channels)

(1) Mechanisms of calcium overload Disorder of Na+ -Ca2+ exchange→ Intracellular Na+↑, H+↑, NE- 1R- PLC-PKC↑ Activation of Na+-H+ exchanger Cellular membrane injury→ permeability↑, membrane phospholipid degradation↑, OFR↑ Injury of mitochondria Catecholamines↑-R glycocalyx

Why dose calcium overload occur during reperfusion Depleted energe Increased permeability of cellular membrance Increased intracellular sodium

(2) Alterations induced by calcium overload Mitochondria function↓→ATP production↓ Activation of membrane phosphatidase→membrane damage Cardiac arrhythmia OFR↑ Myofibril contracture, rupture, cell damage

What are effects of calcium overload Damage mitochondria → ATP production decrease Cause myocardial injury → contraction weakness promote OFR formation → damage aggravation

3. The Role of Leukocyte Blocking microvasculature in the region of reperfusion Adhering to microvascular endothelium through interaction between L-selectin on surface of WBC and ICAM-1 (cell adhesion molecules) on surface of endothelium Damaging tissues and cells in the region of reperfusion through releasing arachidonic acid (AA)  TXA2, lysosomal enzymes etc. And producing OFR in “respiratory burst”.

Accumulation of WBC Progressive Activation SELECTINS INTEGRINS Capture Slow Rolling Firm Adhesion Transmigration Rolling Chemotactic factor Adhesion molecule)

Vascular endothelial cells and neutrophil injury 1. Microvessel injury (1) no-reflow phenomenon (2) Change in blood flow, diameter and permeability of vessel 2. Cell injury OFR, lysoome, cell factors Cell adhesion, accumulation, flow blockage vessel permeability→edema 四 磷酸化合物缺乏 （一）缺乏的机制 1线粒体受损 →ATP生成↓。 2合成ATP的前体物质↓。 （二）缺乏引起再灌注损伤的机制 1钠泵、钙泵功能障碍→钙超载。 2氧自由基↑。 卅四 磷酸化合物缺乏 No reflow

WBC ADHERENCE to ENDOTHELIUM Damaged endothelium NO decrease CAMs upregulation L-selectin – ICAM-1 WBC ADHERENCE to ENDOTHELIUM Releasing OFR, TXA2, lysosomal enzymes Blocking blood flow Damaging tissues and cell NO, nitric oxide; CAM, cell adhesion molecules; TXA2, thromboxane A2

Mechanisms of IRI OFR Ca overload ？ endothelia- neutrophil Alterations in metabolism and energy Ca overload ？ OFR endothelia- neutrophil Ca overload is common way of irreversible death of cells

↓ ↓ ISCHEMIA—REPERFUSION INJURY Excess oxygen Neutrophil Free radicals infiltration ↓ ↓ ISCHEMIA—REPERFUSION INJURY ↑ ↑ Microvascular damage Calcium overload Major mechanisms of ischemia- reperfusion injury

Alterations of metabolism and function during ischemia-reperfusion injury

Heart 1. Cardiac function-heart pump↓ 2. Electrocardiogram-Reperfusion arrhythmia 3. Energy metabolism change in heart 4. Change in cardiac microstructure

Brain 1. Alterations of brain metabolism (energy↓, acidosis, FFA, transmitters) cAMP↑ / cGMP↓ →PL↑ 2. Abnormal electroencephalogram (EEG) (Slow wave, excitatory transmitters inhibitory transmitters↑) 3. Alterations in brain structure (edema, necrosis)

Ischemia-reperfusion injury in other organs (intestine, kidney, bone)

↓ ↓ ISCHEMIA—REPERFUSION INJURY Excess oxygen Neutrophil Free radicals infiltration ↓ ↓ ISCHEMIA—REPERFUSION INJURY ↑ ↑ Microvascular damage Calcium overload Major mechanisms of ischemia- reperfusion injury

1.Vasomotor Responses OFR Calcium Overload WBC ↘ ↓ ↙ ↘ ↓ ↙ Damaged endothelium ↙ ↘ NO, PGI2 release↓ TXA2, ET release↑ ↘ ↙ VASOCONSTRICTION ↓ Aggravating injury

Vascular Sticking WBC Liable to form OFR Calcium Overload WBC Damaged endothelium Vascular Sticking WBC Liable to form permeability↑ platelets to endothelium thrombosis Edema Releasing OFR Blocking blood proteolytic enzymes flow Aggravating injury

Heart Brain Liver Intestine ↓ ↓ ↓ ↓ Shock Cytotoxic Jaundice Mucosal Excess Oxygen Neutrophil Microvascular Calcium Free radicals infiltration damage overload ↓ ↓ ↓ ↓ ISCHEMIA—REPERFUSION INJURY ↓ ↓ ↓ ↓ Heart Brain Liver Intestine ↓ ↓ ↓ ↓ Shock Cytotoxic Jaundice Mucosal Pump failure edema Enlargement necrosis Arrhythmia Neuron death GTP↑ Ulceration Hemorrhage

Pathophysiological basis of prevention and treatment for ischemia-reperfusion injury

ISCHEMIA-REPERFUSION INJURY How to prevent and treat ISCHEMIA—REPERFUSION INJURY ? Relieving ischemic condition as a prerequisite Excess Oxygen Calcium Neutrophil Free radicals overload infiltration ↓ ↓ ↓ ISCHEMIA-REPERFUSION INJURY ↑ ↑ ↑ OFR Calcium WBC Scavenger Antagonist Antiboby

Controlling reperfusion conditions Reflow as early as possible, low pressure, flow, temperature, pH, Na+, Ca2+ 2. Improving metabolism of ischemic tissue. ATP、Cyt.C、quinhydrone 3. Removing free radical 4. Reducing Ca overload 5. Others

Questions 1. What is ischemia-reperfusion injury? 2. What is free radical? 3. What mechanisms of ischemia-reperfusion injury?