09 ir injury

Dept. of PathologyDept. of Pathology

Medical CollegeMedical College

Hunan Normal UniversityHunan Normal University

(( 湖南师范大学医学院病理学教研室湖南师范大学医学院病理学教研室 )) 1

Chapter 9Chapter 9

Ischemia-Reperfusion Ischemia-Reperfusion InjuryInjury

（缺血（缺血 -- 再灌注损伤）再灌注损伤）

22

Ischemia-Reperfusion InjuryIschemia-Reperfusion Injury

①① IntroductionIntroduction

②② EtiologyEtiology

③③ Pathogenesis Pathogenesis

④④ Alterations of Metabolism and Alterations of Metabolism and

FunctionFunction

⑤⑤ Pathophysiological Basis of Prevention Pathophysiological Basis of Prevention

and Treatmentand Treatment

Ischemia

Concept

Injury More injuryReperfusion

“A paradox”

After prolonged ischemia, reestablishment of blood flow (reperfusion) does not relieve ischemic injury; On the contrary, it aggravates the tissue injury.

Oxygen Paradox

Low O2Perfusion Tissue injury

O2-containing fluid

Tissue injury Perfusion

Paradoxes During IR Injury

Calcium Paradox

Ca2+-free fluid Perfusion Tissue injury

Ca2+-containing fluid

Tissue injury Perfusion

Paradoxes During IR Injury

pH Paradox

Acidosis Tissue injury

Correction of Acidosis

Tissue injury Perfusion

Paradoxes During IR Injury

19551955 SewellSewellLigation of dog coronary artery → restore Ligation of dog coronary artery → restore

blood flow → ventricular fibrillation → deathblood flow → ventricular fibrillation → death

19601960 JenningsJennings Myocardial IRI Myocardial IRI (first to name IRI)

19681968 AmesAmes Brain IRIBrain IRI

19721972 Flore Flore Kidney IRIKidney IRI

19781978 Modry Lung IRIModry Lung IRI

19811981 Greenberg Intestine IRIGreenberg Intestine IRI

History of Ischemia-Reperfusion Injury (IRI) Research

88

Ischemia-Reperfusion InjuryIschemia-Reperfusion Injury

①① IntroductionIntroduction

②② EtiologyEtiology

③③ Pathogenesis Pathogenesis

④④ Alterations of Metabolism and Alterations of Metabolism and

FunctionFunction

⑤⑤ Pathophysiological Basis of Prevention Pathophysiological Basis of Prevention

and Treatmentand Treatment

Coronary Artery Bypass Graft (CABG)

Percutaneous Transluminal Coronary Angioplasty (PTCA)

Shock resuscitation (fluid infusion)

Organ transplantation

Thrombolytic therapy

EtiologyIschemia followed by reperfusion

CABG(Coronary Artery Bypass Graft )

Favaloro(1967)

Benetti(1995)

10

PTCA（ Percutaneous Transluminal

Coronary Angioplasty ）

Balloon Stent

11

Factors Influencing IR Injury

①①Duration of ischemia Duration of ischemia

②②Collateral circulationCollateral circulation

③③Dependency on oxygen supplyDependency on oxygen supply

④④Condition of reperfusionCondition of reperfusion

12

Effect of Duration of Ischemia on IRI

Ischemia time(min)

Reperfusion time(min)

Ventricular

tachycardia

()

Ventricular

fibrillation ()

Mortality

()

2 10 0 0 0

5 10 47.6 47.6 25.8

10 10 30.0 40.0 10.0

15 10 9.0 0 0

1414

Ischemia-Reperfusion InjuryIschemia-Reperfusion Injury

①① IntroductionIntroduction

②② EtiologyEtiology

③③ PathogenesisPathogenesis

④④ Alterations of Metabolism and Alterations of Metabolism and

FunctionFunction

⑤⑤ Pathophysiological Basis of Prevention Pathophysiological Basis of Prevention

and Treatmentand Treatment

Pathogenesis of IR Injury

①① Role of OFR/ROSRole of OFR/ROS

②② Calcium overloadCalcium overload

③③ Activation of neutrophilsActivation of neutrophils

Free Radicals

Highly reactive group of atoms, molecules or radicals, which carry unpaired electron in the outer orbit.

Free radicals that contain oxygen.

Properties of free radicalsProperties of free radicalsChemically activeChemically activeHighly oxidativeHighly oxidativeShort half-lifeShort half-life

Oxygen Free Radicals (OFR)

Reactive Oxygen Species (ROS)

A group of chemically reactive molecules containing oxygen.

Oxygen free radicals: O2 . , OH ., LO .

Non-free radicals: 1O2 , H2O2

Classification of ROS

Free Radicals

Oxygen Free Radicals

Reactive Oxygen Species

Non-Free Radicals

(Oxygen-

containing)

Non-Oxygen Free Radicals

O2 .

OH .

LO .

1O2

H2O2

OONO-

L .

Cl . CH3

.

The Relationship Between Free radicals and Reactive Oxygen Species

RReactive specieseactive species Half-lifeHalf-life Half-lifeHalf-life

Hydroxyl radical (Hydroxyl radical (OH)OH)

Alcoxyl radical (ROAlcoxyl radical (RO))

Singlet oxygen (Singlet oxygen (11OO22))

Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))

Peroxyl radical (ROOPeroxyl radical (ROO))

Nitric oxide (Nitric oxide (NO)NO)

Hydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22))

Hydroxyl radical (Hydroxyl radical (OH)OH)

Alcoxyl radical (ROAlcoxyl radical (RO))

Singlet oxygen (Singlet oxygen (11OO22))

Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))

Peroxyl radical (ROOPeroxyl radical (ROO))

Nitric oxide (Nitric oxide (NO)NO)

Hydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22))

1010-9-9 s s

1010-6-6 s s

1010-5-5 s s

00..05 – 105 – 1..00 s s

77 s s

1 – 101 – 10 s s

hours/hours/daysdays

hours/hours/daysdays

1010-9-9 s s

1010-6-6 s s

1010-5-5 s s

00..05 – 105 – 1..00 s s

77 s s

1 – 101 – 10 s s

hours/hours/daysdays

hours/hours/daysdays19

Half-life of Half-life of Some ROSSome ROSHalf-life of Half-life of Some ROSSome ROS

Mechanism of OFR Increase During IR Injury Mechanism of OFR Increase During IR Injury

①① Increased OFR productionIncreased OFR production

②② Decreased OFR clearanceDecreased OFR clearance

Role of ORF/ROS

Injurious Effects of OFRInjurious Effects of OFR

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Generation of OFR

①① Xanthine oxidase pathwayXanthine oxidase pathway

②② Neutrophils pathwayNeutrophils pathway

③③ Mitochondria pathwayMitochondria pathway

Xanthine Oxidase Pathway

Xanthine dehydrogenase

Xanthine oxidase (XO)

ATP

Ischemia

[Ca2++]i↑

ATP

ADP

AMP

Hypoxanthine XanthineO2._H2O2 + +

O2

ReperfusionUric acidO2

._

+ H2O2 +

XOO2

OH.

Ca2++-dependent proteinase

Adenosine

23

Generation of OFR

①① Xanthine oxidase pathwayXanthine oxidase pathway

②② Neutrophils pathwayNeutrophils pathway

③③ Mitochondria pathwayMitochondria pathway

OFR OFR

Respiratory burstRespiratory burst （呼吸爆（呼吸爆发）发）

Activation of neutrophilsActivation of neutrophils

IschemiaIschemia

Neutrophils Pathway

Oxygen consumptionOxygen consumption↑↑

ReperfusionReperfusion OO22

↑↑

Chemoattractants (CChemoattractants (C33, , LTBLTB44) )

Kill pathogenKill pathogen

Damage tissueDamage tissue

OO22

HH22OO22

HOClHOCl

NADPHNADPH oxidaseoxidase

NADPHNADPH

MPOMPO MPOMPO((myeloperoxidasemyeloperoxidase))

NADPNADP++

OHOH

NONO

OO22

OONOOONO--OHOH

Respiratory burst Respiratory burst 

Also called oxidative burst, is the rapid release of OFR Also called oxidative burst, is the rapid release of OFR from WBCs (neutrophils). from WBCs (neutrophils).

25

26

Generation of OFR

①① Xanthine oxidase pathwayXanthine oxidase pathway

②② Neutrophils pathwayNeutrophils pathway

③③ Mitochondria pathwayMitochondria pathway

Generation of Endogenous OFR

O2（ 98%） 4e_+4H

+

2H2O + ATPCytochrome

oxydase

e_

O2e

_+2H+

H2O2 OH.e

_+H+

H2O

e_+H+

H2O（ 1-2%）

_

SODO2. Fe Fe 3 3

Ischemia

Mitochondria Pathway

Ca2+ entering mito

ATP

OO22.._ _

e_

ReperfusionO2

OO22 +O2（ 98

%）4e

_+4H

+

2H2OCytochrome oxydase

e_

O2e

_+2H+

H2O2 OH.e

_+H+

H2O

e_+H+

H2O_O2.

×

(2%)

Mechanism of OFR Increase During IR Injury Mechanism of OFR Increase During IR Injury

①① Increased OFR productionIncreased OFR production

②② Decreased OFR clearanceDecreased OFR clearance

Role of ORF/ROS

Injurious Effects of OFRInjurious Effects of OFR

30

Clearance of OFR

①① Enzymatic clearanceEnzymatic clearance

SOD (Superoxide dismutase)SOD (Superoxide dismutase)

CAT (Catalase)CAT (Catalase)

②② Non-enzymatic clearanceNon-enzymatic clearance

Enzymatic Clearance of OFR

e_

O2e

_+2H+

H2O2e

_+H+

（ 1-2%）_O2. H2O + O2

SOD CAT

Mn-SOD CuZn-SOD

Amyotrophic lateral sclerosis(ALS)

Mutation

Stephen Hawking

32

Clearance of OFR

①① Enzymatic clearanceEnzymatic clearance

②② Non-enzymatic clearanceNon-enzymatic clearance

Non-enzymatic OFR scavengers Non-enzymatic OFR scavengers

Vitamins (Vit C, Vit E)Vitamins (Vit C, Vit E)

CeruloplasminCeruloplasmin

Dimethyl sulfoxide (DMSO)Dimethyl sulfoxide (DMSO)

Allopurinol Allopurinol

Glutathione (GSH)Glutathione (GSH)

H2O2 + 2GSH 2H2O + GSSG

Mechanism of OFR Increase During IR Injury Mechanism of OFR Increase During IR Injury

①① Increased OFR productionIncreased OFR production

②② Decreased OFR clearanceDecreased OFR clearance

Role of ORF/ROS

Injurious Effects of OFRInjurious Effects of OFR

Injurious Effects of OFR

-S-S-

CH3-S-Lipid

crosslinkingO

OHHO

Fatty acid oxidation

Lipid Damage

Cell membrane damageDestruction of membrane structureIncreased permeabilityMembrane protein function ↓

Organelle membrane damageLysosome

Destruction of membrane → release of lysosomal enzymes → autocytolysis

MitochondrionSwelling → Mito dysfunction → ATP ↓

Sarcoplasmic reticulum (SR)SR uptake of Ca2+ ↓ → Ca2+ overload

Consequences of Lipid Damage

Protein Damage

Protein breakage

-S-S-

Protein crosslinking

Lipid-protein crosslinking

OHHO

OHHO

Disulfide bridging

OH

DNA Damage

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Pathogenesis of IR Injury

①① Role of ORF/ROSRole of ORF/ROS

②② Calcium overloadCalcium overload

③③ Activation of neutrophilsActivation of neutrophils

Calcium Overload

During IR, the concentration of cytosolic CaDuring IR, the concentration of cytosolic Ca2+2+

increases obviously, causing cell damage and increases obviously, causing cell damage and

dysfunction. This phenomenon is called “Calcium dysfunction. This phenomenon is called “Calcium

Overload”.Overload”.

Mechanisms of Calcium Overload

NaNa++-Ca-Ca2+ 2+ exchanger dysfunctionexchanger dysfunction

Damage in cell membraneDamage in cell membrane

Damage in organelle (Mito or SR) membraneDamage in organelle (Mito or SR) membrane

Na+-Ca2+

Exchanger

Ca2+ Pump

Ca2+

[Ca2+]e ： 10-3M [Ca2+]i ： 10-7M

Ca2+ channel

MitoSR

Ca2+

Na +

Ca 2+

Ca 2+

SR: Sarcoplasmic reticulum

Ischemia → ATP↓→ Na+-K+ ATPase↓→ [Na+]i↑→ Activate Na+-Ca2+ exchanger

K+

Na+

3Na+

Ca2+

ATP↓

Na+↑Ca2+↑

Mechanism of Na+-Ca2+ Exchanger Dysfunction

Mito SR

IRI

[Ca2+]i ↑

OFR ↑

Proteinase

Destruction Membrane Cytoskeleton

Nuclease

Chr Damage

Na+-Ca2+

ExchangerCa 2+

Na +

Mito dysfunction

Mito oxidation ↓

Consequences of Calcium Overload

Pathogenesis of IR Injury

①① Role of ORF/ROSRole of ORF/ROS

②② Calcium overloadCalcium overload

③③ Activation of neutrophilsActivation of neutrophils

IR injuryIR injury

Activation of Neutrophils

Chemokines(LTs, PAF, Kinin)

Adhesion molecules (integrin, ICAM-1)(integrin, ICAM-1)

Activation of Neutrophils

Granzymes(elastase,

collagenase)

OFR Cytokines

Ischemia injury

Reperfusion injury

Reperfusion

Free Radicals

Ca2+ overload

Neutrophils

Summary of Pathogenesis of IR Injury

4848

Ischemia-Reperfusion InjuryIschemia-Reperfusion Injury

①① OverviewOverview

②② EtiologyEtiology

③③ Pathogenesis Pathogenesis

④④ Alterations of Metabolism and Alterations of Metabolism and

FunctionFunction

⑤⑤ Pathophysiological Basis of Prevention Pathophysiological Basis of Prevention

and Treatmentand Treatment

OO22--

HH22OO22

HOClHOClOOHH

GutGut

Heart & Heart & vesselsvessels

Lungs &Lungs &airwaysairways

Brain &Brain &nervesnerves

49

IR Injury to Important OrgansIR Injury to Important Organs

ArrhythmiaArrhythmiaVentricular fibrillationVentricular fibrillationVentricular TachycardiaVentricular Tachycardia

Myocardial dysfunctionMyocardial dysfunctionCO CO ↓↓Myocardial stunningMyocardial stunning

Reversible reduction of the function of heart Reversible reduction of the function of heart contraction after reperfusion.contraction after reperfusion.

Restored after a few hours or days. Restored after a few hours or days.

Myocardial IR Injury

5151

Ischemia-Reperfusion InjuryIschemia-Reperfusion Injury

①① IntroductionIntroduction

②② EtiologyEtiology

③③ Pathogenesis Pathogenesis

④④ Alterations of Metabolism and Alterations of Metabolism and

FunctionFunction

⑤⑤ Pathophysiological Basis of Prevention Pathophysiological Basis of Prevention

and Treatmentand Treatment

Reduce ischemia

Control reperfusion conditions

Scavenge OFR

Relieve Ca2+ overload

Improve metabolism

- Energy supplementation

- Cell protectors

Prevention and Treatment of IR Injury

Lower pressureLower pressure

Lower flow speedLower flow speed

Lower temperatureLower temperature

Lower pHLower pH

Lower CaLower Ca2+2+ and Na and Na++

↓ ↓ OFR and edemaOFR and edema

↓ ↓ CaCa2+2+ overload overload

↓ ↓ metabolism →↓ energy metabolism →↓ energy consumptionconsumption

Control Reperfusion ConditionsControl Reperfusion Conditions

NS: Normal salineSOD: Superoxide dismutaseDMSO: Dimethyl sulfoxideVerap: Verapamil (Calcium antagonist)

Treatment of IR-induced Myocardial Injury

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