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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
21
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
39
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
Inci
denc
e of
In
cide
nce
of
Vent
ricul
ar F
ibril
latio
n Ve
ntric
ular
Fib
rilla
tion