Ischemia-reperfusion Injury

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Chaper 10
Ischemia-reperfusion Injury
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Introduction
Concept
To recover the blood perfusion after tissue
ischemia aggravates the tissue damage and even
induces irreversible damage. This paradoxical
phenomenon is termed ischemia-reperfusion injury.
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Clinical significance
It has been demonstrated that
ischemia-reperfusion injury may occur in many
organs for example the heart, brain, liver,
kidney, lung, intestine, skeletal muscle and skin
etc..
It has very important practical significance
to reduce or prevent the ischemia-reperfusion
injury, for such as organ transplantation,
microcirculation reperfusion in shock, cardiac
bypass surgery, thrombolysis, etc..
Experimental research found the calcium paradox,
oxygen paradox and pH paradox.
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Calcium paradox With calcium-free solution to
perfuse in vitro heart of rat in 2 minutes, and
then calcium solution infusion, abnormal changes
of the myocardial electric signals, cardiac
function, metabolism and morphologcal structure
occur, this phenomenon known as the calcium
paradox.
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Oxygen paradox with hypoxic solution, to perfuse
tissues and organs or to culture cells in a
certain time under hypoxic conditions, then
return to normal oxygen supply to tissues and
cells, it not only failed to restore the damage,
but more seriously, a phenomenon known as the
oxygen paradox.

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pH paradox metabolic acidosis induced by
ischemia is an important reason of cell function
and metabolic disorder, but quickly corrected
acidosis of ischemic tissue during reperfusion,
will aggravate cell injury, known as the pH
paradox.
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Section 1
Factors and conditions of ischemia -reperfusion
injury
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1, Factors of IRI
Any of the blood reperfusion after tissue and
organ ischemia may cause ischemia reperfusion
injury.
For example, microcirculation reperfusion in
shock, cardiac bypass surgery, myocardial
infarction, thrombolytic therapy,
PTCA(???????????), organ transplantation,
cardio-pulmonary resuscitation,etc..
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2, Conditions of IRI
(1) Duration of ischemia
Too short ischemia period no IRI
Too long ischemia period necrosis of tissue
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(2) Collateral circulation
(3) Dependentcy on oxygen supply
(4) Conditions of reperfusion
Lower reperfusion pressure, Lower
temperature, lower pH, lower concentration of Na
and Ca2 are associated with mild RI.
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Section 2
Mechanisms of ischemia-reperfusion injury
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Contents
1, Effect of free radicals
2, Calcium overload
3, Effect of white cells
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1, Effect of free radicals
1,1. Concept and species of FR
Concept of FR
FR are a highly reactive group of atoms
,radicals and molecules, which carry unpaired
electron in outer orbital.
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Species of FR
(1) Oxygen free radical,OFR
Free radicals induced by oxygen
Superoxide anion radical(O2.-) Hydroxyl radical
(OH.)
OFR
Reactive oxygen species
Singlet oxygen(1O2) Hydrogen peroxide(H2O2)
non OFR
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(2) Lipid free radical
It is an intermediate metabolites produced by
reaction both OFR and polyunsaturated fatty acid
L (????) , LO (?????) , LOO (??????)
(3) Others
CL , CH3 , NO. , etc..
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1,2. Metabolism of FR
(1) Production of ROS
Mitochondria --- the main location of O2.-
production
4e- 4H
Normal O2 ? ? ? H2O ATP
e-
e- 2H
e- H
e- H
Abnormal O2? ? O-2 ? ? H202 ? ? OH ? ?
H20
H20
In addition, O-2 is produced by oxidation
process of Hb, Mb, CA, XO, NADPH oxidase
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Production of OH ---- Fenton reaction
Fe2or Cu2
O-2 H2O2 ? ? ? OH OH- O2
(2) Defense system of anti-oxidation in vivo
Enzyme antioxidant---- SOD?CAT?GSH-Px etc..
Non enzyme antioxidant ---- VitE?VitA? VitC etc..
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1,3. Mechanism of FR generation
(1) Xanthine oxidase pathway
(2) Neutrophils pathway
(3) Mitochondria pathway
(4) Catecholamine auto-oxidation pathway
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1. Xanthine oxidase pathway
ATP
Ca2-dependent proteases
Adenine nucleoside
Hypoxanthine nucleoside
Xanthine

Hypoxanthine
H
2
O2
O2
OH
Reperfusion phase
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(2) Neutrophils pathway
Activated neutrophils
IR
XO?
FR
LTs, C3
NADPH oxidase NADH oxidase
Respiratory burst
OFR
IRI
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(3) Mitochondria pathway
?ATP
?Oxidative phosphorylation
Ischemia hypoxia
?PO2 in cell
?Ca2 in Mit
?One-electron reduction of O2
?Mn-SOD in Mit
?OFR
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(4) Catecholamine auto-oxidation pathway
Ischemia, hypoxia
Activated Sympathetic - adrenal medulla System
?Catecholamine
autooxidation
?OFR
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1,4. Mechanism of IRI caused by FR
(1 ). Enhanced lipid peroxidation of membrane
? Distroying structure of membrane
lipid peroxidation
?unsaturated fatty acid
?fluidity
?Internal flow of Ca2
?permeability
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? Indirectly inhibiting membrane protein function
Lipid-lipid cross-link, Polymerization
Lipid peroxidation
(-) Receptors of M
(-) Na ATPase
(-) G-protein coupled receptor
(-) Ca2 ATPase
(-) Na-Ca2 exchanger
Dysfuction of cellular signal transduction
Cellular edema
Calcium overload
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? Promoting synthesis of FR and bioactive
substance
()Phospholipase C
Lipid peroxidation
()Phospholipase D
Decomposition of membrane phospholipids
arachidonic acid
FR
FR
FR
PG
TXA2
LTs
? Deareased ATP synthesis
Lipid peroxidation of Mit
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(2). Inhibition of protein function
Protein denaturation protein cross-link
Impairment of protein function
For example
?Myocardial contractility
Injury of myocardial fiber protein
Injury of calcium transport protein in SR
abnormal Ca2 regulation
(3).DNA disruption and chromosome aberration
OH
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2, Calcium overload
Concept of Calcium overload
The phenomenon of cellular structure damage
and disturbance of function and metbolism which
caused by intracellular calcium increasing
abnormally due to various factors is termed
calcium overload
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????Ca2????
Ca2
Ca2-ATP-E
Na
Ca2
Ca2
Ca2?
10-3mmol/l
Ca2
10-7mmol/l
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2,1. Mechanism of calcium overload
(1)Abnormity of Na - Ca2 exchange
It is the main pathway of internal flow of Ca2
in IRI
? The higher intracellulr Na activating the
Na/Ca2 exchanger directly
Ischemia? ?ATP??Na pump ??Na in cells ?
Reperfusion?activating Na-Ca2 exchange??Ca2 in
cells
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? The higher intracellulr H activating the
Na/Ca2 exchanger indirectly
Ischemia??H in cells and interstitial fluid ?
Reperfusion??H in interstitial fluid and?H in
cells ?
Activating H-Naexchange ??Na in cells ?
activating Na-Ca2 exchange??Ca2 in cells
? The activated PKC activating the Na/Ca2
exchanger indirectly
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PKC???Na-Ca2???????????
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(2)Biological membrane damage
? Cell membrane injury
A. Ischemia ? destroying cell M
B. Enhanced lipid peroxidation of M
?permeability
C.?Ca2 ?activating phospholipase
? SR membrane ??intake of Ca2 ??Ca2 in cytoplasm
? Mitochandria membrane ? ?ATP ? ?Ca2 ATPase of
M and SR??Ca2 in cytoplasm
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2,2. Mechanism of IRI caused by Ca2 overload
(1) Promoting sythesis of FR
?Ca2in cells??Ca2-dependent proteases??XO??FR
(2) Aggravating acidosis
?Ca2in cells??ATPases? hydrolysis of ATP ??H
(3) Destroying cell membrane
?Ca2in cells?activating phospholipase
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(4) Mitochandria dysfunction
?Ca2in cells??Ca2in Mit ?calcium phosphate
??ATP production
(5) Activating other enzymes
Activating proteases ?decomposition of M and
structure proteins
?Ca2in cells ?
Activating nuclease ?injury of chromosome
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3, Effect of white cells
It has been manifested that the microvessel
and cell damage which mediated by leukocytes play
an important role in IRI.
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3,1. Mechanism of neutrophils increase during IRI
(1) Increased production of adhesion molecules
Selectin ---- P- selectin, L- selectin
Integrin ---- CD11/CD18
Immunoglobulin superfamily ---- ICAM-1, VCAM-1
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(2) Increased production of chemokines
Injury of tissue
leukotriene?prostaglandin?PAF?kinin
neutrophils
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3,2. Mechanism of IRI mediated by neutrophils
(1) Injury of microvessel
? Alteration of hemorheology in microvessel
no-flow phenomenon
? Alteration of microvessel calibre
? Increase of microvessel permeability
(2) Injury of cells
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Endothelium
L-selectin
VSMC
VSMC
VSMC
VSMC
VSMC
Myocyte
Myocyte
Myocyte
Myocyte
Myocyte
Myocyte
Interaction between endothelial cells and
neutrophils.
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?????(no-flow phenomenon)
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Cell necrosis
Ca2
Ca2overload
Ischemia
Inflammatory factors
Cell damage
Reperfusion
Neutrophils
No reflow
O2
?OFR
Tissue recovery
Mechanism of ischemia reperfusion injury
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Section 3
Functional and metabolic changes during IRI
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1, IRI of myocardium
1,1. Myocardial stunning
The recovery of blood perfusion in ischemic
myocardium for a period of time, manifests the
phenomenon of reversible decreased contractile
function, known as myocardial stunning.
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1,2. Reperfusion arrhythmia
Ischemic myocardium manifests arrhythmias in
the process of reperfusion, known as reperfusion
arrhythmia.
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The major manifestations are ventricular
tachycardia and fibrillation
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Mechanism of reperfusion arrhtthmia
? Nonuniformity of action potential duration
? Formation of delayed afterdepolarization after
the action potential
? Changes in electrophysiological characteristics
of myocardium
? Decreased fibrillation threshold
? Decreased NO
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1,3. Alteration of energy metabolism
Damage of mitochandria
?Synthesis of energy
?High-energy phosphate Compounds
Reperfusion
Loss of substrate
1,4. Alteration of myocardial ultrastructure
Alteration of myocardial ultrastructure both
in reperfusion injury and in ischemia injury is
basically the same.
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2, IRI of brain
2,1. Alteration of energy metabolism
?cAMP
Activating phospholipase
IR
Free radicals
?free fatty acids
lipid peroxidation
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2,2. Alteration of amino acid metabolism
?Excitatory amino acids ---- glutamic acid,
Aspartic acid ?
?inhibitory amino acids ---- Alanine???, GABA,

Taurine???, glycin???
2,3. Alteration of histology in brain
Brain edema and necrosis of brain cells
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3, IRI of other organs
3,1. IRI of lung
3,2. IRI of intestine
3,3. IRI of kidney
3,4. IRI of liver
3,5. IRI of skeletal muscle
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Section 4
Prevention and treatment of IRI
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1, Elimination of ischemia causes and an early
recovery of blood flow
2, Controlling conditions of reperfusion
Lower temperature, pressure, pH, flow speed,
Ca2, Na and higher K
3, Improving metabolism of ischemia tissues
ATP, cytochrome C and hydroquinone??
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4, Eliminating free radicals
Vit E,A,C, GSH,NADPH
SOD, CAT, GSH-Px, Ceruloplasmin
Inhibitor of XO -- allopurinol????,
scavenger of OH--DMSO????
5, Lightening Ca2 overload
Calcium channel blockers, inhibitors of Na-Ca2
exchanger
6, Application of inhibitors of neutrophil
Hydroxyurea ???
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7, Application of cell protective agent
Taurine???, Metallothionein?????
8, Others
Ischemia preconditioning, IPreC, ?????
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Ischemia postconditioning, IPostC, ?????
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Question?
1.Ischemia-reperfusion injury, calcium paradox,
oxygen paradox, pH paradox, free radicals,
Reactive oxygen species, Calcium overload,
no-flow phenomenon, Myocardial stunning,
reperfusion arrhythmia,
2. Briefly describe the pathogenesis of IRI
3. Briefly describe the mechanism of OFR
generation
4. Briefly describe the mechanism of IRI caused
by FR .
5. Briefly describe the mechanism of calcium
overload.
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6. Briefly describe the mechanism of IRI induced
by calcium overload.
7. Briefly describe the effects of white cells in
IRI.
8. Briefly describe the mechanism of myocardial
stunning and reperfusion arrhythmia.
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The higher intracellulr H activating the
Na/Ca2 exchanger indirectly
Reperfusion??H
Na
?H
?ATP?
ischemia?
Na
Na
?Ca2
Ca2
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1,4. Injury of oxygen free radical
Target Chemical alteration Physiological alterations
Lipids Peroxidation of double bonds Lipid-lipid cross-linking Membrane permeability changes Impairment of organelle function Receptor conformation changes
Protein Oxidation of double bonds Oxidation of SH groups Enzyme inhibition Protein denaturation Receptor conformation changes
Carbohydrates Oxidation of double bonds Receptor conformation changes Denaturation of glycoprotein
Nucleic acids Oxidation of double bonds Strand scission Base modification Mutations
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Ca2 overload
Ischemia-reperfusion injury of myocardium
induced by Ca2 overload.
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????Ca2????
Ca2
Ca2-ATP-E
H
ATP?
Na2
Ca2
Ca2
Ca2?
10-3mmol/l
10-7mmol/l