Ischemia-Reperfusion Injury and Free Radicals

Jianzhong Sheng MD PhD

• 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

HistoryHistory

• 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.

• 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.

Clinic:

• Shock, DIC microcirculation reperfusion• Coronary reperfusion 、 artery bypass graft• Restoration of blood supply in reimplantatio

n of limb, transplantation of organs.

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.

What is ischemia-reperfusion injury?

In the definition of ischemia-reperfusion injury

3 Key points are: Ischemia for a long time Reestablishment of blood flow More severe injury

Characteristics of ischemia-reperfusion injury:

1. Reversible Irreversible injury2. 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,

1. Duration of ischemia the longer period of ischemia, the more severe injury

2. Severity of ischemia the more grievous ischemia, the more severe injury

3. Speed of reperfusion the faster reperfusion of blood, the more severe injury

4. Ischemia preconditioning increasing tolerance to reperfusion injury

Why are more severe injury induced by reestablishment of blood flow after ischemia?

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

MICROVASCULAR DAMAGE—NO-Reflow Phenomenon

Pathogenesis of ischemia-reperfusion injury

1. Injury of free radicals

O2

(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.

1)Oxygen free radical

2)Lipid radical

(2) Oxygen free radical, OFR

Types：(1)the superoxide anion (O2-)

(2)the hydroxyl radical (OH ·)

(3)singlet oxygen (1O2 )

(4)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 ： 1) Fatty acid radical (L·)

2) Lipid peroxide （ LOO· ）

(4) Others: Cl·, CH3·, NO·

(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

(6) Generation of OFR

O2 + e O2

O2+ 2e + 2H+ H2O

2H2O2

O2 + 3 e + 3H+

HO +

H2OO2 + 4 e + 4H+ 2 H2O

Cytaa3

SOD

SOD, Superoxide dismutase

Haber-Weiss reaction (without Fe3 )

O2－ + H2O2 O2 + OH

+OH

SLOW

hydroxyl radical; ferrum

Fenton-Haber-Weiss reaction

Fe3

O2－ + H2O2 O2 + OH +

OH

FAST

(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)

Peroxydase （ H2O2 ）

Superoxide dismutase

MnSOD CuZnSOD

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

1) Mitochondria pathway

Ca2+entering MT

O2+e↑

O-2↑Hypoxia Mn-SOD ·

Superoxide dismutase

Mn− SOD + O-2 Mn+ − SOD + O2

2) Xanthine oxidase pathway

Xanthine oxidase (XO) 10%

Xanthine dehydrogenase (XD) 90%

Ca+2

Ischemia: ATP comsumption↑ Hypoxanthine ↑

Reperfusion: (1) Ca2+ overload→activating protein kinase

XO

(2) Restoration of O2 supply

xanthine + O·-2+ H2O2

O·-2+ H2O2 +Uric acid

O2

O2

XD

OH ·Effect of XO on formation of OFR

3) Neutrophil pathway

NADH(I)NADPH(II)+ O2 NADPH oxidase

H+ + O-2·+H2O2

NADH oxidase

C3, LTB4

(Complement C3 Leukotriene B4 )

Activating neutrophil Hexose shunt activity↑

cellular respiration ↑

4) Catecholamines

Adr Methyl transferasevanillylmandelic acid (normal)

RemoveStress 80% O2

adrenochrome O-

2·

monoamine oxidase

(8) Alterations induced by OFR

1) lipid peroxidation

a. Alteration of membrane lipid

b. Function inhibition of membrane proteins

c. Enhance of arachidonic acid metabolism

d. Blockage of ATP production in mitochondria membrane

2) Injury of chromosome and nuclear acid

80% induced by OH

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

Destructive effects of OFR:

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

Na + -Ca 2+

exchanger

Ca2+ Binding proteins

Mt

SR

Ca 2+

Ca 2+

Ca2+pumpCa2+

channelCa2+

(1) Mechanisms of calcium overload

1) Disorder of Na+ -Ca2+ exchange→ Intracellular Na+↑, H+↑, NE- 1R- PLC-PKC↑

2) Activation of Na+-H+ exchanger

3) Cellular membrane injury→ permeability↑, membrane phospholipid degradation↑, OFR↑

4) Injury of mitochondria

5) Catecholamines↑-R

Why dose calcium overload occur

during reperfusion

Depleted energe

Increased permeability of cellular

membrance

Increased intracellular sodium

(2) Alterations induced by calcium overload

1) Mitochondria function↓→ATP production↓

2) Activation of membrane phosphatidase→membrane damage

3) Cardiac arrhythmia

4) OFR↑

5) 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 re

perfusion

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

SELECTINS

Progressive Activation

INTEGRINS

CaptureSlow 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 blockagevessel permeability→edema

No reflow

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

OFRCa overload

endothelia-neutrophil

？

Alterations in metabolism and energy

Ca overload is common way of irreversible death of cells

Mechanisms of IRI

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 ar

rhythmia 3. Energy metabolism change in hea

rt 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)

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

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

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 injur

y

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

1. 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?