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
• 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
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.
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 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,
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?

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

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+ H2O2
SOD
Cytaa3
O2 + 3 e + 3H+ HO + H2O
H2O2
O2 + 4 e + 4H+ 2 H2O

SOD, Superoxide dismutase

 Haber-Weiss reaction (without Fe3 )

O 2  + H 2O 2
－
O2 + OH +OH

SLOW

hydroxyl radical; ferrum

 Fenton-Haber-Weiss reaction

3
Fe
O 2  + H 2O 2
－
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)
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

Hypoxia Mn-SOD  ·
-
O 2↑
Superoxide dismutase
2) Xanthine oxidase pathway

Xanthine oxidase (XO) 10%

Ca+2

Xanthine dehydrogenase (XD) 90%

Ischemia: ATP comsumption↑ Hypoxanthine ↑

(1) Ca2+ overload→ O2

Reperfusion: activating protein kinase

xanthine + O·-2+ H2O2

XD XO

(2) Restoration of O2 O2
supply

O·-2+ H2O2 +Uric acid

Effect of XO on formation of OFR
OH ·
 3) Neutrophil pathway

Activating
C3, LTB4 neutrophil Hexose shunt activity↑
(Complement C3
Leukotriene B4 )

cellular respiration ↑

NADH oxidase
NADH(I) H +O-
2·+H2O2
+
NADPH(II) + O2 NADPH oxidase
 4) Catecholamines 

Methyl transferase
Adr vanillylmandelic acid (normal)
monoamine oxidase

Stress 80% O2
Remove
O-2·
adrenochrome
 (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

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
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
Ca 2+

Ca 2+

Ca2+ Ca2+ Ca2+pump

channel Binding
Ca2+
proteins
SR
Na + -Ca 2+
exchanger
Mt
(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
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
Capture
Rolling Slow Firm
Rolling Adhesion Transmigration

SELECTINS
INTEGRINS

Chemotactic
Adhesion molecule)
factor
 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 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

 Mechanisms of IRI
Alterations in metabolism
and energy

Ca overload ？
OFR

endothelia-
neutrophil

Ca overload is common way of irreversible death of cells

 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)
 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
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
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?