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Introduction
l 1955，Sewell，ventricular fibrillation after
缺血再灌注损伤 block coronary artery and then unblock

Ischemia-Reperfusion Injury (I-RI) l 1960，Jennings，Ischemia-Reperfusion Injury

l other organs, including brain(1968),
LIU Yongming kidney(1972), lung(1978), intestine(1981), liver,
pancreas…
Department of Pathophysiology
l Clinical phenomenon: bypass surgery, shock
School of Basic Medical Sciences
treatment, organ transplantation, thrombolysis,
Wuhan University Percutanueous Transluminal Coronary
Angioplasty (PTCA)

Concept When do cells die during ischemia-reperfusion?

• Ischemia Injury:
Ischemic death
decreased blood perfusion lead to injury of 80
Reperfusion
Reperfusion death???
histocytes and tissue damage 60
Cell Death (%)

40

• Ischemia-Reperfusion Injury(I/RI) ： 20

restoration of blood flow after prolonged 0

Ischemia
1 2

reperfusion
3 4

ischemia aggravates the tissue damage even

more

The sequence of events during ischemia

and reperfusion
Reperfusion Injury

• Usually reperfusion injury more severe than ischemia

during the ischemic period.

• Restoration of blood flow to the ischemic limb, heart

or transplanted organ, relevance to the practice of
Vascular and Cardiac Surgery, Transplant Surgery…

• oxygen paradox, calcium paradox, pH paradox

• Characterized by cellular edema, intracellular Ca2+

overload, disruption of lipid membranes, and
perturbations in mitochondrial structure and function.

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Influence factors of I/RI Pathogenesis of I/RI

• Duration of ischemia:
1. Reactive oxygen species (ROS)
5-20min
• Conditions of reperfusion fluid: 2. Calcium overload
solution: O2 , Ca2+，H+，K+，Na +
3. Leukocyte-endothelium interactions
lower temperature and velocity
• Functional state of organs before ischemia:
dependency on oxygen (heart, brain)

1. Reactive Oxygen Species Concept of free radical

(oxygen paradox) Free Radical:
atom or molecule which containing an
unpaired electron in outer orbital
A. What’s the free radical
B. Where free radical come from
C. How free radical lead to injury

An electron has a negative charge, a proton has a positive charge

Classification of free radical Oxygen Free Radicals(OFR)

Most free radicals of biological and medical derived

from oxygen (also nitrogen or carbon centered Three different type molecules:
radicals):
• Superoxide anion O2-.
üReactive Oxygen Species(ROS):
O2•(superoxide anion) ，OH•(hydroxyl radical) ， • Hydroxyl radical .OH
H2O2(hydrogen peroxide)，´O2(singlet oxygen)，
• Hydrogen peroxide H2O2
üOxygen-derived Free Radicals(OFR): O2• ，OH•
üLipid-derived Free Radicals: L• , LO• , LOO•

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Free Radical Generation

e- e- +2H+ e- +H+ e- +H+

H2O2 GTX
Cat
O2 O2
●

H2O2 OH H2O
SOD
O2 H2O
O2- Fe2+
Mito
H2O Mitochondria pathway
cells NADH Ox

XO
·OH Direct injury Fe3+
O2•–＋ H2O2 OH •
Energy (ATP) Cu2+
Lipids DNA Proteins
Fenton reaction pathway
Apoptosis

ROS: Sources and roles

O2•– Superoxide Dismutase( SOD)

OH • Catalase(CAT)
H2O2

Free radical antioxidant

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Mechanism of OFR generation

1. Endothelium pathway：
xanthine dehydrogenase（XD）→ xanthine oxidase（XO）

ATP→ADP →AMP
XD
ischemia ↓Ca2+
XO –
hypoxanthine xanthine+O2 • + H2O2
O2 XO

reperfusion –
Uric acid+ O2 • +H2O2

OH•

2. Leukocytes pathway：
ØDamage of oxygen free radicals
respiratory burst
§ lipid peroxidation(脂质过氧化）
Activation of O2 oxygen free ①attack membrane phospholipids → decrease
neutrophils radical membrane fluidity, increase permeability, and loss
NADPH oxidase
of membrane integrity, [Ca2+ ]i ↑
② depress Na+ pump、Ca2+ pump：[Na+ ]i↑、
In general, to kill the microorganism [Ca2+ ]i↑
③interrupt singal tranduction
④seriously damage to mito，SR and lysosome

ØDamage of oxygen free radicals

MDA(malondialdehyde)

OH •
§ Protein:
direct injury：oxidation, cause dimer
indirect injury: reaction of troponin to Ca2+ ↓

§ DNA
base hydroxylation、breakdown of DNA
aging and congenital disease

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Oxygen Free Radicals

Short Summary (I)
• Xanthine oxidase which is activated in ischemic
endothelial cells to generate superoxide radicals.
• Neutrophils also generate oxygen free radicals.
• These toxic moieties are rapidly generated at the onset
of reperfusion and cause widespread damage to
cellular macromolecules.
• Peroxidation of lipid membranes, protein degradation,
nucleic acid damage, cytochrome inactivation and
neutralization of nitric oxide.
• Most damaging effect is on lipid membranes, impairs
normal fluidity and permeability of cell membranes
leading to cellular edema, massive Ca2+ and Na+
overload and cell lysis.

Oxygen Free Radicals 2. Calcium Overload (钙超载)

Short Summary (II)
(Calcium paradox)
• Oxygen free radical scavengers and antioxidants have
been shown both experimentally and clinically to calcium overload：
ameliorate reperfusion injury. concentration of intercelluar calcium increases
• Natural protective enzyme systems to reduce free obviously to higher degree which is associated
radical damage include superoxide dismutase, with cell injury
catalase, and glutathione peroxidase.
• Most important endogenous antioxidant is glutathione. • Metabolic pathways of [Ca2+ ]i
N-acetylcysteine is an artificial glutathione precursor. • Pathogenesis of calcium overload
• Detrimental effects of calcium overload

Calcium Homeostasis CALCIUM FLOW IN THE CELL

intracellular Endoplasmic reticulum

IP3 Metabotropic
Ca2+ PLC
VDCC ATP receptor
Ca2+ DG
ROCC Ca2+ Ca2+
ADP+Pi ATP Ca2+ AD Ca2+ Agonist-operated
Ca2+ Pump CYTOSOL P channel (NMDA)
Ca2+
ER /SR ER：endoplasmic reticulum
SR：sarcoplasmic reticulum Ca2+ Ca2+ Ca2+ Voltage-operated
Mt Calciosome
channel
Na+-Ca2+ exchanger
Ca2+ Pump Voltage
Ryano 受体 ∆Ψm + H+ 2Na
IP3 受体 _ + Ca2+ Extracellula
1Ca2+ Ca2+ uniporter r matrix
Na+
3Na+ Ca2+ u voltage-dependent Ca2+
Mitochondrion megachannel
calcium channel (VDCC) <1.5 KDa
u receptor-operated

calcium channel (ROCC)

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Pathogenesis of calcium overload

Calcium
1.2 mM

extracellular0.6 • Na+-Ca2+ exchange ：

0.0

direct activation: intracellular sodium,

intracellular
indirect activation: Na+-H+
mitochondrial
• increased permeability of cellular membrane：
glycocalyx injuryed;
• mitochondrial dysfunction
Normal Ischemia Reperfusion Delayed
death

3. leukocyte

•activation, margination and aggregation

of PMNs after reperfusion • Occlusion of microvascular lumen

• Sudden and large increase in oxygen consumption

Causes: adhesion molecule; chemotatic
and generation of a series of active oxygen species
factor; mediators of inflammation
• Release of arachidonic acid: TXA2, PAF
•Role of leukocyte in I/R I

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No-Reflow Phenomenon
• no-reflow is the phenomenon occurring when
removal of coronary occlusion does not lead to Pathogenesis:
restoration of coronary blood flow • ① vaso-endothelial cell damage；
• no-reflow phenomenon is caused by cellular
swelling, intravascular aggregation, and the • ② occlusion of microvascular
leakage of intravascular fluid into the interstitial • ③ mediators of inflammation
space (May et al, 1978)
• Recent animal studies ：no-reflow phenomenon
provokes an inflammatory response in
postcapillary venules that can be minimized by
inhibiting the transendothelial migration of
inflammatory cells

No-Reflow Phenomenon Mechanisms of No-Reflow

I/RI to vital organs

Heart
• Reperfusion arrhythmia：
• Decrease of myocardial
contractility:myocardial stunning
• Others：ATP↓, cell swelling, contraction
band, apoptosis

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Reperfusion arrhythmia myocardial stunning

•Incidence:50%-80%
Contractile dysfunction in the stunned myocardium
•ventricular tachycardia could be due to prior calcium overloading that could
ventricular fibrillation ( VF ) have caused a shift in the calcium sensitivity of
contractile apparatus in myocardium
•Animal experiments:
canine MI : 10-20min , then R：1-5min
[Ca2+ ]i＜ 100nM
＜ 15min injury to cardiac muscle is mild
＞ 100nM
＞ 45min electric action of cardiac muscle disappear
＝1μM时

myocardial stunning I/R I to vital organs

at 1992，Kusuoka proposed that:

Ca2+
overload
Cal’modulin ↑ breakdown of •MODS?
contractile apparatus

Reaction of
Stunned myocardium contractile apparatus
to Ca2+↓

Ischemia Preconditioning Ischemic Preconditioning

• The size of an infarct resulting from a 40-min occlusion of a
1. Effective!!
branch of a dog’s coronary artery could be greatly reduced if
2. Multiple controlled factor the heart were subjected to 4 brief periods of 5 min of
3. Memory period: ischemia and 5 min of reperfusion prior to sustained ischemia.
- 2 hours in anesthetized animals The heart adapted itself within minutes to become resistant to
- 4 hours in conscious animals ischemia-induced infarction.
4. Disease status: • Murry CE et al. Preconditioning with ischemia: a delay of
lethal cell injury in ischemic myocardium. Circulation 74:1124–
- diabetes, hypercholesterolemia,
1136, 1986.
hypertension, aging
5. Mechanism

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Cardioprotections by PC Microcirculation Protections by PC

• Blunt the impaired endothelial-

• Limit infarct size dependent relaxation.

• Mitigate myocardial stunning • Ameliorate capillary plugging,

leukocyte adhesion, and emigration.
• Improve Arrhythmias
• Reduce venous protein leakage effects
of prolonged ischemia.

Ischemia Preconditioning
• Acute preconditioning (classical preconditioning) another Sounds:
– within ~2 h
brief ischaemia in one tissue confers resistance to subsequent
– protein synthesis-independent sustained ischaemic insults in another tissue
( Basic scientific experiments and preliminary clinical trials in
• Delayed preconditioning (ischemic tolerance) humans)

– 24 h - 72 h after the initial insult Reperfusion injury.

– altered gene expression→synthesis of Plast Reconstr Surg. 2006 Mar;117(3):1024-33. Review
proteins (antioxidant enzymes, NO
synthase, etc.).

THERAPEUTIC INTERVENTIONS Ischemia Postconditioning

1. Hypothermia and Intraarterial Flushing • Postconditioning protects against endothelial

ischemia-reperfusion injury in the human forearm
2. Preconditioning
3. Postconditioning
4. Antithrombotic Agents
5. Inhibition of Leukocytes
6. Free Radical Scavengers

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Methods for Postconditioning Ischemia Postconditioning

1. Effective
2. Cycle length mattered less than the timing of
the initiation
3. injury suffered during reperfusion is largely
responsible for the IR-induced endothelial
dysfunction
4. postconditioning and ischemic preconditioning
have common signaling pathways

FMD : Flow-mediated Dilation; I : Ischemia; R:Reperfusion

THERAPEUTIC INTERVENTIONS Inhibition of Leukocytes

1. Hypothermia and Intraarterial Flushing • The interruption in the binding of leukocytes

2. Preconditioning to endothelium attenuates reperfusion injury in
experimental models
3. Postconditioning
4. Antithrombotic Agents à clinical trial using antibody against the
5. Inhibition of Leukocytes CD11/CD18 integrin receptor failed to demonstrate a
clinical advantage in reducing infarct size in heart
6. Free Radical Scavengers
patients after primary angioplasty

THERAPEUTIC INTERVENTIONS Free Radical Scavengers

• Recombinant human bovine superoxide dismutase
1. Hypothermia and Intraarterial Flushing • Vitamins A, C, and E
-A+E
2. Preconditioning
- E + iloprost
3. Postconditioning • Allopurinol
4. Antithrombotic Agents - xanthine oxidase inhibitor
àreduces the formation of reactive oxygen species
5. Inhibition of Leukocytes # a clinical study :
6. Free Radical Scavengers 60 patients with reimplanted thumbs
àallopurinol 300 mg/day for 5 days resulted in
less inflammation and a faster recovery of thumb
sensation

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Conclusion
Principles of prevention
• Attempts to bypass this complexity have led to a
search for the early “upstream” initiating events, rather
and treatment
than the “downstream” cascading events.
ØRestoring perfusion of tissue in time
• Pharmacologic interventions to reduce
ØAdjust the reperfusing solution and conditions
the adverse effects of ischemia-reperfusion injury
low: speed, temperature, pH, sodium and calcium
• Reduce the no-reflow phenomenon by diminishing the
ØScavenge of free radicals
leukocyte-endothelium interaction at the postcapillary
SOD, CAT, GSH, Vit.C, beta - carotene
venule
ØPrevention of calcium overload
• Reducing platelet aggregation
calcium antagonist（Ca拮抗剂）
• Reducing microthrombi formation

scavenge free radicals Summary

• Reperfusion through thrombolysis or percutaneous coronary
angioplasty (PTCA) is standard treatment in impending acute
• Low molecular weight scavenger: myocardial infarction. Although restoration of blood flow to the
jeopardized myocardial area is a prerequisite for myocardial
glutathine peroxidase（GSH-PX）, salvage, reperfusion may lead to additional tissue injury named
“reperfusion injury”. Not all of the ischemic organs suffer
catalase（CAT）,VitA，VitC，VitE, from reperfusion injury, it depends on the duration of ischemia,
reduced glutathione（GSH） condition of reperfusion fluid and the conditions of organs
before ischemia.
• Enzyme: SOD：O2•– • The mechanism of ischemia reperfusion injury is complex,
including production of free radicals, calcium overload,
CAT、POD：H2O2 interaction between endothelial cells and neutrophils and altered
energy metabolism. Once the oxygen free radicals formed, they
may result in reperfusion injury by lipid peroxidation, inhibition
of proteins function and disruption of nucleic acid and
chromosome.

Summary KEY POINT

• Calcium overload can cause damage of cells through activation
of Ca2+-dependent enzymes, dysfunction of mitochondrion and • Concept
continual contraction of myocardium.
• Different organ has different manifestation of reperfusion injury. • Mechanisms
For the heart, it includes arrhythmia, reversible contractile
dysfunction, myocardial stunning, endothelial dysfunction and • Effect
cell death.
• People are now focus on the possibility of preventing
reperfusion injury by various methods such as shorten ischemia
time, control reperfusion conditions, supply energy and use
some pharmacological agents such as inhibitors of neutrophil,
adenosine, calcium antagonists or calcium channel blocker and
cell protectors.

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