1. Ischemia-Reperfusion Injury and Free Radicals Jianzhong Sheng MD PhD

2. 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

3. The injury of ischemic cardiac muscles was
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.
证实再灌注会引起心肌超微结构不可逆性坏死，包括爆发性水肿，组织结构崩解，收缩带形成和线粒体内磷酸钙颗粒形成。

4. 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.
通过几十年的观察研究，人们逐渐认识了这种缺血、恢复血流灌注后反常的有害现象，对其进行了较为深入的研究。

5. Clinic: Shock, DIC microcirculation reperfusion
Coronary reperfusion、artery bypass graft
Restoration of blood supply in reimplantation of limb, transplantation of organs.

6. 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.

7. 3 Key points are: In the definition of ischemia-reperfusion injury
Ischemia for a long time
Reestablishment of blood flow
More severe injury

8. 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

9. Etiology of ischemia-reperfusion injury
Cause
Ischemia followed by reperfusion

10. Which factors are involved in reperfusion injury
Generally speaking,
Duration of ischemia the longer period of ischemia, the more severe injury
Severity of ischemia the more grievous ischemia, the more severe injury
Speed of reperfusion the faster reperfusion of blood, the more severe injury
Ischemia preconditioning increasing tolerance to reperfusion injury

11. 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

12. Pathogenesis of ischemia-reperfusion injury

13. 1. Injury of free radicals

14. (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.
Oxygen free radical
Lipid radical

15. (2) Oxygen free radical, OFR
Types：
the superoxide anion (O2-)
the hydroxyl radical (OH ·)
singlet oxygen (1O2 )
hydrogen peroxide (H2O2)

16. (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：
Fatty acid radical (L·)
Lipid peroxide（LOO·）
(4) Others: Cl·, CH3·, NO·
氧化还原过程中产生的具有高活性的一系列中间产物

17. (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

18. O2  SOD (6) Generation of OFR O2 + e Cytaa3 O2 + 3 e + 3H+ 2 H2O
HO + H2O
H2O2
2 H2O
O2 + 4 e + 4H+
SOD, Superoxide dismutase

19. Haber-Weiss reaction (without Fe3 )
O2－ + H2O O2 + OH +OH 
SLOW
hydroxyl radical; ferrum

20. Fenton-Haber-Weiss reaction
O2－ + H2O O2 + OH +OH 
FAST

21. Cysteine, Vit C, glutathione
(6) Elimination of oxygen free radicals
1）Small MW scavenging agents
Dihydrocoenzyme II
Cysteine, Vit C, glutathione
Vit E、 Vit A

22. 2）Enzymatic scavenging agents
Catalase (CAT)
Superoxide dismutase
MnSOD
CuZnSOD
Peroxydase （H2O2）

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

24. 1) Mitochondria pathway
Ca2+entering MT
O2+e↑
Mn− SOD + O Mn+ − SOD + O2
O-2↑
Hypoxia Mn-SOD 
Superoxide dismutase
Pm O2 呼吸链受抑 NADH蓄积供电子

25. 2) Xanthine oxidase pathway
Xanthine oxidase (XO) 10%
Xanthine dehydrogenase (XD) 90%
Ca+2

26. Effect of XO on formation of OFR
Ischemia:
ATP comsumption↑
Hypoxanthine ↑
(1) Ca2+ overload→
activating protein kinase O2
Reperfusion:
xanthine + O·-2+ H2O2 XD XO
(2) Restoration of O2
supply O2
O·-2+ H2O2 +Uric acid
Effect of XO on formation of OFR
OH ·

27. H+ + O-2·+H2O2 3) Neutrophil pathway NADH(I) NADPH(II) + O2 Activating
C3, LTB4
(Complement C3
Leukotriene B4 )
Hexose shunt activity↑
cellular respiration ↑
NADH oxidase
NADH(I)
NADPH(II)
H+ + O-2·+H2O2 
+ O2
NADPH oxidase

28. 4) Catecholamines  Adr Remove O-2· adrenochrome Methyl transferase
vanillylmandelic acid (normal)
monoamine oxidase
Stress 80% O2
Remove
O-2·
adrenochrome

29. (8) Alterations induced by OFR
1) lipid peroxidation
Alteration of membrane lipid
Function inhibition of membrane proteins
Enhance of arachidonic acid metabolism
Blockage of ATP production in mitochondria membrane
4造成生物膜损害

30. 2) Injury of chromosome and nuclear acid
80% induced by OH

31. 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

32. How to maintain Intracellular calcium
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

33. 2. Calcium overload Ca2+ Binding proteins Mt SR Ca2+pump Ca2+ channel
Na + -Ca 2+ exchanger
Ca2+ Binding proteins Mt SR
Ca 2+
Ca2+pump
Ca2+
channel
1 钠-钙交换过程： 钙跨膜转运机制顺着某些离子的浓度梯度弥散。 2 影响交换的因素： 决定因素：细胞外Na+浓度 影响细胞外Na+浓度的因素： Na+-K+交换 “钠-钙交换过程是可逆的。
1 钙泵（calcium pump)
（耗能）
2 钠-钙交换(Na+-Ca2+ exchange)
（间接耗能）
3 钙通道（calcium channels)

34. (1) Mechanisms of calcium overload
Disorder of Na+ -Ca2+ exchange→ Intracellular Na+↑, H+↑, NE- 1R- PLC-PKC↑
Activation of Na+-H+ exchanger
Cellular membrane injury→ permeability↑, membrane phospholipid degradation↑, OFR↑
Injury of mitochondria
Catecholamines↑-R
glycocalyx

35. Why dose calcium overload occur
during reperfusion
Depleted energe
Increased permeability of cellular
membrance
Increased intracellular sodium

36. (2) Alterations induced by calcium overload
Mitochondria function↓→ATP production↓
Activation of membrane phosphatidase→membrane damage
Cardiac arrhythmia
OFR↑
Myofibril contracture, rupture, cell damage

37. What are effects of calcium overload
Damage mitochondria →
ATP production decrease
Cause myocardial injury →
contraction weakness
promote OFR formation →
damage aggravation

38. 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”.

39. Accumulation of WBC Progressive Activation SELECTINS INTEGRINS
Capture
Slow Rolling
Firm Adhesion
Transmigration
Rolling
Chemotactic
factor
Adhesion molecule)

40. 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
四 磷酸化合物缺乏
（一）缺乏的机制
1线粒体受损 →ATP生成↓。
2合成ATP的前体物质↓。
（二）缺乏引起再灌注损伤的机制
1钠泵、钙泵功能障碍→钙超载。
2氧自由基↑。
卅四 磷酸化合物缺乏
No reflow

41. WBC ADHERENCE to ENDOTHELIUM
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

42. Mechanisms of IRI OFR Ca overload ？ endothelia- neutrophil
Alterations in metabolism
and energy
Ca overload ？
OFR
endothelia-
neutrophil
Ca overload is common way of irreversible death of cells

43. ↓ ↓ ISCHEMIA—REPERFUSION INJURY
Excess oxygen Neutrophil
Free radicals infiltration
↓ ↓ ISCHEMIA—REPERFUSION INJURY
↑ ↑
Microvascular damage Calcium overload
Major mechanisms of ischemia- reperfusion injury

44. Alterations of metabolism and function during ischemia-reperfusion injury

45. Heart 1. Cardiac function-heart pump↓
2. Electrocardiogram-Reperfusion arrhythmia
3. Energy metabolism change in heart
4. Change in cardiac microstructure

46. 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)

47. Ischemia-reperfusion injury in other organs (intestine, kidney, bone)

48. ↓ ↓ ISCHEMIA—REPERFUSION INJURY
Excess oxygen Neutrophil
Free radicals infiltration
↓ ↓ ISCHEMIA—REPERFUSION INJURY
↑ ↑
Microvascular damage Calcium overload
Major mechanisms of ischemia- reperfusion injury

49. 1.Vasomotor Responses OFR Calcium Overload WBC ↘ ↓ ↙
↘ ↓ ↙
Damaged endothelium
↙ ↘
NO, PGI2 release↓ TXA2, ET release↑
↘ ↙
VASOCONSTRICTION ↓
Aggravating injury

50. Vascular Sticking WBC Liable to form
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

51. Heart Brain Liver Intestine ↓ ↓ ↓ ↓ Shock Cytotoxic Jaundice Mucosal
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

52. Pathophysiological basis of prevention and treatment for ischemia-reperfusion injury

53. 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

54. 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

55. Questions 1. What is ischemia-reperfusion injury?
2. What is free radical?
3. What mechanisms of ischemia-reperfusion injury?