3. Introduction
Ischemia-reperfusion injury is a complex phenomenon involving not only
intracellular injury processes but also an injurious inflammatory response.
Both the intracellular injury processes and the injurious events of the inflammatory
response are interconnected in pathogenetic networks.
Ischaemia reperfusion (IR) injury is a clinical entity with a major contribution to the
morbidity and mortality of liver surgery and transplantation.
4. Introduction (Cont.)
Hepatic ischaemia reperfusion (IR) injury can be identified either in interventional
settings causing direct liver ischaemia such as liver surgery and transplantation or
in the setting of systemic hypoxaemia (e.g. respiratory failure) or shock states
followed by resuscitation (e.g. haemorrhage, sepsis).
5. Definitions
Ischaemia is a reduction or absence of blood supply to an organ resulting in a lack of
oxygen and vital nutrients in tissues.
Warm ischaemia occurs with interruption of blood flow at body temperature (37 °C)
and develops in situ during liver surgery, transplantation or systemic shock.
Cold ischaemia occurs during cold (4 °C) ex vivo organ preservation.
Cold ischaemia is usually coupled with warm ischaemia during liver transplantation
surgery.
F. Radu-Ionita et al. (eds.), Liver Diseases,2020
6. Definitions
Reperfusion is the restoration of blood supply to an ischaemic organ.
Ischaemia-reperfusion (IR) injury is the cellular damage after reperfusion of
previously viable ischaemic tissues.
F. Radu-Ionita et al. (eds.), Liver Diseases,2020
8. Intracellular Events
•Cellular acidosis occurs in the initial ischaemic period as a result of cellular
hypoxia and a shift from aerobic to anaerobic respiration.
•ATP depletion and accelerated glycolysis within mitochondria as well as lactic acid
production.
•pH values return to normal which enhances pH-dependant activation of harmful
enzymes such as proteases and phospholipidases .
•This phenomenon has been termed the pH paradox
9. Intracellular Events (Cont.)
•Intracellular Ca2+ overload occurs due to a failure of membrane pumps and
release from endoplasmic reticulum Ca2+ stores.
•Ca2+ is taken up by the mitochondria to act as a buffer for the increase in
cytosolic levels.
•Important Ca2+ dependant enzymes involved in apoptosis such as calpains,
protein kinase C and phospholipidase C are also activated.
11. Intracellular Events (Cont.)
•ROS production and oxidative stress are the hallmark of liver IR injury
pathogenesis.
•ATP depletion in the ischaemic phase leads to anaerobic respiration and an
increase in ATPdegradation products such as adenosine, hypoxanthine and
xanthine.
•At early reperfusion, the rate of oxygen delivery exceeds that of cellular activity
returning to aerobic pathways.
•This results in the production of damaging oxygen free radicals (superoxides,
hydrogen peroxide and reactive nitrogen species).
14. Intracellular Events (Cont.)
•Mitochondrial dysfunction is initiated by a lack of oxygen which interrupts
oxidative phosphorylation and ATP production.
•Lack of ATP disrupts mitochondrial electrolyte homeostasis
•Furthermore, at reperfusion the mitochondria can be a source of toxic ROS
production.
• Activated phospholipidase and protease enzymes cause direct damage to the
mitochondria membrane leading to membrane instability and mitochondrial
permeability transition.
15. Innate Immune Response
•DAMPs are damage associated molecular patterns; ‘danger signalling molecules’
that can initiate and perpetuate a noninfectious inflammatory response.
•DAMPs interact with pattern recognition receptors (PRR) such as Toll-like
receptors (TLR) on Kupffer cells, dendritic cells, and neutrophils to initiate an
inflammatory process.
•Complement activation plays an important role in both local and remote IR
injury acting directly via the formation of membrane attack complexes (MAC)
and indirectly by cytokine and chemokine activation.
16. Cellular Response
•Kupffer Cells are the liver resident macrophages and form the earliest cellular
response in liver ischaemic injury.
•Kupffer cells are activated during ischaemia and early stages of reperfusion via the
complement system and become a powerful source of cytokine (TNF-alpha and IL-
1β) and ROS production.
•This leads to LSEC activation and expression of adhesion molecules such as ICAM-1
and VCAM-1 which in turn enhance circulating leukocyte chemotaxis, adhesion
and transmigration.
17. Cellular Response (Cont.)
•Neutrophils are recruited to the liver after reperfusion by a complex network of
chemokines released from KCs and LSECs such as macrophage inflammatory
protein-2 (MIP2).
•Neutrophils cause cellular injury by releasing matrix metalloproteases (MMP)
and myeloperoxidases (MPO) which are potent oxidants.
•The neutrophil oxidative burst supersedes KCs as the main source of ROS
production in the later stages of IR injury which causes direct damage to
hepatocytes, LSECs and the extracellular matrix (ECM).
18. Cellular Response (Cont.)
•CD4+ T cells are recruited to the liver after reperfusion and play an important
role in the adaptive immune response to liver IR.
•Natural killer (NK) T cells contribute to neutrophil activation via release of
interferon gamma (IFNg) and IL-17.
•CD4+ T cells expressing αβ TCR can inhibit neutrophil recruitment and oxidative
burst.
•Platelets are activated and adhere to LSECs within minutes of reperfusion
through interaction with adhesion molecules (ICAM-1, VCAM-1 and E-selectin)
and ECM degradation products such as fibrinogen.
•Platelet adhesion and aggregation leads to reduced microcirculatory perfusion.
21. Microcirculatory Failure
•A combination of platelet and leukocyte accumulation as well as direct damage
to hepatocytes, LSECs and vasoconstriction in early reperfusion results in a
reduction in sinusoidal diameter and reduced flow.
•Some areas even have no flow despite reperfusion of the liver—this is referred
to as the ‘no reflow’ phenomenon.
-Imbalance of vasoactive substances
-Endothelial glycocalyx (GXL) degradation
22. Microcirculatory Failure
•Vasoconstriction occurs due to an excess of endothelin-1 (ET-1) production in
early reperfusion secondary to KC activation.
•ET-1 is a powerful vasoconstrictor and excess levels result in micro and
macrovascular reduction in blood flow to the liver.
•Nitric oxide (NO), released by vascular endothelial cells in response to shear
stress, on the other hand is a vasodilator and promotes cell survival through
inhibition of caspase activities.
•NO also regulates microcirculatory vascular tone and inhibits platelet
aggregation.
23.
24. Microcirculatory Failure
•Endothelial glycocalyx (GXL) degradation has recently been implicated in liver IR.
•This is a thin and fragile layer of proteoglycans and glycosaminoglycans on the
luminal surface of all blood vessels including the liver sinusoids.
•It plays an important role in vascular permeability, endothelial-leukocyte/platelet
interaction (inflammation and coagulation) as well as mechanotransduction (NO
production).
25. Aetiology
•A temporary reduction in blood supply to the liver causes IR injury.
•This can be due to a systemic reduction or local cessation and restoration of
blood flow.
•Systemic causes include severe hypotension and shock followed by
resuscitation—the so called ‘shock liver’ that occurs in trauma and sepsis.
•Liver hypoperfusion can also occur in patients who recover from a cardiac arrest
or undergo cardiopulmonary bypass
26. Aetiology (Cont.)
•Local cessation and restoration of liver blood supply occurs with temporary
‘inflow occlusion’ applied to control bleeding in liver surgery .
•Routinely occurs with liver transplantation during organ procurement until the
donor organ is revascularised in the recipient.
•Portal vein resection and reconstruction may also involve temporary clamping of
the portal vein and results in a degree of liver IR injury
30. Prevention and Treatment
•There is currently no accepted treatment for liver IR injury.
•Several pharmacological agents and surgical techniques have been beneficial in
reducing markers of hepatocyte injury in experimental liver IR
32. Intermittent Clamping
•Inflow occlusion or portal triad clamping (PTC) can be continuous or
intermittent;
•Intermittent clamping (IC) increases parenchymal tolerance to ischaemia.
•IC permits longer total ischaemia times for more complex resections.
•Alternating between 15 min of inflow occlusion and 5 min reperfusion cycles
can be performed safely for up to 120 min total ischaemia time.
33. Ischaemic Preconditioning (IPC)
•Brief periods of ischaemia followed by reperfusion was protective against
further sustained ischaemia.
•IPC involves a short period of ischaemia (10 min) and reperfusion (10 min)
intraoperatively
•It allows continuous ischaemia times of up to 40 min without significant liver
dysfunction.
34. Remote Ischaemic Preconditioning (RIPC)
•RIPC involves preconditioning a remote organ prior to ischaemia of the target organ.
•It has been shown to be reduce warm IR injury to the liver in experimental studies.
•Major liver resection for colorectal liver metastasis used a tourniquet applied to the
right thigh with 10 min cycles of inflation-deflation to induce IR injury to the leg for
60 min.
•A reduction in post-operative transaminases and improved liver function was shown
without the use of liver inflow occlusion.
35. Pharmacological Agents
•Antioxidants are either free radical scavengers or inhibit specific pathways in
ROS production.
•Allopurinol for example is a xanthine oxidase inhibitor .
•Vitamin E acts as a radical scavenger while α-lipoic acid has a transition metal
resulting in chelation with ROS.
•Melatonin is an endogenous antioxidant and reduced TNF-α and iNOS
production.
36. Pharmacological Agents
•Anti-inflammatory agents such as methylprednisolone has been extensively
studied
•It reduces hepatocellular apoptosis and inflammatory mediator release as well
as reducing post-operative transaminases.
•Pharmacological preconditioning with volatile anaesthetic agents such as
sevoflurane or isoflurane 30 mins prior to warm ischaemia in liver resections
reduces postoperative liver dysfunction, especially in those with liver steatosis
37. Liver Transplantation
•Organ resuscitation is the process of improving the viability and function of
marginal donor organs
•Surgical and pharmacological interventions to reduce IR injury are eagerly
pursued to optimise high risk donor livers for transplantation.
•The DRI has been combined with preservation and recipient factors such as cold
ischaemia times and model for end-stage liver disease (MELD) scores to better
predict graft failure.
39. Donor Optimisation
•Donor co-morbidities and damage sustained during the final illness of donors
affect the quality of the donor liver.
•A triphasic process involving: first a Cushing’s reflex of hypertension and
bradycardia, followed by a massive release of catecholamines resulting in a
transient hypertension, tachycardia and myocardial impairment.
40. Normothermic Regional Perfusion (NRP)
•DCD donation involves a prolonged warm ischaemia time.
•legal requirements prevent cannulation and heparinisation of the donor for at
least 5 min after confirmation of death.
•This technique involves delivering oxygenated cold perfusion using an
extracorporeal membrane oxygenation circuits (ECMO) to the abdominal
organs in situ by cannulation of the iliac vessels and the abdominal aorta.
41. Preservation and Resuscitation
Preservation Solutions
•Designed to mitigate the cellular and molecular damage that occurs during
ischaemia
•The various compositions largely consist of electrolytes, buffers, impermeants
and metabolites (ROS scavengers and nutrients).
•There are different electrolyte compositions to reflect intra- or extracellular
ratios of Na+ and K+ with added calcium, chloride and magnesium.
42. Preservation and Resuscitation
•Buffers (e.g. bicarbonate) counteract changes in pH whilst free radical
scavengers (e.g. glutathione) reduce ROS formation.
•Preservation solutions combined with cooling to reduce oxygen and metabolic
demand improves organ viability ex vivo.
•Static cold storage (SCS) involves rapid flushing of the organs in situ with
preservation fluids and after retrieval surgery, the liver is submerged in a sterile
bag containing the same solution and placed on ice for storage during transport.
43. Hypothermic Machine Perfusion (HMP)
•Reduces metabolic activity and oxygen demand with supplemental oxygen in the
perfusion solution.
•Flow of the perfusion fluid helps remove toxic metabolites produced during ischaemia.
•Although flow triggers shear-dependant endothelial protective mechanism such as
nitric oxide production, high flow pressures and prolonged perfusion cause injury to the
pressure sensitive liver sinusoidal endothelial cells.
•An optimal perfusion pressure and duration is yet to be determined.
•endischaemic HOPE is applied for a short period prior to implantation at the recipient
center after SCS in transportation
46. Normothermic Machine Perfusion (NMP)
•This requires a constant supply of nutrients and oxygenated perfusion at body
temperature (37 °C).
•Several NMP circuits have been designed to incorporate pumps, a blood
reservoir, a heat exchanger and an oxygenator.
•NMP has been shown to improve bile production and reduce markers of
hepatocellular injury as well as a reduction in platelet aggregation at reperfusion
47. Recipient Strategies
•Washout Techniques
•The toxic metabolites, electrolytes and inflammatory mediators are washed out
into the systemic circulation once the graft is revascularised.
•leads to development of haemodynamic instability and post-reperfusion
syndrome.
•Washout can be performed using preservation solutions, crystalloids or colloids
like human albumin solution, on bench or in situ prior to venous reperfusion, in
antegrade or retrograde fashion
48. Remote Ischaemic Preconditioning (RIPC)
•Direct intraoperative ischaemic preconditioning of the liver by intermittent
inflow occlusion in the recipient during transplantation is challenging.
•Direct IPC by portal triad clamping in the donor liver during retrieval surgery has
been performed in DBD donors.
49. Future Perspectives
•Hepatic IR injury remains the main cause of morbidity and mortality in liver
surgery and transplantation.
•Therapeutic options to treat or prevent liver IR are limited.
•Recent advances in our understanding of the immunological responses and
endothelial dysfunction in the pathogenesis of liver IR injury may pave the way
for the development of new and more effective and targeted pharmacological
agents.
•With the advent of machine perfusion, there is a great opportunity for re-
conditioning or resuscitating marginal donors with the aim of increasing the
donor pool and reducing waiting list deaths.
50. Conclusion
•Liver IR injury develops as a result of a complex network of inflammation and
endothelial activation resulting in cell death.
•Liver IR injury is common in liver surgery and transplantation and remains the
main cause of morbidity and mortality.
•Several treatments have shown benefit in experimental IR, however none have
been translated into routine clinical practice.
•Promising recent developments in pharmacological agents and machine
perfusion of organs are currently being investigated
