Ischemia Reperfusion Injury

Slide 1 · Ischemia–Reperfusion Injury

Cell injury mechanisms and clinicopathologic consequences

Restored flow: cellular rescue and paradoxical damage

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

By the end of the lecture, distinguish:

Ischemic injury from injury generated after restoration of flow.

The four proposed mechanisms of reperfusion-associated damage.

How oxidants, calcium, inflammation and complement converge on cell death.

Why restoring perfusion remains essential despite its potential for added injury.

Slide 3 · Reperfusion can rescue—and injure—the same tissue

Potential benefit

Paradoxical consequence

Reoxygenation supports metabolism in viable cells.

Blood flow brings needed substrates and removes accumulated metabolites.

Reversibly injured cells may recover.

New damaging processes begin with reperfusion.

Previously salvageable cells may be lost.

Net tissue damage includes ischemic and reperfusion components.

Reperfusion injury means additional damage attributable to the restoration phase.

Slide 4 · Perfusion maintains more than oxygen delivery

Oxygen

Supports mitochondrial oxidative phosphorylation.

Metabolic substrates

Maintains cellular energy generation.

Metabolite clearance

Limits accumulation of products that inhibit glycolysis.

Circulating components

Can also deliver leukocytes and complement during reperfusion.

Slide 5 · Ischemia and hypoxia are not synonymous

Feature

Hypoxia with maintained flow

Ischemia

Blood flow

Maintained

Reduced or obstructed

Oxygen availability

Reduced

Reduced due to impaired flow

Glycolytic substrate delivery

May continue

Compromised

Removal of metabolites

Continues

Impaired

Typical injury

May be less rapid

Often more rapid and severe

Slide 6 · Why ischemia produces faster injury

Reduced blood flow

Oxygen and substrate delivery fall

Oxidative phosphorylation fails

Mitochondrial ATP production decreases

Anaerobic compensation is limited

Substrates depleted; Metabolites accumulate

Energy-dependent systems fail

Cell injury progresses toward necrosis

The shortage is both oxygen and usable metabolic support.

Slide 7 · The chronology of ischemic cell injury

Early

ATP decreases; Ion homeostasis begins to fail.

Potentially reversible

Cell and endoplasmic-reticulum swelling develops.

Prolonged injury

Membrane and mitochondrial dysfunction progress.

Irreversible

Membrane breakdown and necrosis occur.

Sequence shown conceptually; Cellular outcomes depend on the severity and duration of ischemia.

Slide 8 · Mitochondrial respiration is an early casualty

Ischemia

Reduced intracellular oxygen

Electron transport

Oxidative phosphorylation slows or ceases

ATP depletion

Energy-dependent cellular processes weaken

Cell dysfunction

Ion transport and protein synthesis suffer

This pathway explains injury even before reperfusion occurs.

Slide 9 · ATP depletion first disrupts ion pumping

Low ATP

Na⁺/K⁺-ATPase activity falls

Ion imbalance

Na⁺ accumulates; Intracellular K⁺ declines

Osmotic influx

Water moves into the cell

Cell swelling

Early reversible structural change

Cell swelling is a consequence of energy failure, not proof of irreversibility.

Slide 10 · Cell swelling signals an initially reversible phase

What develops

Why it matters

Intracellular sodium increases.

Osmotically driven water accumulation expands cells.

Endoplasmic reticulum may dilate.

The plasma membrane can remain functionally recoverable.

Prompt restoration of adequate perfusion may permit recovery.

Further mitochondrial and membrane damage threatens viability.

Slide 11 · Anaerobic glycolysis offers limited rescue

Low oxygen

Greater dependence on glycolytic ATP

Glycogen consumption

Cellular reserves are used

Lactate and phosphates

Acidity increases; Intracellular pH falls

Impaired enzyme activity

Compensation becomes insufficient

Reduced flow prevents adequate substrate replenishment and waste removal.

Slide 12 · Energy failure eventually becomes irreversible

Progression during ischemia

Pathologic endpoint

ATP depletion persists or worsens.

Mitochondrial and lysosomal membranes are injured.

Cellular integrity cannot be maintained.

Loss of membrane integrity culminates in necrosis.

Restoring flow cannot revive cells already irreversibly injured.

Other reversibly injured cells may still be at risk during reperfusion.

Slide 13 · Reversibility determines the possible benefit

Ischemic tissue at the moment flow returns

Reversibly injured cells

Potential recovery if adequate perfusion is restored

Irreversibly injured cells

Necrosis is not reversed by blood flow

Vulnerable salvageable cells

May undergo additional damage during reperfusion

Slide 14 · Two processes coexist after restoration of flow

Restoration of homeostasis

New injury pathways

Oxygen and substrates become available.

Recoverable cells may restore ATP generation.

Metabolic waste can be cleared.

Oxidants are generated during reoxygenation.

Calcium dysregulation is aggravated.

Inflammatory and complement pathways can intensify damage.

Slide 15 · Four proposed reperfusion-injury mechanisms

Oxidative stress

Reactive oxygen and nitrogen species rise.

Calcium overload

Cytosolic and mitochondrial Ca²⁺ accumulation worsens.

Inflammation

Danger signals, cytokines and neutrophils amplify injury.

Complement activation

Antibody-associated complement activation adds damage.

Slide 16 · Reoxygenation can create a burst of oxidants

Oxygen returns

Reperfused cells are exposed to O₂

Incomplete oxygen reduction

Reactive oxygen species are formed

Antioxidant buffering is impaired

Ischemic cells are more susceptible

Macromolecules are damaged

Membrane, protein and DNA injury increase

The oxygen required for recovery can also participate in oxidative injury.

Slide 17 · Oxidants arise in multiple reperfused cell types

Leukocytes

Activated inflammatory cells generate oxidants.

Endothelial cells

Previously damaged cells can produce reactive species.

Parenchymal cells

Injured resident cells participate in oxidant generation.

Antioxidant defenses

Reduced capacity during ischemia raises vulnerability.

Slide 18 · Oxidative stress is a balance, not a single molecule

Amount of reactive species in injured tissue

Increased generation

Reoxygenation, leukocytes and damaged cells

Decreased removal

Compromised scavenging and antioxidant defenses

Net accumulation

Oxidative stress and cellular macromolecule injury

Slide 19 · ROS and reactive nitrogen species converge on injury

Reactive oxygen species

Reactive nitrogen species

Oxygen-derived oxidants include superoxide and hydroxyl radical.

Hydrogen peroxide is a reactive oxygen species but not a free radical.

Incomplete oxygen reduction increases oxidant burden.

Nitric oxide can participate in radical reactions.

Superoxide can react with nitric oxide to form peroxynitrite.

These species can damage membrane lipids, proteins and DNA.

Slide 20 · Principal reactive species: Essential distinctions

Species

Biochemical distinction

Injury relevance

Superoxide (O₂•⁻)

Oxygen-derived radical

Precursor of additional oxidants

Hydrogen peroxide (H₂O₂)

ROS; not itself a radical

Can generate more reactive products

Hydroxyl radical (•OH)

Very reactive oxygen radical

Damages lipids, proteins and DNA

Peroxynitrite (ONOO⁻)

Reactive nitrogen-derived oxidant

Can oxidatively damage macromolecules

Slide 21 · Lipid peroxidation undermines membranes

ROS attack

Unsaturated membrane lipids

Lipid radicals / peroxides

Propagation of chain reaction

Membrane injury

Loss of structural and functional integrity

Cell damage

Plasma and organelle membranes are affected

Lipid damage can propagate beyond the initial oxidative reaction.

Slide 22 · Oxidative protein changes impair cell function

Enzyme modification

Oxidation can disturb catalytic sites.

Protein conformation

Oxidation alters folding and stability.

Cross-linking

Protein–protein bonds may be modified.

Proteostasis burden

Abnormal proteins may undergo increased degradation.

Slide 23 · Reactive species can also damage nuclear DNA

Oxidant exposure

ROS react with nucleic acids

DNA lesions

Strand breaks, cross-links and adducts

DNA damage response

Cell-cycle arrest or repair may occur

Failure of adaptation

Severe injury can contribute to cell death

DNA injury is one of several macromolecular effects; It is not a specific test for reperfusion injury.

Slide 24 · Activated leukocytes are an oxidant source

Tissue injury

Inflammatory signals attract leukocytes

Neutrophil activation

Oxidant-producing systems are engaged

ROS production

Inflammatory oxidant burst develops

Bystander effects

Host membranes and proteins may be injured

A response meant to clear damaged tissue can aggravate local injury.

Slide 25 · Depleted antioxidant defenses increase susceptibility

Normal protection

After ischemia

ROS are produced in normal cellular metabolism.

Enzymatic and nonenzymatic systems neutralize oxidants.

Protection depends on scavenging near oxidant sources.

Protective buffering may be compromised.

New ROS production can exceed available removal.

Oxidative damage therefore rises during reoxygenation.

Slide 26 · Enzymatic antioxidant defenses act at different steps

Defense

Principal role

Teaching implication

Superoxide dismutases

Convert superoxide into H₂O₂ and O₂

Affects the superoxide burden

Catalase

Converts H₂O₂ into water and oxygen

Detoxifies hydrogen peroxide

Glutathione peroxidase

Reduces H₂O₂ using glutathione

Links ROS removal to redox reserve

Antioxidants / metal binding

Scavenge radicals or restrict metal catalysis

Lower generation and propagation of oxidative damage

Slide 27 · Oxidants increase susceptibility to further injury

Reperfusion

Oxygen again available

Oxidative stress

Reactive species increase

Membrane injury

Ion gradients and organelles suffer

Cell vulnerability

Additional damage becomes more likely

Membrane injury can heighten susceptibility to further oxidant-associated damage.

Slide 28 · Cytosolic calcium is normally tightly regulated

Normal organization

Injury state

Free cytosolic Ca²⁺ is kept much lower than extracellular Ca²⁺.

Endoplasmic reticulum and mitochondria store much intracellular calcium.

Controlled Ca²⁺ signals regulate cell function.

Excess cytosolic calcium is toxic.

Ischemia releases calcium from internal stores.

Later membrane injury permits additional calcium influx.

Slide 29 · Calcium loading begins before reperfusion

Ischemic insult

Homeostatic processes weaken

Internal stores

Calcium released into cytosol

Membrane dysfunction

Additional extracellular Ca²⁺ enters

Mitochondrial uptake

Organelle calcium loading rises

Calcium overload is initiated by ischemia and is exacerbated by reperfusion.

Slide 30 · Reperfusion magnifies pre-existing Ca²⁺ overload

Injured cell exposed to restored perfusion

Damaged plasma membrane

Allows additional Ca²⁺ influx

ROS-injured sarcoplasmic reticulum

Aggravates intracellular Ca²⁺ dysregulation

Ca²⁺-loaded mitochondria

Become vulnerable to permeability transition

Slide 31 · The sarcoplasmic reticulum links ROS to Ca²⁺

Reoxygenation

ROS production increases

Sarcoplasmic reticulum injury

Calcium regulation deteriorates

More cytosolic Ca²⁺

Exacerbates calcium overload

Organelle dysfunction

Additional injury follows

This coupling is especially relevant to understanding the reperfusion process in muscle cells.

Slide 32 · Mitochondrial calcium overload promotes pore opening

Excess Ca²⁺

Accumulates within mitochondria

Permeability transition

Mitochondrial pore opening is favored

Membrane potential loss

Oxidative phosphorylation fails

ATP depletion

Necrotic injury can progress

Mitochondrial permeability transition is a mechanistic link between calcium and energy failure.

Slide 33 · Calcium overload perpetuates ATP depletion

Initial ischemia

ATP becomes limited

Ion handling fails

Calcium control deteriorates

Mitochondrial Ca²⁺

Permeability transition is favored

Further ATP loss

Energy failure deepens

Additional ATP loss further impairs ion handling and calcium regulation.

Slide 34 · Excess Ca²⁺ activates destructive enzymes

Enzyme class

Main target

Damage caused

Phospholipases

Membrane phospholipids

Membrane disruption

Proteases

Membrane / cytoskeletal proteins

Structural degradation

Endonucleases

DNA and chromatin

Nuclear fragmentation

ATPases

Cellular ATP stores

Accelerated energy depletion

Slide 35 · ROS and calcium pathways are mechanistically coupled

ROS → membrane

Oxidative damage facilitates disordered Ca²⁺ influx.

ROS → intracellular stores

Sarcoplasmic-reticulum injury increases calcium dysregulation.

Ca²⁺ → mitochondria

Permeability transition weakens ATP synthesis.

Common endpoint

Energy failure and membrane injury reinforce cell death.

Slide 36 · Reperfusion injury can be sterile inflammation

Trigger without an infection

Tissue consequence

Dead and damaged cells release danger signals.

Resident macrophages can secrete cytokines.

Hypoxic tissue cells display adhesion-related signals.

Circulating leukocytes are recruited to injured tissue.

Recruited neutrophils add to local damage.

The inflammatory response extends injury beyond the initial insult.

Slide 37 · Danger signals connect cell death to inflammation

Ischemic tissue injury

Damaged / dead cells appear

Danger signals

Host-response pathways are activated

Resident immune cells

Macrophages secrete cytokines

Leukocyte recruitment

Neutrophils move toward injured tissue

Inflammation follows tissue damage and can increase it.

Slide 38 · Resident macrophages contribute to recruitment

Local sentinel cells

Resident macrophages detect injury-associated signals.

Cytokine output

Inflammatory mediators are released.

Vascular communication

Cytokine activity supports leukocyte recruitment.

Amplification

Infiltrating leukocytes subsequently increase injury.

Slide 39 · Endothelial adhesion facilitates leukocyte influx

Hypoxic tissue and endothelium

Adhesion-molecule expression increases

Blood flow resumes

Circulating neutrophils arrive

Adhesion and extravasation

Leukocytes leave the vascular space

Tissue accumulation

Inflammatory injury intensifies

Vascular reopening supplies the cellular participants of inflammation.

Slide 40 · Recruited neutrophils cause secondary injury

Why neutrophils are recruited

Why their arrival can be harmful

Cytokines and adhesion molecules signal tissue injury.

Neutrophils enter the reperfused tissue.

Their normal role includes response to damaged tissue.

Activated leukocytes release reactive products.

Inflammatory activity damages viable neighboring cells.

Net injury includes both primary ischemia and secondary inflammation.

Slide 41 · Reperfusion supplies oxygen and inflammatory cells

Restoration of blood flow

Oxygen and metabolic substrates

Recovery of salvageable cells

Leukocytes and plasma proteins

New cellular and humoral injury pathways

Damaged microenvironment

Inflammatory signals favor localized amplification

Slide 42 · Neutrophil recruitment has experimental support

What Robbins reports

Proper interpretation

Experimental antibodies blocking cytokines or adhesion molecules reduce neutrophil extravasation.

These interventions had beneficial effects in reperfusion-injury models.

This supports a causal role for recruited neutrophils.

It does not establish an approved clinical antibody regimen.

Experimental protection must not be equated with proven patient benefit.

Slide 43 · Complement can be activated by ischemic-tissue IgM

Ischemic tissue

Certain IgM antibodies may deposit

Blood flow resumes

Complement proteins gain access

Complement engagement

Complement system becomes activated

Cell injury and inflammation

Existing damage is exacerbated

The reason for preferential IgM deposition is unknown.

Slide 44 · Complement amplifies more than one injury process

Antibody deposition

Some IgM preferentially localizes to ischemic tissue.

Complement proteins

Restored circulation supplies the humoral components.

Inflammatory amplification

Activated complement augments local inflammation.

Additional injury

Tissue damage can increase after blood flow returns.

Slide 45 · Complement: Distinguish claim from uncertainty

Supported in Robbins

Not established by this passage

IgM may deposit in ischemic tissues.

Why the IgM preferentially deposits.

Complement can bind after reperfusion.

A universal mechanism in every patient or organ.

Activation may worsen injury and inflammation.

A clinically proven complement-blocking therapy.

Slide 46 · The four mechanisms act as an interconnected network

Oxidants

Injure membranes and calcium-regulating compartments.

Ca²⁺ overload

Drives permeability transition and ATP loss.

Neutrophils

Generate additional oxidants and inflammatory damage.

Complement

Contributes to tissue injury and inflammatory amplification.

Slide 47 · Multiple pathways converge on a few lethal endpoints

Oxidative stress + Ca²⁺ overload + inflammation + complement

Mitochondrial dysfunction

Failure of oxidative phosphorylation; ATP depletion

Membrane injury

Disturbed compartment barriers and enzyme leakage

Cell death

Additional necrosis in already vulnerable tissue

Slide 48 · Necrosis reflects failure of cellular integrity

Interpretation of tissue damage:

Irreversible ischemic damage already commits some cells to necrosis.

Reperfusion injury can cause additional cells to lose viability.

Mitochondrial ATP failure and membrane damage are convergent mechanisms.

Histologic changes reflect cell death, but routine morphology alone does not apportion damage between the two phases.

Slide 49 · Ischemia versus reperfusion: Causal attribution

Question

Ischemic phase

Reperfusion phase

Primary trigger

Reduced perfusion

Restoration of perfusion

Principal energy effect

Less oxidative phosphorylation

Recovery or continued mitochondrial failure

Distinct contributors

Substrate depletion, metabolite buildup

Reoxygenation ROS, Ca²⁺ aggravation

Inflammatory contribution

Danger signals begin

Neutrophil and complement pathways intensify

Cell viability

Depends on insult severity/duration

Recovery and new injury can coexist

Slide 50 · Myocardial infarction provides a clinical setting

Before flow is restored

After restoration of flow

Coronary ischemia jeopardizes oxygen-dependent ATP generation.

Viable cells may be reversibly injured.

Other cells may already be irreversibly damaged.

Salvageable myocardium has an opportunity to recover.

Oxidative, calcium and inflammatory mechanisms may injure additional cells.

Reperfusion injury does not negate the need to restore perfusion.

Slide 51 · Cerebral infarction provides a second setting

Ischemic cerebral tissue

After restored cerebral perfusion

Reduced blood flow compromises oxygen and metabolic substrate delivery.

Mitochondrial dysfunction and ATP depletion threaten cell survival.

The extent of irreversible injury varies with the ischemic insult.

Recovery is possible in still-viable tissue.

Oxidative stress and inflammatory recruitment may worsen injury.

Cerebral infarction as clinically relevant to reperfusion injury.

Slide 52 · Restoring blood flow remains the therapeutic priority

Goal of restoring perfusion

Save salvageable tissue

Support surviving cells before irreversible injury occurs

Recognize a trade-off

Reperfusion may contribute additional cellular injury

Avoid false inference

A secondary hazard does not make ongoing ischemia desirable

Slide 53 · Experimental mechanisms are not clinical directives

Observation from the chapter

Legitimate inference

What cannot be concluded

Antibody blockade limits neutrophil influx in experiments

Neutrophils contribute mechanistically

Routine antibody treatment is effective in patients

Calcium overload favors mitochondrial pore opening

Mitochondria are an injury hub

A specific calcium-targeted therapy is established

Oxidants cause macromolecular damage

Oxidative stress is an injury pathway

Any antioxidant necessarily improves clinical outcome

Slide 54 · Hypoxic adaptation is distinct from reperfusion injury

HIF-1 during hypoxic stress

Reperfusion injury

A response to reduced oxygen availability.

Supports glycolysis, survival pathways and new vessel formation.

Described in the adjacent ischemic-cell-injury section.

Arises with restoration of blood flow.

Includes ROS, Ca²⁺, inflammation and complement activation.

Cannot be reduced to a failure of one hypoxic adaptive pathway.

Slide 55 · Case: The reperfused myocardium

Clinical reasoning scenario

A patient with coronary arterial obstruction receives treatment that restores myocardial blood flow. Despite successful restoration of perfusion, injury may extend beyond cells already lost during ischemia.

Identify two mechanisms that could increase cell loss after reperfusion.

Slide 56 · Case analysis: Separate injury phases

Damage already present

Additional reperfusion injury

Ischemia reduced oxidative phosphorylation.

ATP depletion and ion-pump failure injured cells.

Prolonged damage may have produced irreversible necrosis.

ROS can oxidize membranes and proteins.

Calcium overload favors mitochondrial permeability transition.

Inflammatory cells and complement can amplify damage.

Slide 57 · Case: After cerebral blood flow returns

Clinical reasoning scenario

Cerebral perfusion is re-established after a period of ischemia. Some cells recover, while inflammatory cells appear in the injured region and oxidative stress increases.

Why is the presence of recovered perfusion not proof that all remaining injury was caused by the original ischemic period?

Slide 58 · Case analysis: A causal explanation

Initial ischemia

ATP loss; Cell injury

Flow restored

O₂ and circulating components return

Secondary mechanisms

ROS + Ca²⁺ + neutrophils + complement

Final result

Recovery in some cells, additional death in others

The contribution of each mechanism need not be identical in every tissue.

Slide 59 · What cannot be diagnosed from this excerpt alone

Keep the evidentiary boundary clear:

No tissue-specific histologic criterion uniquely identifies reperfusion injury.

No diagnostic biomarker cutoff or validated test is specified here.

No proportion of final infarct size attributable to reperfusion is supplied.

Mechanistic plausibility does not establish the efficacy of a particular treatment.

Slide 60 · Integrating the chronology and mechanistic pathways

Reduced flow

Substrate and O₂ deficiency; ATP falls.

Cell injury

Calcium dysregulation, organelle and membrane damage.

Reflow

O₂ and plasma/circulating cells return.

Amplification

ROS, Ca²⁺, neutrophils and complement add injury.

Sequence shown conceptually; Cellular outcomes depend on the severity and duration of ischemia.

Slide 61 · Mechanistic knowledge check

1. Why does ischemia usually injure faster than isolated hypoxia?

It compromises substrate delivery and metabolite clearance as well as oxygen.

2. What does mitochondrial Ca²⁺ overload favor?

Opening of the permeability transition pore with ATP failure.

3. What finding supports a role for neutrophils?

Experimental benefit from limiting cytokine / adhesion-molecule-mediated extravasation.

Slide 62 · Five durable points for clinical pathology

Synthesis of the mechanism:

Reperfusion is necessary to salvage reversibly injured ischemic tissue.

A subset of reperfused cells can incur additional injury rather than recover.

Oxidants, intracellular Ca²⁺, inflammation and complement are principal proposed mechanisms.

Mitochondrial failure and membrane damage are important convergent endpoints.

Mechanistic evidence must be separated from established clinical interventions.

Slide 63 · Reference

Robbins, Cotran & Kumar: Pathologic Basis of Disease. 11th edition. 2026.

Chapter 2: Cell Injury, Cell Death, and Adaptations.

Sections: Mechanisms of Cell Injury; Oxidative Stress; Disturbance in Calcium Homeostasis; Hypoxia and Ischemia; Ischemia-Reperfusion Injury.