Role of free radicals/oxidative stress/ biochemical changes in ischemic reperfusion cell injury md pathology exam question robbins based answer
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Role of free radicals, oxidative stress, and biochemical changes in ischemia-reperfusion cell injury
Definition
Ischemia-reperfusion injury is the paradoxical worsening of cell injury and death when blood supply is restored to an ischemic but still viable tissue. Reperfusion is necessary to salvage tissue, but it can kill additional cells that could otherwise have recovered. It is important in myocardial infarction, cerebral infarction, organ transplantation, and limb ischemia.
Robbins, Cotran & Kumar Pathologic Basis of Disease, p. 50, 67.
Robbins, Cotran & Kumar Pathologic Basis of Disease, p. 50, 67.
1. Free radicals and oxidative stress
Free radicals
Free radicals are highly reactive chemical species with an unpaired electron. The most relevant are reactive oxygen species (ROS):
- Superoxide anion: O₂•¯
- Hydrogen peroxide: H₂O₂
- Hydroxyl radical: •OH, the most reactive ROS
- Peroxynitrite: ONOO¯, formed from nitric oxide and superoxide
Oxidative stress means excess production of ROS or inadequate removal by cellular antioxidant systems.
Why ROS rise during reperfusion
When oxygenated blood returns to ischemic cells, a sudden supply of O₂ reaches cells with damaged mitochondria. These mitochondria cannot reduce oxygen completely during oxidative phosphorylation, resulting in a burst of ROS.
ROS are generated by:
- Damaged mitochondria in ischemic parenchymal cells
- Activated neutrophils/macrophages recruited to reperfused tissue, via NADPH oxidase respiratory burst
- Damaged endothelial cells
- Xanthine oxidase and other oxidases
- Iron-catalyzed conversion of H₂O₂ to •OH by the Fenton reaction:
[ H_2O_2 + Fe^{2+} \rightarrow Fe^{3+} + OH^- + \bullet OH ]
During ischemia, intracellular antioxidant defenses such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione may be depleted or impaired. Thus, the cell is particularly vulnerable when oxygen returns.
Robbins, Cotran & Kumar Pathologic Basis of Disease, p. 48-49, 67.
2. Mechanisms of ROS-mediated cell injury
A. Lipid peroxidation of membranes
ROS attack polyunsaturated fatty acids in cell membranes.
Affected membranes include:
- Plasma membrane
- Mitochondrial membrane
- Endoplasmic reticulum membrane
- Lysosomal membrane
Consequences:
- Increased membrane permeability
- Loss of ionic gradients
- Leakage of cellular enzymes and proteins
- Mitochondrial dysfunction and failure of ATP generation
- Lysosomal enzyme leakage, causing autodigestion of the cell
Lipid peroxidation is self-propagating because the damaged lipid can itself form another free radical.
B. Protein oxidation and fragmentation
ROS oxidize amino acid side chains and cause:
- Protein cross-linking
- Protein fragmentation
- Enzyme inactivation
- Damage to ion pumps, cytoskeletal proteins, receptors, and membrane transport proteins
- Increased membrane permeability and failure of cellular homeostasis
C. DNA damage
ROS induce DNA strand breaks and base modifications.
This can trigger:
- Activation of DNA repair pathways
- Cell-cycle arrest
- Apoptosis if damage is severe
- Necrosis when injury is overwhelming and ATP is depleted
Thus, ROS can cause both necrosis and apoptosis.
3. Other biochemical changes that worsen reperfusion injury
A. Intracellular calcium overload
During ischemia, ATP depletion causes failure of ATP-dependent Ca²⁺ pumps. Therefore intracellular Ca²⁺ begins to rise.
On reperfusion, calcium influx is further increased because of:
- Plasma membrane injury
- ROS-mediated injury to the sarcoplasmic reticulum/endoplasmic reticulum
- Persistent pump failure due to mitochondrial damage
Raised cytosolic Ca²⁺ causes:
- Activation of phospholipases → membrane phospholipid breakdown
- Activation of proteases → cytoskeletal and membrane protein degradation
- Activation of endonucleases → DNA and chromatin fragmentation
- Activation of ATPases → further ATP depletion
- Increased mitochondrial Ca²⁺ → opening of mitochondrial permeability transition pore
B. Mitochondrial permeability transition
High mitochondrial Ca²⁺ plus ROS opens the mitochondrial permeability transition pore.
This leads to:
- Loss of mitochondrial membrane potential
- Failure of oxidative phosphorylation
- Severe fall in ATP
- Increased mitochondrial swelling and membrane injury
- Release of cytochrome c and other proapoptotic proteins
- Apoptosis or irreversible necrotic cell death
C. Inflammation and neutrophil-mediated injury
Ischemic cells release damage-associated signals. Hypoxic endothelium and parenchymal cells express adhesion molecules, and resident macrophages release cytokines.
After reperfusion:
- Neutrophils enter the tissue
- Neutrophils release ROS, proteases, and inflammatory mediators
- Endothelial injury and microvascular dysfunction worsen
- More parenchymal cells are injured
D. Complement activation
Some IgM antibodies may deposit in ischemic tissues. When blood flow returns:
- Complement proteins bind these antibodies
- Complement is activated
- Inflammation and cell injury are amplified
Robbins, Cotran & Kumar Pathologic Basis of Disease, p. 67.
Flowchart for exam
Ischemia
↓
ATP depletion + acidosis + Na+/K+ pump failure + initial Ca2+ rise
↓
Reperfusion / reoxygenation
↓
Burst of ROS/RNS from damaged mitochondria, endothelium, and neutrophils
↓
Lipid peroxidation + protein damage + DNA damage
↓
Membrane injury + mitochondrial dysfunction + lysosomal leakage
↓
Further Ca2+ influx and mitochondrial permeability transition
↓
ATP depletion, cytochrome c release, inflammation, complement activation
↓
Apoptosis and necrosis of cells that were initially reversibly injured
Short note conclusion
Free radicals are central mediators of ischemia-reperfusion injury. Reoxygenation causes excessive generation of ROS in mitochondria, endothelial cells, and infiltrating leukocytes, while ischemia impairs antioxidant defenses. ROS cause lipid peroxidation, protein denaturation, DNA damage, membrane disruption, mitochondrial failure, calcium overload, and activation of inflammatory and complement pathways. These mechanisms transform potentially reversible ischemic injury into irreversible injury, apoptosis, and necrosis.
A recent systematic review of ischemia-reperfusion injury also supports oxidative stress as a major mechanism, although organ-specific pathways and therapies differ recent review.
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