Mechanisms of Ischemic Mechanisms of Ischemic Brain DamageBrain Damage

Jenn MejillaJenn Mejilla

2 Hypothesis of Brain Ischemia2 Hypothesis of Brain Ischemia

Calcium HypothesisCalcium Hypothesis

Excitotoxic HypothesisExcitotoxic Hypothesis

Calcium HypothesisCalcium Hypothesis

Massive Ca+2 entry into cells leads to cell Massive Ca+2 entry into cells leads to cell deathdeath Ca+2 catalyzed the breakdown of structural Ca+2 catalyzed the breakdown of structural

components of cells (membrane lipids and components of cells (membrane lipids and cytoskeletal proteins).cytoskeletal proteins).

Agonist-receptor interactions at the motor end Agonist-receptor interactions at the motor end plate caused necrosis of the target, plate caused necrosis of the target, innervated by cholinergic fibers.innervated by cholinergic fibers.

When this general hypothesis was applied to the nervous system, it was assumed that calcium entering dendritic cells, caused necrosis of selectively vulnerable neurons by ischemia or hypoxia, hypoglycemic coma, and status epilepticus.

Calcium was assumed to enter cells by way of voltage-sensitive calcium channels, which are abundant at the basal dendrites of cells with a tendency to epileptogenic firing.

Calcium MetabolismCalcium Metabolism

Presynaptic depolarization causes Ca+2 Presynaptic depolarization causes Ca+2 to enter the cytoplasm of the presynaptic to enter the cytoplasm of the presynaptic endingsendings

Followed by release of glutamate. This Followed by release of glutamate. This activates two types of ionotropic glutamate activates two types of ionotropic glutamate receptors- AMPA and NMDA.receptors- AMPA and NMDA.

(AMPA =amino-3-hydroxy-5-methol-4-isoazole propionic acid)(AMPA =amino-3-hydroxy-5-methol-4-isoazole propionic acid)

(NMDA = N-methyl –D- aspartate)(NMDA = N-methyl –D- aspartate)

•When glutamate activates the AMPA receptor, a channel is opened that allows the passage of Na+, K+ and H+. When Na+ enters down its electrochemical gradient, it depolarizes the membrane. This allows the influx of Ca+2 by way of any voltage-sensitive calcium channels that may be localized to the postsynaptic membranes of the dendrites and cell body (eg. L and T types)

•In addition, it relieves the Mg+2 block of the NMDA gated channel, allowing Ca+2 to enter this high-conductance, unselective cation channel.

•The excitatory event is terminated by reuptake of glutamate into presynaptic vesicles and into glial cells.

• Ca+2 entry via NMDA receptors has special pathophysiologic significance:• NMDA receptor-gated channel has a high calcium conductance• The channels or calcium ions they conduct are in contact with cell structures that are vulnerable to the increase in intracellular Ca+2.

1. When Ca+2 ions enter cells by way of NMDA receptor-gated channels, they are more prone to trigger the production of ROS

, reactive oxygen species, such as H2O2, O2-, OH.

2. Postynaptic calcium influx stimulates neuronal NO synthase, allowing for the simultaneous appearance of O2- and NO in postsynaptic structures.

Excitotoxic HypothesisExcitotoxic Hypothesis

Described in 1981Described in 1981 Excitatory amino acid-related toxicity led to Excitatory amino acid-related toxicity led to

neuronal cell death in tissue slices or primary neuronal cell death in tissue slices or primary neuronal cell cultures.neuronal cell cultures.

It was initially argued that glutamate activation It was initially argued that glutamate activation of AMPA receptors leads to an influx of Na+, of AMPA receptors leads to an influx of Na+, Cl- and water- which causes osmolytic cell Cl- and water- which causes osmolytic cell damage.damage.

Later, results showed that the osmolytic Later, results showed that the osmolytic damage was reversible, but the influx of damage was reversible, but the influx of calcium caused a delayed type of damage.calcium caused a delayed type of damage.

It is now clear that a single Ca+2 exposure It is now clear that a single Ca+2 exposure can lead to secondary compromise of can lead to secondary compromise of Ca+2, suggesting a delayed failure of Ca+2, suggesting a delayed failure of calcium regulation.calcium regulation.

Glutamate and Calcium Triggered Glutamate and Calcium Triggered EventsEvents

Enhanced LipolysisEnhanced Lipolysis

Altered Phosphorylation of ProteinsAltered Phosphorylation of Proteins

Enhanced production of reactive oxygen Enhanced production of reactive oxygen and reactive nitrogen species.and reactive nitrogen species.

Enhanced LipolysisEnhanced LipolysisIschemia leads to lipolysis because ATP and Ischemia leads to lipolysis because ATP and cytidine triphosphate are no longer present to cytidine triphosphate are no longer present to catalyze the resynthesis of phospholipids, once catalyze the resynthesis of phospholipids, once they are broken down, and because calcium they are broken down, and because calcium activates enzymes, degrading phospholipids to activates enzymes, degrading phospholipids to biologically active compounds such as FFA’s biologically active compounds such as FFA’s and lysophospholipids.and lysophospholipids.

FFA’s and lysophospholipids are mediators of FFA’s and lysophospholipids are mediators of membrane dysfunction b/c they make act as membrane dysfunction b/c they make act as ionophores and uncoupling agents.ionophores and uncoupling agents.

Once reperfusion is initiated, the oxidative meta-Once reperfusion is initiated, the oxidative meta-

bolism of arachidonic acid accumulated during the bolism of arachidonic acid accumulated during the ischemia leads to the formation of ischemia leads to the formation of cyclooxygenase and lipooxygenase products- cyclooxygenase and lipooxygenase products- active in triggering inflammatory responses.active in triggering inflammatory responses.

Altered Phosphorylation of ProteinsAltered Phosphorylation of Proteins

Ca+2 is an important modulator of the Ca+2 is an important modulator of the phosphorylation state of many proteins. When phosphorylation state of many proteins. When proteins are phosphorylated and proteins are phosphorylated and dephosphorylated, their functions are altered. dephosphorylated, their functions are altered. So, when calcium concentration is So, when calcium concentration is transient,particularly when Ca+2 is excessive transient,particularly when Ca+2 is excessive and sustained, membrane function and and sustained, membrane function and metabolic activities alteration can cause harmful metabolic activities alteration can cause harmful effects.effects.

Production of ROS and NOSProduction of ROS and NOS

Ischemia with reperfusion leads to the Ischemia with reperfusion leads to the production of ROS . These free radicals give production of ROS . These free radicals give rise to lipid peroxidation, protein oxidation and rise to lipid peroxidation, protein oxidation and DNA damage.DNA damage.

Oxygen radicals and NO, together, exert toxicityOxygen radicals and NO, together, exert toxicity NO has important role in brain ischemia. NO has important role in brain ischemia.

3 types3 types n-NOS and e-NOS (calcium dependent and constitutively n-NOS and e-NOS (calcium dependent and constitutively

expressed)expressed) i-NOS (expressed by activated macrophages and neutrophils)i-NOS (expressed by activated macrophages and neutrophils)

Neuronal NOS is involved in synaptic signalling; however, under ischemic conditions, it mediates celldeath.

The same is true for i-NOS.

Therefore, the production of NO by the calcium-Dependent n-NOS may be detrimental because it Allows additional and toxic ROS to be formed.

Dissolution of the CytoskeletonDissolution of the Cytoskeleton

Increase in intracellular Ca+2 activates Increase in intracellular Ca+2 activates proteases that break down neurofilaments and proteases that break down neurofilaments and contribute to the disassembly of microtubules. contribute to the disassembly of microtubules.

This breakdown cause serious problems in This breakdown cause serious problems in intracellular communication, which depends on intracellular communication, which depends on the integrity of the cytoskeleton as well as cause the integrity of the cytoskeleton as well as cause damage to the mitochondria of cells.damage to the mitochondria of cells.