Molecular and Cellular Mechanisms of Ischemia-Induced Neuronal Death

Focal Ischemia

Focal ischemia in humans is most commonly caused by ischemic stroke due to a vascular occlusion in the cerebral circulation. ^, It is the fifth leading cause of death in the United States and the primary cause of disabilities in adults. Of the approximately 600,000 new victims each year, nearly 30% die and 20%–30% become severely and permanently disabled. Neurologic deficits resulting from focal ischemia include paralysis and abnormalities in coordination, sensory function, and language ability.

The tissues at risk are at the core or center of the stroke, which contains cells that receive essentially no blood, and the penumbra or surrounding region, which includes cells that receive reduced blood. Cells in the core die from several overwhelming causes and probably cannot be salvaged by any treatment short of immediate clot removal. Although the infarct starts in the ischemic core, at its maximum, it encompasses both the core and penumbra, generally by 6–24 hours after induction of permanent ischemia. The duration of the ischemic episode determines the extent or grade of damage in rats. At 10–20 minutes after initiation of focal ischemia, only a few scattered dead neurons are observed in the core. The infarct appears in the striatum and cortex when the ischemia is 60 minutes and keeps increasing with cumulative occlusion time, reaching maximal with an occlusion duration of 3 hours. The mechanisms underlying cell death in the core are complicated, but they most certainly include glutamate receptor–mediated necrotic cell death (see later).

Models of Global Ischemia

Global ischemia can be produced by permanent occlusion of the vertebral arteries and transient occlusion of the common carotid arteries (in rats) or by transient occlusion of the common carotid arteries (in rats and mice) followed by reperfusion or transient occlusion of the common carotid arteries (in gerbils). The most commonly used models of global ischemia are (1) the four-vessel occlusion (4-VO) model in rats ( Fig. 5.1A ) ^, ; (2) the two-vessel occlusion (2-VO; also known as temporary bilateral common carotid occlusion) model in gerbils ^, or mice ^, ; and (3) 2-VO in combination with hypotension in rats. ^, The duration of global ischemia is typically short—in the order of 5–20 minutes. During the ischemic episode, blood flow to the entire brain is immediately reduced to less than 1%, and adenosine triphosphate (ATP) is depleted in cells throughout the brain.

Four-VO in rats is a well-established and widely used model of global ischemia, in which neuronal death is primarily restricted to pyramidal neurons of the hippocampal CA1 and does not manifest itself until 3 days after insult. ^, Briefly, the vertebral arteries of rats are exposed and subjected to permanent electrocauterization. The common carotid arteries are exposed and isolated with a 3-0 silk thread, and the wound is sutured. Twenty-four hours later, the wound is reopened, and the common carotid arteries are subjected to temporary occlusion with surgical clasps (4 minutes for sublethal ischemia and 10 minutes for global ischemia). When the carotid arteries are occluded, blood flow is typically reduced to less than 3% of normal in the hippocampus, striatum, and neocortex. ^,

Models of Focal Ischemia

Focal ischemia is the animal model that most closely approximates stroke or cerebral infarction in humans. It is typically performed on rats and mice and is produced experimentally by middle cerebral artery occlusion (MCAO). Arterial occlusion can be permanent (i.e., the arterial blockade is maintained throughout the experiment) or transient (the occlusion is for up to 3 hours, followed by reperfusion). The occlusion can be either proximal or distal (see later). These procedures induce a necrotic core of cells that are irreversibly damaged and a penumbra of cells that can be rescued (see Fig. 5.1B ).

Proximal MCAO is most commonly induced by ligating the common carotid and external carotid arteries, followed by the insertion of a suture into the internal carotid artery. The coating part of the suture is supposed to be beyond the origin of the posterior communicating artery and past the origin of the MCA. The center of the core is the region in which blood flow is reduced to less than 15% and encompasses the lateral portion of the caudate-putamen and the parietal cortex. The penumbra, the area in which blood flow is reduced to less than 40%, comprises the remainder of the neocortex, the entorhinal cortex, and medial caudate-putamen. In distal MCAO, blood flow to the basal ganglia is not interrupted, and the damage is restricted to the neocortex. This type of occlusion can be induced surgically with a clip or thrombotic clots in combination with transient unilateral occlusion of the common carotid arteries. ^, The reduction of blood flow in the core and penumbra is similar to that in the proximal model.

In addition to MCAO, focal ischemia can also be induced by a model of hypoxia/ischemia.

This model combines permanent unilateral occlusion of a common carotid artery with transient systemic hypoxia, such that oxygen flow to the brain is reduced to 3% in adults or to 8% in neonates. After 15–30 minutes of hypoxia, infarct occurs in the cortex, striatum, and hippocampus.

Necrosis

Necrosis, or necrotic cell death, is morphologically characterized by cell swelling, swelling of mitochondria and other organelles, the rupture of the plasma membrane, and subsequent release of intracellular contents. ^, The nucleus exhibits pyknosis and irregular clumping of the chromatin (peripheral chromatolysis), a pattern that contrasts sharply with the sparse, regularly shaped and uniformly distributed aggregates of chromatin observed in apoptosis.

Necroptosis

Necroptosis is a type of regulated cell death that exhibits both characteristics of necrosis and apoptosis. Necroptosis is a caspase-independent necrotic cell death pathway that is regulated by receptor-interacting protein 1 (RIP1) kinase and its downstream mediator RIP3 kinase. ^{, ,} Among the proposed necroptosis signaling pathways, the tumor necrosis factor (TNF)-TNF receptor 1 (TNFR1)–mediated pathway is the most prominent. Briefly, upon binding of TNF to TNFR1, TNFR-associated death domain (TRADD) is formed, which is termed Complex I. Then, the death domain receptors engage intracellular signaling cascades that activate the obligatory serine/threonine kinase known as RIP1. RIP1 activates the nuclear factor (NF)-κB signaling pathway to promote cell survival and inflammation ( Fig. 5.2A ). The FAS-associated death domain (FADD), RIPK3, FLIPs, and procaspase 8 form the Complex II, which regulates both apoptosis (Complex IIa) and necroptosis (Complex IIb). In Complex IIa, the long isotype of FLIP (FLIP _L ) and procaspase 8 inactivate RIPK1 and RIPK3 by forming a heterodimeric caspase, thus preventing necroptosis. Meanwhile, Complex IIa results in apoptosis by activating caspase 8, which mediates the executioner caspases caspase 3 and caspase 7 signaling pathways (see Fig. 5.2B ).

In Complex IIb, acetylated RIP1 dissociates from Complex I to form an unstable connection between RIP1 and RIP3. However, studies indicate the interaction with deacetylase enzyme sirtuin-2 (SIRT2) and RIP3 are required to deacetylate RIP1. Deacetylated RIP1 becomes a prominent part of Complex IIb, which directly leads to necroptosis (see Fig. 5.2C ). ^, Activated RIP3 can steer TNF-induced apoptosis toward necroptosis or even full-blown necrosis, in part through disruption of energy metabolism, generation of reactive oxygen species (ROS), nitroxidative stress by nitric acid, ^, increases in intracellular Ca ²⁺ , activation of Ca ²⁺ -dependent noncaspase proteases such as calpains and cathepsins, activation of cyclophilin D, opening of the mitochondrial membrane transition pore (MOMP), and mitochondrial release of poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1).

In the final stage of necrotic death, swollen cells are internalized by a process known as macropinocytosis, in which only parts of the cell are taken up by phagocytes. Necrosis is considered the predominant form of neuronal death in models of focal ischemia and ischemic stroke in humans. Genetic ablation of cyclophilin D, which abolishes necrotic cell death, or pharmacologic inhibition of necrosis by necrostatin 1, a small-molecule inhibitor of RIP1, affords a significant reduction of infarct volume and improves neurologic outcome after focal ischemia. These observations implicate RIP1 as a potential therapeutic target in ischemic stroke.

Apoptosis

Caspase-Dependent Apoptosis

Caspases are a family of structurally related cysteine proteases and are the initiators and executioners of apoptotic processes. ^, The human genome encodes 13–14 distinct caspases; of these, caspases 2, 3, 6, 7, 8, 9, and 10 predominantly function in cell death, whereas the others are involved in regulating immune responses. Caspases are classified into “initiator caspases” (caspases 2, 8, 9, and 10), which integrate upstream apoptotic stimuli, and “effector caspases” (caspases 3, 6, and 7), which are activated by initiator caspases and cleave an array of diverse cellular targets.

The caspase cascades can be activated by either extrinsic or intrinsic pathways. In the extrinsic or death receptor–dependent pathway, apoptosis is initiated when injurious stimuli such as ischemia activate CD95-Fas (receptor for CD95-Fas ligand), a member of the TNFR–nerve growth factor superfamily of death domain receptors that includes TNFR1, CD95-Fas, and TRAIL receptor ( Fig. 5.3 ). ^{, ,} Within seconds of its activation, CD95-Fas forms a cytosolic death-inducing signaling complex (DISC) with its adapter proteins, which act via their death domains to bind CD95-Fas and via their death effector domain to recruit procaspase 8 ( Fig. 5.4 ). ^, DISC catalyzes the cleavage and inactivation of RIP1 and RIP3 and activation of procaspase 8 to generate caspase 8. The extrinsic pathway can connect to the intrinsic pathway when caspase 8 cleaves the Bcl-2 family protein Bid to generate truncated Bid (tBid), which translocates to the mitochondria, where it initiates permeabilization of the outer mitochondrial membrane and initiates the mitochondrial pathway of apoptosis. Therefore the extrinsic pathway may activate a caspase-dependent cell death execution pathway via one of the following routes: (1) death receptor mediated and triggered the caspase 8 (or 10)-caspase 3 pathway; (2) death receptor mediated and triggered the caspase-8-tBid-MOMP-caspase 9-caspase 3 pathway; or (3) ligand deprivation–induced dependence receptor signaling followed by activation of the caspase 9-caspase 3 pathway. ^,

In the intrinsic or mitochondrial pathway, apoptosis is initiated when cell death stimuli activate pro-death Bcl-2 family proteins that in turn permeabilize the mitochondrial outer membrane, leading to the release of mitochondrial proteins such as cytochrome c , second mitochondria-derived activator of caspases (Smac), and apoptosis-inducing factor (AIF) into the cytoplasm ( Fig. 5.5 ). ^{, ,} Once in the cytoplasm, cytochrome c binds ATP to activate the apoptotic protease activating factor 1 (Apaf-1), which oligomerizes and recruits procaspase 9 to form the caspase-activating complex or “apoptosome” ( Fig. 5.6 ). Activated caspase 9 then cleaves procaspase 3 to generate the active “effector” caspase 3. Caspase 3 promotes cell death by proteolytic cleavage of downstream target proteins, including poly (ADP-ribose) polymerase, nuclear lamins, DNA-dependent protein kinase, ICAD (the inhibitory subunit of the DNA ladder-inducing endonuclease CAD), and many others, endowing cells with the morphologic characteristic of apoptosis. DNA fragmentation and other events result in cellular disintegration, followed by engulfment of fragmented cells by surrounding cells. ^{, , ,} Thus the apoptosome enables cytochrome c to start a caspase cascade of proteolysis independently of ligand-activated death receptors.