Molecular mechanisms of injury progression Figure

In the border zone of permanent focal ischemia or in the ischemic territory after transient vascular occlusion, cellular disturbances may evolve that cannot be explained by a lasting impairment of blood flow or energy metabolism. These disturbances are referred to as molecular injury, where the term "molecular" does not anticipate any particular injury pathway (Figure 1.8). The molecular injury cascades (Figure 1.8) are interconnected in complex ways, which makes it difficult to predict their relative pathogenic importance in different ischemia models. In particular, molecular injury induced by transient focal ischemia is not equivalent to the alterations that occur in the core or the penumbra of permanent ischemia. Therefore, the relative contribution of the following injury mechanisms differs in different types of ischemia.

Acidotoxicity: during ischemia oxygen depletion and the associated activation of anaerobic glycolysis cause an accumulation of lactic acid which, depending on the severity of ischemia, blood glucose levels and the degree of ATP hydrolysis, results in a decline of intracellular pH to levels between 6.5 and below 6.0. As the severity of acidosis correlates with the severity of ischemic injury, it has been postulated that acidosis is neurotoxic. Recently, evidence has been provided that ASICs (acid-sensing ion channels) are glutamate-independent vehicles of calcium flux, and

Spreading depression

Spreading depression

CYTOTOXIC EDEMA

EXCITOTOXICITY

Glu CALCIUM OVERLOAD

Signal transduction

CYTOTOXIC EDEMA

EXCITOTOXICITY

Glu CALCIUM OVERLOAD

Membrane-damage

Leukocyte infiltration

INFLAMMATION

Membrane-damage

Signal transduction

"V

Gene expression ER STRESS RESPONSE

Protein synthesis inhibition

Stress protein expression MITOCHONDRIAL PERMEABILITY TRANSITION

Energy failure PermeabilityCytochrome C Enzymetransition release NECROSIS

induction

Caspase 3

FREE RADICALS

Inflammation mediators

APOPTOSIS

NAD depletion

Microglia activation y

Inflammation mediators

APOPTOSIS

Microglia activation

Figure 1.8. Schematic representation of molecular injury pathways leading to mitochondrialfailure and the endoplasmic reticulum 16 stress response. Injury pathways can be blocked at numerous sites, providing multiple approaches for the amelioration of both necrotic _ and apoptotic tissue injury.

that blockade of ASICs attenuates stroke injury. This suggests that acidosis may induce calcium toxicity, and that this effect is the actual mechanism of acido-toxicity [68].

Excitotoxicity: shortly after the onset of ischemia, excitatory and inhibitory neurotransmitters are released, resulting in the activation of their specific receptors. Among these neurotransmitters, particular attention has been attributed to glutamate, which at high concentrations is known to produce excito-toxicity [69]. The activation of ionotropic glutamate receptors results in the inflow of calcium from the extracellular into the intracellular compartment, leading to mitochondrial calcium overload and the activation of calcium-dependent catabolic enzymes. The activation of metabotropic glutamate receptors induces the IP3-dependent signal transduction pathway, leading inter alia to the stress response of endoplasmic reticulum, and by induction of immediate-early genes (IEG) to adaptive genomic expressions. At high concentration, glutamate results in primary neuronal necrosis. However, following pharmacological inhibition of ionotropic glutamate receptors, an apoptotic injury mechanism evolves that may prevail under certain pathophysiological conditions. The importance of excitotoxicity for ischemic cell injury has been debated, but this does not invalidate the beneficial effect of glutamate antagonists for the treatment of focal ischemia. An explanation for this discrepancy is the above-described pathogenic role of peri-infarct depolarizations in infarct expansion. As glutamate antagonists inhibit the spread of these depolarizations, the resulting injury is also reduced.

Calcium toxicity: in the intact cell, highly efficient calcium transport systems ensure the maintenance of a steep calcium concentration gradient of approximately 1:10 000 between the extra- and the intracellu-lar compartment on the one hand, and between the cytosol and the endoplasmic reticulum (ER) on the other. During ischemia anoxic depolarization in combination with the activation of ionotropic glutamate and acid-sensing ion channels causes a sharp rise of cytosolic calcium [70]. At the onset of ischemia this rise is further enhanced by activation of metabotropic glutamate receptors which mediate the release of calcium from endoplasmic reticulum (ER), and after recovery from ischemia by activation of transient receptor potential (TRP) channels which perpetuate intracellular calcium overload despite the restoration of ion gradients (Ca2+paradox) [71]. The changes in intracellular calcium activity are highly pathogenic: prolonged elevation of cytosolic calcium causes mito-chondrial dysfunction and induces catabolic changes, notably by activation of Ca2+-dependent effector proteins and enzymes such as endonucleases, phospholipases, protein kinases and proteases that damage DNA, lipids and proteins. The release of calcium from the ER evokes an ER stress response, which mediates a great number of ER-dependent secondary disturbances, notably inhibition of protein synthesis. Calcium-dependent pathological events are therefore complex and contribute to a multitude of secondary molecular injury pathways.

Free radicals: in brain regions with low or intermittent blood perfusion, reactive oxygen species (ROS) are formed which produce peroxida-tive injury of plasma membranes and intracellular organelles [72]. The reaction with nitric oxide leads to the formation of peroxynitrate, which also causes violent biochemical reactions. Secondary consequences of free radical reactions are the release of biologically active free fatty acids such as arachi-donic acid, the induction of endoplasmic reticulum stress, the induction of mitochondrial disturbances and fragmentation of DNA. The latter may induce apoptosis and thus enhance molecular injury pathways related to mitochondrial dysfunction. The therapeutic benefit of free radical scavengers, however, is limited, as recently documented by the therapeutic failure of the free-radical-trapping agent NXY-059 [73].

Nitric oxide toxicity: nitric oxide (NO) is a product of NO synthase (NOS) acting on argenin. There are at least three isoforms of NOS: eNOS is constitutively expressed in endothelial cells, nNOS in neurons and the inducible isoform iNOS mainly in macrophages. Pathophysiologically, NO has two opposing effects [74]. In endothelial cells the generation of NO leads to vascular dilatation, an improvement of blood flow and the alleviation of hypoxic injury, whereas in neurons it contributes to glutamate excitotoxicity and - by formation of peroxynitrate - to free-radical-induced injury. The net effect of NO thus depends on the individual pathophysiological situation and is difficult to predict.

Zinc toxicity: zinc is an essential catalytic and structural element of numerous proteins and a secondary messenger which is released from excitatory synapses during neuronal activation. Cytosolic zinc

overload may promote mitochondrial dysfunction and generation of reactive oxygen species (ROS), activate signal transduction pathways such as protein kinase C or enhance glutamate toxicity by inhibiting GABAA channels and blocking excitatory amino acid transporters. However, zinc may also exhibit neuro-protective properties, indicating that cells may possess a specific zinc set-point by which too little or too much zinc can promote ischemic injury [75].

Inhibition of protein synthesis: a robust molecular marker for the progression of ischemic injury is inhibition of protein synthesis, which persists throughout the interval from the onset of ischemia until the manifestation of cell death [37]. It is initiated by the ischemia-induced release of calcium stores from the endoplasmic reticulum (ER), which results in ER stress and various cell biological abnormalities such as misfolding of proteins, expression of stress proteins and a global inhibition of the protein-synthesizing machinery [76]. The latter is due to the activation of protein kinase R (PKR), which causes phosphorylation and inactivation of the alpha subunit of eukaryotic initiation factor eIF2. This again leads to selective inhibition of polypeptidepol chain initiation, disaggregation of ribosomes and inhibition of protein synthesis at the level of translation.

Other consequences of ER stress are ubiquination and trapping of proteins which are crucial for cellular function, and SUMOylation (i.e. conjugation with the small ubiquitin-like modifier, SUMO), which causes suppression of most transcription factors. The former is presumably the reason for the irreversibility of translation arrest because protein aggregates include components of the translation complex [77]. Obviously, persistent inhibition of protein synthesis is incompatible with cell survival but, as the interval between onset of ischemia and cell death greatly varies, other factors are also involved.

Mitochondrial disturbances: the concurrence of an increased cytosolic calcium activity with the generation of reactive oxygen species leads to the increase in permeability of the inner mitochondrial membrane (mitochondrial permeability transition, MPT), which has been associated with the formation of a permeability transition pore (PTP). The PTP is thought to consist of a voltage-dependent anion channel (VADC), the adenine nucleotide translocator (ANT), cyclophilin D and other molecules. The increase in permeability of the inner mitochondrial membrane has two pathophysiologically important consequences. The breakdown of the electrochemical gradient interferes with mitochondrial respiration and, in consequence, with aerobic energy production. Furthermore, the equilibration of mitochondrial ion gradients causes swelling of the mitochondrial matrix, which eventually will cause disruption of the outer mitochondrial membrane and the release of pro-apoptotic mitochondrial proteins (see below). Ischemia-induced mitochondrial disturbances thus contribute to delayed cell death both by impairment of the energy state and by the activation of apoptotic injury pathways [78].

A large number of biochemical substrates, molecules and mechanisms are involved in the progression of ischemic damage.

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