Role of oxidative Stress in Cerebral Ischemia-reperfusion Injury

Transcription

1 Role of oxidative Stress in Cerebral Ischemia-reperfusion Injury Evidences obtained from the past two decades showed that there is an involvement of reactive oxygen species in cerebral ischemia (Christophe and Nicolas 2006). Thrombolytic reperfusion or reoxygenation during spontaneous perfusion provides oxygen as a substrate for various enzymatic reactions in the subcellular organelles or cytosolic compartments and mitochondria that will further increase oxidative stress and add to the cellular damage (Kabakov et al. 2003). 2.1 Potential mechanisms for radical production Mitochondrial dysfunction The primary function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. ROS generated from mitochondria mediate signaling through p53 (Huang et al. 2008). Through mitochondrial intermembrane space, molecular signaling returns to mitochondria and triggers the release of critical apoptotic activators and effectors of cell death, such as apoptosis inducing factor or cytochrome C in mitochondria-dependent apoptosis (Kroemer et al. 2007). Mitocondria releases the ROS, which is implicated in acute brain injuries such as stroke and neurodegeneration. p53 interface to apoptosis and cell-cycle progression. This indicates that p53 is the master regulator of cell death by inducing apoptosis. Apoptosis related gene expression was controlled with specific transcription factors of p53 gene encodes NOXA (NADPH oxidase activator 1), BAX (bcl 2 associated X protein), PUMA (p53-upregulated modulator of apoptosis) and p53aip1 (p53 acetate-induced protein 1) are the p53 products (Niizuma et al. 2009). They directly act on mitochondria and induce apoptosis, p53-induced 20

2 protein with a death domain (PIDD) is also a product of p53 and activates caspase-2, resulting in the activation of mitochondria-dependent apoptosis (Niizuma et al. 2009). Recent studies demonstrate that p53 is involved in neuronal death that occurs with stroke and neurodegeneration (Morrison et al. 2000; Niizuma et al. 2009; Culmsee et al. 2003; Jung et al. 2011). Over-expression of copper/zincsuperoxide dismutase (SOD1) down-regulate PUMA, suggesting a functional relationship between oxidative stress and the p53 signaling pathway. The role of mitochondrial dysfunction and oxidative stress are determinants of neuronal death after stroke and neurodegeneration, focusing on BAX, PUMA, PIDD, transcription-independent p53 translocation and SOD1 over-expression (Niizuma et al. 2009) Free fatty acid metabolites Intra cellular influx of calcium and depletion of ATP are the postulated consequences during cerebral ischemia in its initiating pathways involving in the breakdown of accumulated free fatty acids and break down of lipid membrane constituents. Calcium influx activates phospholipase-c in ATP- poor environment. Alternately, in ischemia, the concentration of camp increases through the release of potassium, adenosine and catecholamines which turns to activation of phospholipase A. The free fatty acid levels are more in ischemia due to breakdown of lipid membranes by the catalytic actions of these enzymes. In ischemia, rapid accumulation of free fatty acids occur particularly, arachidonic acid. It is the major source of superoxide free radical. Arachidonic acid causes brain injury through several mechanisms. 21

3 Na + -K + -adenosine triphosphatase inhibition elicit arachidonic acid efflux causing cerebral edema. Arachidonic acid may cause release of neurotransmitters and inhibit reuptake mechanisms Arachidonic acid readily intercalates into lipid membranes and produces changes in packing of lipid molecules Reperfusion initiates the metabolism of accumulated arachidonic acid via lipoxygenase and cyclooxygenase pathways (Rao et al. 1999, 2000). They have to produce prostaglandins, thromboxanes and superoxides. The prostaglandins may be responsible for vascular slugging and alter vascular reactivity. Brain injury may be caused directly through oxygen radicals and indirectly through vascular paralysis (Rao et al. 1999, 2000) Purine metabolites Cerebral ischemia-reperfusion injury leads to degradation of ATP levels through generation of toxic oxygen metabolites. With the onset of ischemia, brain s adenine nucleotides are metabolized to nucleosides and purine bases. Therefore ischemia is allocated to a rapid rise in interstitial concentration of adenosine and hypoxanthine. Furthermore, the adenosine metabolized to inosine, hypoxanthine and eventually as a substrate for xanthine oxidase pathway. In reperfusion, xanthine oxidase pathway results in production of oxygen radicals. High levels of oxygen radicals lead to increase of xanthine oxidase activity (Nijveldt et al. 2001). Under normal conditions, the brain tissue has low levels of xanthine oxidase. However, the conversion of xanthine dehydrogenase to xanthine oxidase by calcium-activated proteases occurs in ischemia-reperfusion condition 22

4 (Nijveldt et al. 2001). Thus the xanthine oxidase pathway plays an important role for the production of superoxide in endothelial cells during reperfusion Uncoupling of NOS Nitric oxide synthases (NOSs), particularly endothelial NOS (enos) is a potential source of superoxides under pathological conditions of cerebral ischemia-reperfusion. Under normal circumstances, these enzymes transfer electrons from a heme group in the oxygenase domain to the substrate L-arginine to form L-citrulline. 5,6,7,8-tetrahydrobiopterin (BH4) serves as a cofactor in this process. If the availability of either BH4 or L arginine decreases, enos switches from a coupled state, in which it generates NO to an uncoupled state in which the electrons from the heme reduce oxygen to form superoxide (Chen et al. 2011). The superoxide anion reacts with nitric oxide to produce peroxynitrite, which impairs endothelium-dependent vascular relaxation by reducing NO and may also oxidise BH4. This causes a deficiency of BH4 and will further promote uncoupling of NOS. Furthermore, ischemia induces inos, an enzyme responsible for producing NOS in nanomolar concentration (as compared to the physiological picomolar ones). These high concentrations of NOS are cytostatic and cytotoxic for fungi, bacteria, protozoa, or tumoral cells and are also cytotoxic for the neurons because they inhibit key mitochondrial enzymes like NADH-ubiquinone oxydoreductase, NADH-succinate oxydoreductase and aconitase (Cuzzocrea et al. 2001) Polymorphonuclear leukocytes There is some evidence that polymorphonuclear leucocytes contribute to cerebral ischemia-reperfusion injury. It has been proposed that ischemia leads to activation of cerebral vasculatures trapping leucocytes, which may result in the 23

5 release of chemotaxic factors (leukotrienes) during reperfusion. Activated leukocytes may impair microvascular circulation and mechanical obstruction of capillaries. Leukocytes interact with platelets, involved in the metabolism of the arachidonic acid and produce cyclooxygenase and lipoxygenase byproducts, including oxygen radicals (Htun et al. 2006) The role of calcium in the production of free radicals Calcium is an important mediator of ischemic brain damage whether due to global or focal ischemia by generating free radicals. The influx of free cytosolic calcium leads to activation of lipases, proteases, kinases, phosphatases and endonucleases. They develop harmful metabolic cascade events in ischemia, such that, activation of protein kinase C have synergistic effects on superoxide release by endothelial cells. As described above, increased intracellular calcium in ischemia activates phospholipases and liberates free fatty acids (arachidonic acid). In reperfusion, arachidonic acid is metabolized by the cyclooxygenase and lipoxygenase with the production of oxygen radicals and vasoconstrictive prostanoids. Calcium influx into the vascular smooth muscle may also have an additive effect along with vasoconstricting prostanoids and contribute to post ischemic hypoperfusion. Calcium also plays an important role in the activation of the protease calpain (Atsma et al. 1995), which cleaves a peptide bond in xanthine dehydrogenase to form xanthine oxidase, thereby contributing to the increased formation of free radicals (Schurr et al. 1992) Potential sites of radical-induced injury Vascular endothelium and parenchyma have potential pathways for radical production (Traystman et al. 1991). It is not clear whether these sites are the main sources of radical production during ischemia-reperfusion. Experimentally, 24

6 cultured endothelial cells release free radicals mainly by the xanthine oxidase pathway and by the metabolism of prostanoids. While examination of brain homogenates and mitochondrial preparations showed the parenchymal radical production. However, the endogenous anti oxidative defenses are troubled through over production of free radicals by pro-oxidant enzymes and mitochondria. In ischemic brain tissue, inactivation of detoxification system leads to consumption of anti oxidants and failure to refill anti oxidants. In vitro studies characterized the DNA damage by ROS, suggesting that different ROS affect DNA in different ways: hydrogen peroxide does not react with DNA bases at all the hydroxyl radical generates a multiplicity of products from all four DNA bases, while superoxide selectively attacks guanine (Traystman et al. 1991). 25

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