Mechanism of Ischemic brain injury

Brain requires a continuous and steady flow of glucose and oxygen to undergo oxidative phosphorylation for energy production because brain has no store of energy and deprivation occur in minutes only (Durukan & Tatlisumak., 2007). Normal cerebral blood flow (CBF) is typically in the range of 45-50 ml/min/100g. (Dearden, 1985). When CBF falls to < 7 ml/min/100g irreversible cellular damage occur. The ischemic core is surrounded by region of moderate ischemic zone called ischemic penumbra (IP) with a CBF ranging from 7 to 17 ml/min/100g (Baron, 1999), which remains metabolically active but electrically silent (Astrup et al., 1981).
Decline in cerebral blood flow causes a number of important cellular changes. These include impaired energy metabolism, a measurable decrease in ATP production, increase in intracellular calcium level, loss of ion homeostasis, excitotoxicity, activation of enzyme such as nitric oxide synthatase, cyclooxygenase, phospholipase A2, protein kinase C, massive production of ROS, BBB disruption, edema and apoptosis (Chan, 2001).

1. Energy failure
The first consequence of CBF reduction is the depletion of substrate, particularly oxygen and glucose that causes loss of aerobic glycolysis and anaerobic glycolysis supervenes.  Anaerobic glycolysis produces much less ATP per molecule of glucose and yields an excess amount of lactic acid. Excess amount of lactic acid induce temporary opening of Ca2+ conduction channel resulting large and sustain increase in neuronal Ca2+. (Nedergaard, 1995). Less ATP production leads to perturbation of the Na+/K+-ATPase, and Ca2+/H+-ATPase pumps and reversal of Na+-Ca2+ transporter (Phan et al., 2002) resulting elevation of intracellular Na+, Ca2+, Cl- and extracellular K+ levels. Increase intracellular Na+ causes rapid influx of water from extravascular compartment into cell produce edema called cytotoxic edema.

2. Glutamate mediated excitotoxicity

•    Calcium overload
Calcium present extracellularly at a concentration 10,000 times greater than the intracellularly. With the energy depletion, membrane potential is lost and consequently neurons and glia depolarize (Dirnagl et al., 1999). After depolarization, large amount of glutamate are released into the extracellular compartment from presynaptic neurons. Glutamate activates NMDA, AMPA, and Metabotropic glutamate receptors. The channel within the NMDA receptor is blocked in a voltage-dependent manner by magnesium ion (Mg2+), which is dislodged by depolarization induced by activation of AMPA receptor (Nowak et al., 1984). NMDA receptor is the predominant route of Ca2+ flux during glutamatergic transmission. AMPA and kainite receptors are generally involved in Na+ conductance. Besides ionotropic receptor, glutamate also stimulates a family of metabotropic receptors that activate second messenger pathway resulting in the release of Ca2+.
The elevation in Ca2+ initiate a series of pathobiochemical processes such as activation of proteases, lipases, phospholipases, protein kinases, cyclooxygenase, synthase, calpain, and endonucleases, xanthine oxidase, calcium dependent nitric oxide synthase, leading to the breakdown of the cell membrane, cytoskeleton and the genomic DNA that lead to neuronal degeneration (Dirnagl et al., 1999).
As a consequence of phospholipase activation, the production of free fatty acids (FFA's) including the potent prostaglandin inducer, arachidonic acid (AA). The degradation of the membrane by phospholipases almost certainly damages membrane integrity, further reducing the efficiency of calcium pumping and leading to further calcium overload and a failure to regulate intracellular calcium levels (Farber et al., 1981).  The production of AA as a result of FFA release causes the production of thromboxane and leukotrienes. Both these compounds are profound tissue irritants, which can cause platelet aggregation, clotting, vasospasm, and edema ( McIlvoy, 2005).
An influx of Ca2+ through NMDA receptors may lead to the activation of nNOS and the release of NO. Release of NO further leads to the formation of superoxide (O2 -), peroxynitrite (ONOO-) and hydroxyl (OH•) radicals (Beckman et al., 1990). ONOO- causes mitochondrial dysfunction during severe hypoxia–ischemia results in increased generation of oxygen free radicals leading to prompt dysfunction of cellular membrane causing necrosis.

•    Spreading depression
Cellular injury within the penumbral region occur from recurrent waves of depolarization called spreading depression wave originate at the interface between ischemic core and penumbra. Spreading depression consumes energy and increases infarct volume. (Hartings et al., 2003). The most likely source for these spreading depression wave is the elevated extracellular K+ level and increase in the glutamate release at the boundaries between the ischemic core and the penumbra (Dijkhuizen et al., 1999).

3. Oxidative stress
During ischemia, the hydrolysis of ATP to AMP leads to an accumulation of hypoxanthine (Tien & Aust., 1982). Increased intracellular calcium enhances the conversion of xanthine dehydrogenase to xanthine oxidase . After reperfusion and reintroduction of oxygen, Xanthine oxidase may produce xanthine and superoxide from Hypoxanthine and oxygen (Lo et al., 2003;). Oxygen radicals are also produced during enzymatic conversions, such as the cyclooxygenase-dependent conversion of arachidonic acid to prostanoids (Lo et al., 2003).  Under ischemic conditions, formation of mitochondrial permeability transition pore occurs and leads to a burst of free oxygen radicals and the release of proapoptotic molecules (Mergenthaler et al., 2004).
Reactive oxygen species react irreversibly with several cellular constituents such as proteins, double bonds of phospholipids, and nuclear DNA and cause lipid peroxidation, membrane damage, dysregulation of cellular processes, and mutations of the genome. Cell damage causes aberrations in ion homeostasis, cell signaling, and gene expression. Oxygen radicals serve as important signaling molecules that trigger inflammation and apoptosis (Dirnagl et al., 1999).

4. BBB disruption
BBB disruption occurs due to mechanical or hypoxic damage of vascular endothelium, toxic damage of inflammatory molecules and free radicals and mainly due to destruction of the basal lamina by matrix metalloproteinases which leads to vasogenic edema, influx of toxic substances, inflammation and presumably hemorrhagic complications. (Gartshore et al., 1997)

5. Inflammatory reactions
Inflammatory events, which are initiated at the blood micro vessel interface few hours after the onset of ischemia, underlie the transition from ischemic to inflammatory injury. Major players in the inflammatory injury are cytokines (IL-1, IL-6, TNF-α, and TGF-β), adhesion molecules (selectins, integrins, and immunoglobulins), eicasanoids and inducible neuronal nitric oxide synthase (iNOS), which are produced immediately after the onset of ischemia and contribute to irreversible damage. These molecules are produced by endothelial cells, astrocytes, microglial cells and leukocytes (granulocytes, monocyte/macrophages, lymphocytes).
Cell adhesion molecules including selectins, integrins and proteins of immunoglobulin gene family mediate cell to cell interaction for leukocyte migration. Selectins get upregulated in endothelial cells and leukocytes after focal cerebral ischemia and reperfusion (Huang et al., 2000). This polymorphonuclear (PMN) leukocyte get accumulated in the microvessels of penumbral region, leads to additional disruption of microcirculation and subsequent brain injury. Adhesion molecules of immunoglobulin gene family (intracellular adhesion molecule-1 and -2 (ICAM-1 and -2) and vascular cell adhesion molecule-1 (VCAM-1)) promote leukocyte adhesion to endothelial cells. (Soriano et al., 1996).
Similarly, cytokines such as IL-1, IL-6 and TNF-α are also important mediators of the inflammatory reactions in cerebral ischemia (del Zoppo et al., 2000). IL-1 induce release of arachidonic acid, enhancement of NMDA excitotoxicity and stimulation of nitric oxide synthase (Huang et al., 2006) and TNF-α induce the expression of adhesion molecules, which promotes leukocyte adherence and accumulation.
In addition, iNOS and cyclooxygenase-2 (COX-2) have been also suggested to play important role in inflammation through production of oxygen free radicals and prostanoids (Yokota et al., 2004).

6. Necrosis
Most brain lesions that develop after cerebral ischemia due to reduction of CBF evolve from an initial stage of reversible to an infarct or an area where most neurons become necrotic and then spread towards periphery of occluded artery (Garcia et al., 1995). Necrosis occurs due to loss of osmotic homeostasis in response to physiological trauma such as acute anoxia, a sudden shortage of nutrients, or extreme physical or chemical injury.
The energy failure or drastic decrease in cellular ATP and glucose level occur due to rapid decrease in glucose uptake after ischemia. The decreased ATP levels affect the activity of Na+–K+ ATPase, which consumes up to 70% of cellular ATP. The rise in cellular Na+ due to a decreased activity of Na+–K+ ATPase, causes Ca2+ influx through a Na+–Ca2+ exchanger and Ca2+–Mg2+ ATPase. The increase in intracellular Na+,K+ and Ca2+  causes a rapid influx of water from the extracellular compartment to cell. The cell swells as they take up water (Rathmell & Thompson, 1999) termed cytotoxic edema. Cytotoxic edema begins approximately 30 min to 1 hr after the ischemic insult. If reperfusion of the ischemic area then takes place, rupture of the plasma membrane cause leakage of macromolecules and water into the extravascular space and consequent inflammatory response, termed vasogenic edema (Bradley, 1987). Vasogenic edema begins to develop after 6 hr. The severity and duration of the ischemia determines whether cells may undergo a rapid “necrotic” death due to lysis, or a slower and controlled deterioration, culminating in “apoptotic” death.

7. Apoptosis
Two pathway of apoptosis
1.    Mitochondrial pathway of apoptosis 
2.    Receptor-mediated pathway of apoptosis

1. Mitochondrial pathway of apoptosis 
 
Cerebral ischemia and reperfusion generate ROS within mitochondria, which then signal the release of cytochrome c from mitochondria to the cytosolic compartment. cytochrome c is a water-soluble peripheral membrane protein of mitochondria and an essential component of the mitochondrial respiratory chain ( Perez-Pinzon et al., 1999)
Cytochrome c interacts with the Apaf-1 and deoxyadenosine triphosphate forming the apoptosome, which leads to activation of caspase-9. Caspase-9, an initiator of the cytochrome-c-dependent caspase cascade, then activates caspase-3 followed by other downstream caspase-2, -6, -8, and –10 (Slee et al., 1999) Caspase-3 activates DNase (CAD) and The downstream caspases cleave many substrate proteins including poly (ADP-ribose) polymerase (PARP) (Chan et al., 1998). Substrate cleavage causes DNA damage and subsequently apoptotic cell death.
The second mitochondria-derived activator of caspase (Smac), is also released by apoptotic stimuli and binds IAPs, thereby promoting activation of caspase-3( Chai et al.,2002). The inhibitor-of-apoptosis protein (IAP) family suppresses apoptosis by preventing the activation of procaspases and also by inhibiting the enzymatic activity of active caspases.
The Bcl-2 family proteins play a crucial role in intracellular apoptotic signal transduction by regulating permeability of the mitochondrial permeability transition pore (MPTP), from where cytochrome c is released. (Shi et al., 2001) Among these proteins, Bax, Bcl-XS, Bak, Bid, and Bad are proapoptotic. They eliminate the mitochondrial membrane potential by affecting the PTP and facilitating the release of cytochrome c.       

2. Receptor-mediated pathway of apoptosis
                                    
The death receptor pathway of apoptosis is initiated by members of the death receptor family, such as the Fas receptor and the tumor necrosis factor (TNF) receptor. In the Fas receptor pathway, the extracellular Fas ligand (FasL) first binds to a receptor and an adaptor molecule, Fas-associated death domain (FADD) protein, then activates procaspase-8 (Hengartner et al., 2000). Subsequently, caspase-8 activates caspase-3, and effector caspase cleaves PARP and activates CAD, leading to DNA damage and cell death. Increased expression of Fas and FasL was observed in the ischemic region after focal cerebral ischemia.
In the middle of this pathway, Caspase-8 is also able to activate one of the Bcl-2 family proteins Bid, and to initiate the mitochondrial pathway of apoptosis. (Li et al., 1998)

8. Necroptosis
Neuronal cell death caused by ischemic injury is known to contain a substantial non-apoptotic component. Such pathway has been initiated by Death receptor (DRs) due to the conditions that are not optimal for apoptosis but suitable for death pathway, which they subsequently described as “necroptosis” (Degterev et al., 2005)
It is established that DR mediated extrinsic pathway of apoptosis proceeds through activation of caspase-8. However, necroptosis although initiated by DR, follow an intermediate route through RIP that serves as a bifurcation point separating necroptosis from other DR-dependent pathway of apoptosis. RIP is a serine/threonine kinase and identified as a Fas-interacting protein. It contains three domains, including a N-terminal kinase domain, an intermediate domain and a C-terminal death domain through which it binds to TRADD (TNF receptor associated death domain), a TNF receptor I associated cytoplasmic adapter protein (TNFRI) and induces apoptosis (Stanger et al., 1995).

9. Free radical generation
During ischemia, the hydrolysis of ATP via AMP leads to an accumulation of hypoxanthine. Increased intracellular calcium enhances the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). Upon reperfusion and reintroduction of oxygen, XO may produce superoxide and xanthine from hypoxanthine and oxygen  Even more damaging free radicals could conceivably be produced by the metal catalyzed Haber-Weiss reaction as follows

O2- + H2O ----Fe3 ------> O2 + OH-+ OH-

Iron, the transition metal needed to drive this reaction, is present in abundant quantities in bound form in living systems in the form of cytochromes, transferrin, hemoglobin and others. Anaerobic conditions have long been known to release such normally bound iron. Indirect experimental confirmation of the role of free iron in generating free-radical injury has come from a number of studies which have confirmed the presence of free-radical breakdown products such as conjugated dienes [Bromont et al; 1989] and low molecular weight species of iron [Babes CF et al; 1985].

During reperfusion and re-oxygenation, significantly increased levels of several free-radical species that degrade cell and capillary membranes have been postulated:

 O2-, OH-, and free lipid radicals (FLRs). O2- may be formed by the previously described actions of XO and/or by release from neutrophils which have been activated by leukotrienes.

Re-oxygenation also restores ATP levels, and this may in turn allow active uptake of calcium by the mitochondria, resulting in massive calcium overload and destruction of the mitochondria [Safer p et al; 1985]All cell types normally produce free radicals and other oxidants but their production and elimination rates are equal and therefore they are not noxious under normal, physiological circumstances. However, several types of environmental stresses and neuronal insults lead to the formation of reactive oxygen and nitrogen species. These reactive species include nitric oxide (NO), superoxide radical anion (O2−), hydrogen peroxide (H2O2), hydroxyl free radical (OH) and Peroxynitrite (ONOO−) (Beckman et al., 1990). During cerebral ischemia, free radicals and other oxidant species causes damage to lipids, DNA and proteins leading to neuronal death. They also responsible for brain edema and breakdown of the blood-brain barrier.

•    Superoxide radical anion (O2−)

When supply of oxygen and glucose in the brain is interrupted, a cascade of events takes place, leads to depletion of energy source in the form of ATP. Less ATP production leads to perturbation of the Na+/K+-ATPase, and Ca2+/H+-ATPase pumps andreversal of Na+-Ca2+ transporter (Phan et al., 2002) resulting disrupts ionic gradients across the membranes. This causes an increase in extracellular K+ as well as intracellular Na+, Cl−, and Ca2+ into the cells.

During cerebral ischemia, increase intracellular Ca2+ activates phospholipase A2 (PLA2). PLA2 liberates unsaturated fatty acid arachidonic acid and free radicals via the cyclooxygenase and lipoxygenase pathways. Cyclooxygenase catalyzes the addition of two O2 molecules to arachidonic acid to produce the prostaglandin PGG2, that is rapidly peroxidized to PGH2 with a simultaneous release of superoxide radical anion (Lipton ,1999). Expression of inducible isoform of cyclooxygenase, (COX-2) leads to the eicosanoid accumulation and superoxide radical anion production ( Nakayama et al., 1998).

Other pro-oxidant enzymes that generate superoxide radical anion are xanthine oxidase and NADPH oxidase. During ischemia, the hydrolysis of ATP to AMP leads to an accumulation of hypoxanthine. Increased intracellular calcium enhances the conversion of xanthine dehydrogenase to xanthine oxidase. After reperfusion and reintroduction of oxygen, Xanthine oxidase may produce xanthine and superoxide from Hypoxanthine and oxygen (Lo et al., 2003) On the other hand, the oxidation of NADPH by NADPH oxidase in infiltrated neutrophils in the brain parenchyma after ischemia constitutes an additional source of superoxide radical anion (Lipton ,1999).

The most important source of superoxide radical anion in vivo in normal, healthy aerobic cells is the mitochondrial electron-transport chain. The mitochondrial respiratory chain consists of a series of electron carriers that are arranged spatially according to their redox potentials and organized into four complexes (Darley-Usmar et al., 1994). Complex I (NADH coenxyme Q reductase) and complex II (succinate dehydrogenase) accepts electrons from the Krebs cycle electron carrier NADH and FADH2 and passes them to coenzyme Q (ubiquinone).UQ passes electrons to complex III (cytochrome bc1 complex), which passes them to cytochrome c (cyt c). Cyt c passes electrons to Complex IV (cytochrome c oxidase), which uses the electrons and hydrogen ions to reduce molecular oxygen to water.In the course of the redox reactions, a proton electrochemical potential gradient has been established across the membrane. This proton electrochemical potential gradient used by complex V (ATP synthase) for the synthesis of ATP. Some of the electrons (approximately 2 to 5%) passing through these carriers leak to O2 producing an univalent reduction to give O2- (Chance et al., 1979). This formation of O2− and H2O2 takes place mainly at complex I (NADPH dehydrogenase) and at complex III (ubiquinone-cytochrome b-C1) (Boveris & Chance., 1973),

Reactive oxygen species are also produced as a result of opening the MPT pore. Excessive mitochondrial Ca2+ accumulation after the elevation of intracellular Ca2+ caused by glutamate receptor overactivation open the MPT pore ( Brustovetsky et al., 2002) because Ca2+ binds and activates Ca2+ binding sites on the matrix side of the MPTP.  MPT can allow antioxidant molecules to exit mitochondria, reducing the organelles ability to neutralize ROS. MPT also causes the mitochondria to become permeable to molecules smaller than 1.5 kDa and draw water in by increasing the organelle’s osmolar  load. This may lead mitochondria to swell and may cause the outer membrane to rupture, releasing cytochrome c. Due to loss of cytochrome c, a important component of electrone transport chain can lead to escape of electrone from chain, which can then reduce the molecules and form free radical (Sato et al, 1993).

•    Nitric oxide (NO)
Nitric oxide is a physiological messenger in the central nervous system that is formed from L-arginine and O2 in a reaction catalyzed by the NO synthase (Knowles & Moncada, 1994). The three isoforms of nitric oxide synthase (NOS) have been characterized in brain cells (Knowles & Moncada, 1994).

1.    nNOS or NOS I  -present in neuron under normal physiological condition
2.    eNOS or NOS II  -present in endothelium under normal physiological condition
3.    iNOS or NOS III  - expressed in macrophages, kupffer cells, neutrophils, fibroblast, vascular smooth muscle, endotheliumin in response to pathological stimuli.

The elevation in intracellular Ca2+ after the ischemic insult activates nNOS and produces  NO. In parallel, an up-regulation of nNOS mRNA and an increase of nNOS immunoreactivity in neurons have been found (Zhang et al., 1994). 10 min after ischemia, nNOS activity increases and returns to normal after 60 min .After ischemia develops, nNOS activity down-regulated and nNOS-stained neurons are lost. This down-regulation may be due to loss of cytosolic proteins due to plasma membrane disruption and inhibition of nNOS activity by  NO itself. (Colasanti et al., 1995). eNOS activity and expression is also up-regulated (Nagafuji et al.,1994).
NO is also synthesized by a Ca2+-independent NOS isoform (iNOS) that is expressed by inflammatory factors such as cytokines and/or lipopolysaccharide (LPS) (Knowles & Moncada.. 1994). Cerebral ischemia is associated with a marked inflammatory reaction that results in the expression and release of several cytokines that activate the expression of iNOS in different cell such as polymorphonuclear neutrophils, astrocytes, vascular cells, in the injured brain (Iadecola et al., 1996) The expression is maximal at 12–48 h after the onset of ischemia
Activation of NF-κB has been required for the expression of iNOS gene (Xie et al., 1994). During ischemia, NF-κB activation occurs due to several stimuli such as cytokines, interferon-γ and especially due to calcium-dependent activation of NF-κB  via activation of NMDA receptors by glutamate. (Cardenas et al., 2000).  Another possibility is that glutamate leads to the expression of iNOS, by directly activating the transcription factor NF-κB (Guerrini  et al.,1995;).

•    Peroxynitrite (ONOO-)
An important source of oxidative stress-mediated brain damage is formation of peroxynitrite, a powerful oxidant that results from the reaction between  NO and O2 − and which is known to promote neuronal damage following ischemia (Bolannos & Almeida, 1999)

•    Other oxidant species
Several species derived from superoxide radical anion mediate most of its toxicity. First, is its reaction with  NO to generate peroxynitrite (Radi et al., 2002). In addition, other reaction products of superoxide radical anion, mainly the hydroxyl radical ( OH), are also very reactive and more likely to mediate toxic effects (Halliwell & Gutteridge, 1984). Formation of  OH derives from the Fenton reaction, by which H2O2 formed from O2 − dismutation would react with Fe2+ to yield Fe3+, OH−, and  OH. This reaction would be favored by free iron and decreased pH that prevails during and after ischemia. However, this reaction is very slow and therefore superoxide radical anion would be largely metabolized by superoxide dismutase and degraded by  NO to form peroxynitrite (Beckman, 1994).


•    Free radical and lipid peroxidation

Description of reaction are given below:

Reaction 1: The superoxide anion radical is formed by the process of reduction of molecular oxygen mediated by NAD(P)H oxidases and xanthine oxidase or non-enzymatically by redox-reactive compounds such as the semi-ubiquinone compound of the mitochondrial electron transport chain.
Reaction 2: Superoxide radical is dismutated by the superoxide dismutase (SOD) to hydrogen peroxide.
Reaction 3: Hydrogen peroxide is most efficiently scavenged by the enzyme glutathione peroxidase (GPx) into the water and molecular oxygen using glutathione as electrone donor.
Reaction 4: Some transition metals (e.g. Fe2+, Cu+ and others) can breakdown hydrogen peroxide to the reactive hydroxyl radical (Fenton reaction).
Reaction 5: The hydroxyl radical is react with components of the DNA molecule, damaging both the purine and pyrimidine bases. (Halliwell & Gutteridge, 1999).
Reaction 6: The hydroxyl radical can abstract an electron from polyunsaturated fatty acid (LH) to give rise to a carbon-centred lipid radical (L ).
Reaction 7: The lipid radical (L ) can further interact with molecular oxygen to give a lipid peroxyl radical (LOO ).
Reaction 8: Lipid hydroperoxides can react fast with Fe2+ to form lipid alkoxyl radicals (LO ), or much slower with Fe3+ to form lipid peroxyl radicals (LOO ).
 Reaction 9: Lipid alkoxyl radical (LO ) undergoes cyclisation reaction to form a six-membered ring hydroperoxide.
Reaction 10: Six-membered ring hydroperoxide udergoes further reactions (involving β-scission) to from 4-hydroxy-nonenal.
Reaction 11: A peroxyl radical react by cyclisation to produce a cyclic peroxide.
Reaction 12: This radical can then either be reduced to form a hydroperoxide (reaction not shown) or it can undergo a second cyclisation to form a bicyclic peroxide which after coupling to dioxygen and reduction yields a molecule structurally analogous to the endoperoxide.
The side chains of all amino acid residues of proteins, in particular cysteine and methionine residues of proteins are susceptible to oxidation by the action of ROS/RNS . Oxidation of cysteine residues may lead to the reversible formation of mixed disulphides between protein thiol groups (–SH) and low molecular weight thiols. (Dalle-Donne et al., 2005).
Advanced glycation end products (AGEs) is a class of complex products. They are the results of a reaction between carbohydrates and free amino group of proteins. The intermediate products are known as Amadori, Schiff Base and Maillard products, named after the researchers who first described them (Dalle-Donne et al., 2005). Most of the AGEs are very unstable, reactive compounds and the end products are difficult to be completely analysed.