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Albeit the underlying mechanism is consistent, the severity of ischemia, tempo- ral and spatial heterogeneity may influence the specific condition of neuronal cell death. In the early stages of cerebral infarction, caspase-8 and caspase-1 are involved in the early apoptosis, contributing to the core. However, caspase-9 is related to the secondary expansion of the lesion in the penumbral area [18].
B-Cell Leukemia/Lymphoma 2 (Bcl-2) Family
The B-cell leukemia/lymphoma 2 (Bcl-2) family has the role of maintaining the integrity of the mitochondrial membrane. It has three subfamilies according to the molecular structure [19–21]. The first subtype is antiapoptotic protein, including Bcl-2, Bcl-xl (B-cell lymphoma-extra large) and Bcl-w. Proapoptotic protein is the second subtype, for instance, Bax (Bcl-2-associated X protein) and Bak (Bcl-2 homologous antagonist killer). The last is Bcl-2 homology domains 3 (BH3) domain protein including Bad (Bcl-2-associated death promoter), Bid (BH3 interacting- domain death agonist), Bim (Bcl-2-interacting mediator of cell death), Noxa and p53 [22]. Cerebral ischemia and reperfusion lead to intracellular stress originating from the mitochondria, the endoplasmic reticulum and the nucleus. The proteins from Bcl-2 family are sensitive to these stress factors after cerebrovascular events.
Tumor Necrosis Factor Receptor (TNFR) Superfamily
The Tumor necrosis factor receptor (TNFR) superfamily includes Fas and TNFR1 [23]. Fas is also called CD95 or Apo1. The Fas ligand (FasL) is a homotrimer, con- stituting microaggregate on the surface of cells. Caspase-8 is activated by death- inducing signaling complex (DISC), of which Fas is an important part.
Other Molecules
Besides, there are still other potential molecules that participate in the post-stroke apoptosis. Nuclear factor-Y transcription factor (NF-YC) [24], Secretory phospholipase A2 (sPLA2) [25], Bim [26], Numb [27] have been suggested to be correlated with apoptosis of neurons by experiments.
2.2 Pathways Related to Apoptosis of CIRI
2.2.1 Intrinsic Pathway
The stimulation by glutamate of N-methyl-d-aspartate (NMDA), amino-3-hydroxy- 5-methyl-isoxazolpropionic acid (AMPA) receptors, or acid-sensing ion channels (ASICs) causes high-level intracellular calcium after cerebral ischemia [28, 29].
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Then, the increased cytosolic calcium activates calpains and induces the cleavage of Bid. The truncated Bid (tBid) interacts with apoptotic proteins such as Bad and Bax at the mitochondrial membrane, which is called heterodimerization [30]. On the other hand, antiapoptotic Bcl-2 interacts with apoptotic proteins and neutralizes their effects. The above process involved Bax and Bcl-2 is the critical event in the mito- chondrial-mediated pathway [31]. Mitochondrial transition pores (MTP) are opened after the heterodimerization. Cytochrome c (Cytc) is released from the pores into the cytosol. Then an apoptosome is constituted by Cytc, procaspase-9 and apoptotic protein-activating factor-1 (Apaf-1) [31]. The apoptosome plays the role of activating caspase family. Activated caspase-3 by caspase-9 exert the ultimate effect of nDNA damage and apoptosis through cleaving nDNA repair enzymes such as poly ADP- ribose polymerase (PARP). By contrast, apoptosis-inducing factor (AIF) mediates cell death by a caspase-independent method, which is also released from the pores and translocates rapidly to the nucleus. Phosphorylation and activation of p53 can also mediates the neuronal apoptosis by damaging DNA [32, 33]. Noticeably, sec- ondary reperfusion injury carrying superoxide anions, can also cause DNA damage.
2.2.2 Extrinsic Pathway
There is considerable evidence from animal studies indicating that brain ischemia triggers the extrinsic apoptotic signaling cascade. Due to the initiating effect of death receptors on the plasma member, the extrinsic pathway is also named receptor- mediated pathway. The extracellular Fas ligand (FasL) binds to Fas death receptors (FasR), which triggers the recruitment of the Fas-associated death domain protein (FADD) [31]. FADD binds to procaspase-8 to create a death-inducing signaling complex (DISC), which activates caspase-8 [34]. Activated caspase-8 either medi- ates cleavage of Bid to truncated Bid (tBid), which integrates the different death pathways at the mitochondrial checkpoint of apoptosis, or directly activates caspase- 3. At the mitochondrial membrane tBid interacts with Bax, which is usually neutralized by antiapoptotic B-cell leukemia/lymphoma 2 (Bcl-2) family proteins Bcl-2 or Bcl-xL. Dimerization of tBid and Bax leads to the opening of mitochondrial transition pores (MTP), thereby releasing cytochrome c (Cytc), which execute cas- pase 3-dependent cell death.
2.3 Significance of Apoptosis in CIRI
The molecular mechanisms of apoptosis after stroke enlighten the exploration of neu- roprotective agents. Ischemic preconditioning in animals triggers activation of cas- pase-3 downstream and upstream of its target caspase-activated DNase (CAD) to prevent neuronal death [35]. Furthermore, enhanced formation of Apaf-1/caspase-9 complex is observed in the rat hippocampus 8–24 h after ischemia [36, 37]. Cao et al.
have cloned a rat gene product, a specific Apaf-1 inhibitor of the Apaf-1/caspase- 9
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pathway that can be neuroprotective in CIRI [12, 35]. Therefore, Apaf-1 signaling pathway may be a legitimate therapeutic target for the treatment of ischemic brain injury [38]. Fas/FasL system acts as apoptosis inducer and triggers pro- inflammatory cytokine production, while the hematopoietic growth factor, erythropoietin (EPO) inhibits apoptosis and protects from ischemic neuronal damage [39]. These findings indicate that death receptors are critically engaged in the apoptosis induction after ischemia in the adult brain and that their suppression may improve the neuronal survival after ischemic injury [12, 40]. FTY720, another antiapoptotic agent, success- fully decreased cleaved Caspase-3 expression by activation of sphingosine 1-phos- phate-1 in rats after cerebral artery occlusion [41]. In global cerebral ischemia in the gerbils, treatment with a purified medicinal herb called baicalin remarkably promoted the expression of BDNF and inhibited the expression of caspase-3 at mRNA and protein levels [42]. Additionally, it is reported that different concentrations of normo- baric oxygen can inhibit the apoptotic pathway by reducing caspase-3 and -9 expres- sion, thereby promoting neurological functional recovery after CIRI [43]. These are various neuroprotective agents on the animal models and they are potential therapeu- tic targets in future clinical pharmacological research.