Keywords

1 Introduction

When blood flow to the brain is interrupted, cells undergo a series of molecular events which include excitotoxicity, mitochondrial dysfunction, acidotoxicity, ionic imbalance, oxidative stress and inflammation. These molecular events can lead to cell death and irreversible tissue injury [1, 2]. The fate of brain cells following cerebral ischemia depends upon the severity of the insult and vulnerability of the neurons. The severity of ischemia depends on the extent of cerebral blood flow (CBF) reduction that determines the degree and deprivation of oxygen and glucose from the cells, however a particular threshold do exist for various kinds of pathophysiologic tissue events. Moreover, the high sensitivity of the brain to blood flow changes and dependence on continuous blood flow are critical factors that make the brain particularly more vulnerable to ischemia. Under physiological conditions, the normal CBF is maintained around 50–60 mL/100 g/min but during cerebral ischemia due to declining CBF, the ripples of damage spread from the center towards the periphery forming a gradient in such a way that maximum damage (infarction) is at the center (core). The CBF in this region falls to <7 mL/100 g/min and within minutes of a focal ischemic stroke occurring, the core of brain tissue exposed to the most dramatic blood flow reduction is fatally injured and subsequently undergoes necrotic cell death. The ischemic core is surrounded by region of moderate ischemic zone called ischemic penumbra (IP), with a CBF ranging from 7 to 17 mL/100 g/min [3], which remains metabolically active but electrically silent [4]. The ischemic penumbra region may comprise as much as half the total lesion volume during the initial stages of ischemia, and represents the region in which there is opportunity for salvage via poststroke therapy. Recent research has revealed that many neurons in the ischemic penumbra or peri-infarct zone may undergo delayed cell death, and thus they are potentially recoverable for some time after the onset of stroke.

This delayed cell death modal was usually called programmed cell death (PCD), which was different from the necrotic cell death that has been considered merely as an accidental uncontrolled form of cell death. PCD is defined as regulated cell death mediated by an intracellular program, which is a basic biological phenomenon that plays an important role during development, preservation of tissue homeostasis, and elimination of damaged cells. There were three major morphologies of programmed cell death in the ischemic injury, including type I, apoptosis; type II, autophagy; and type III, programmed necrosis (known as necroptosis) [5, 6].

Type I—apoptotic cell death—acts as part of a quality control and repair mechanism by elimination of unwanted, genetically damaged, or senescent cells, and as such is critically important for the development of organisms. Highly conserved in both plants and animals, it is also the cell death mechanism best characterised at both genetic and biochemical levels [7]. Type II—autophagic cell death—is a catabolic process conserved among all eukaryotes from yeast to mammals; it is a mechanism by which organelles are removed. Autophagic cell death is the primary degradation mechanism for long lived proteins, and thus maintains quality control for proteins and organelles to enhance survival under conditions of scarcity or starvation [8]. Type III—programmed necrosis [6, 9]—appears as a distinct entity, not by exclusive engagement of selected effectors, but rather, by combinatorial use of the effectors shared with other cell death outcomes.

PCD displays several cellular phenotypes affecting various intracellular organelles and membranes, and the cell nucleus. For example, the well characterised processes of cytoplasmic and chromatin condensation, nuclear fragmentation, membrane blebbing, and formation of membrane bound apoptotic bodies are part of apoptosis. Autophagy involves the formation of a double membrane vesicle which encapsulates cytoplasm and organelles, and fuses with lysosomes, thus resulting in the degradation of the vesicle contents. Programmed necrosis is characterised by the presence of swelling organelles followed by the appearance of “empty” spaces in the cytoplasm that merge and make connections with the extracellular space. The plasma membrane is fragmented, but the nucleus is relatively preserved (Fig. 5.1).

Fig. 5.1
figure 1

Morphological (electron microscope) features of autophagic, apoptotic and necrotic cells. (a) Normal, (b) autophagic, (c) apoptotic (d) and necrotic cells. Whereas the morphologic features of apoptosis are well defined, the distinction between necrotic and autophagic death is less clear. The bioenergetic catastrophe that culminates in cellular necrosis also stimulates autophagy as the cell tries to correct the decline in ATP levels by catabolizing its constituent molecules. Thus, vacuolation of the cytoplasm is observed in both autophagic cells (b) and in cells stimulated to undergo programmed necrosis (d). By contrast, ATP levels are maintained in normal (a) and apoptotic cells (c) consistent with the limited number of autophagic vacuoles in their cytoplasm. The scale bar represents 1 mm (From “Death by design: apoptosis, necrosis and autophagy” by Aimee L Edinger and Craig B Thompson [10])

Full size image

Up to now, the only available therapeutic strategy for ischemic stroke is to reopen an occluded artery by thrombolytic therapy to restore perfusion to the ischemic area during the first few hours, procedure which in itself can sometimes induce secondary damage, so this delayed cell death modalities “programmed cell death” after CIRI provide us a extended therapeutic time window, which is an important research direction in the ischemic stroke study.

2 Apoptotic Cell Death in CIRI

The morphology of apoptotic cells is characterized by vacuoles containing cytoplasm and intact organelles which are named apoptotic bodies. Before the loss of cell membrane integrity, the dying cell is gradually shrinking and absorbed by phagocytic uptake. To date, research indicates that there are two main apoptotic pathways: the intrinsic and extrinsic pathways [11, 12]. The former is also called the mitochondrial pathway because the disruption of mitochondria is pivotal in the process, which leads to the release of the cytochrome C and the downstream activation of caspases. The other pathway, referred as the extrinsic pathway, receptors can be activated by specific ligands that bind to cell surface death receptors.

There are lots of other factors influencing post-stroke apoptosis, including age and gender [13, 14]. It is said that immature brains are more sensitive to the induction of apoptosis because caspase-3 is activated much more in immature brains than in those of adults [13]. Besides, sex hormone exposure may lead to higher risk of cerebral ischemia for women. The pathways of cell death differ in sexual dimorphism, as caspase-dependent pathway is more involved in female whereas AIF translocation is more important in males [14].

It is a common physiological death mechanism in ischemic stroke; but it also causes further impairment under certain pathological conditions. Energetic stress is the consequence of cerebral ischemia, and then reperfusion is accompanied by abrupt ionic shifts and considerable oxidative stress. During above physiopathologic process, apoptosis plays a key role of the neurons.

2.1 Molecular Biology Mechanism of Apoptosis After Ischemic Stroke

2.1.1 Molecules Related to Apoptosis of Neurons

2.1.1.1 Caspase Family

There are totally 14 caspase proteins identified by researchers, and among them at least eight proteins participate in the cell apoptosis. Caspase related to apoptosis can be classified into two types, the trigger and the executor. Caspase-8, Caspase-9 and Caspase-10 belong to the triggers while Caspase-3 and Caspase-7 are the executors [15]. Caspase-3 has been identified as a key mediator of apoptosis in animal models of ischemic stroke [12]. Activation of Caspase 3 requires assembly of a large multimeric complex comprising Caspase 9, APAF1, and cytochrome c. Caspase-3 cleaves many substrate proteins, including poly (ADP-ribose) polymerase (PARP) [16]. PARP inactivation after cleavage by caspase-3 leads to DNA injury and subsequently to apoptotic cell death [17]. In brief, Caspase-3 and Caspase-7 are the main participate and executor when apoptosis is activated after ischemic brain injury.

Albeit the underlying mechanism is consistent, the severity of ischemia, temporal 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].

2.1.1.2 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 [19,20,21]. 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.

2.1.1.3 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, constituting microaggregate on the surface of cells. Caspase-8 is activated by death-inducing signaling complex (DISC), of which Fas is an important part.

2.1.1.4 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]. 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 mitochondrial-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, secondary 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 mediates 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 caspase 3-dependent cell death.

2.3 Significance of Apoptosis in CIRI

The molecular mechanisms of apoptosis after stroke enlighten the exploration of neuroprotective agents. Ischemic preconditioning in animals triggers activation of caspase-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 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, successfully decreased cleaved Caspase-3 expression by activation of sphingosine 1-phosphate-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 normobaric oxygen can inhibit the apoptotic pathway by reducing caspase-3 and -9 expression, thereby promoting neurological functional recovery after CIRI [43]. These are various neuroprotective agents on the animal models and they are potential therapeutic targets in future clinical pharmacological research.

3 Necroptosis in CIRI

Necrosis was classified as non-programmed necrotic death previously which has been described as a response of extreme stress. However, in recent years, there is strong evidence to confirm that part of necrosis also contained program control, therefore proposed new concept as programmed necrosis or named necroptosis. Necroptosis are all classified as programmed cell death based on morphological and biochemical features [6, 44]. This phenomenon was observed in the ischemic stroke model.

3.1 Signal Pathway of Necroptosis

Caspase inhibition cannot blocked tumor necrosis factor (TNF) induced cell death completely, but rather switch to cell fate to necrotic death signal pathway like apoptosis [45, 46]. TNFα is the major trigger of necroptosis, which has capable of initiating caspase-8-dependent apoptosis and RIPK1 kinase-dependent necroptosis [47]. Caspase-8 plays a critical regulatory role in the switch. When FADD-caspase-8-FLIP complex functions inhibited, the cell death pathway switches from apoptosis to typical necroptosis features [48,49,50,51].

TNF-α induced necroptosis is the mostly intensively investigated. TNF receptor 1 (TNFR1) ligation leads to the recruitment of TRADD, TRAF2 and cIAP1/2, which is named as complex I [52]. The complex I activating death-inducing TNFR1 complex II via cylindromatosis (CYLD) [53]. In necrotic signal pathway, receptor-interacting kinase 1 (RIP1 or RIPK1) was the first molecule identified as the core components of the necroptotic machinery [54]. When RIPK1 and RIPK3 phosphorylated, then formed a necrosome through their homotypic interaction motif (RHIM) domains, and activates their kinase activities [55]. This RIPK1–RIPK3 interacts with mixed-lineage kinase domain-like (MLKL) phosphorylation [56]. Downstream of the necrosome are two splice variants of PGAM5, PGAM5S and PGAM5L. PGAM5L binds to the necrosome is not affected by the presence of the necrosis inhibitor necrosulfonamide (NSA). However, the binding of PGAM5S is blocked by NSA. Furthermore, mitochondrial fragmentation caused by the mitochondrial phosphatase PGAM5S recruited the mitochondrial fission factor Drp1 may up-regulate ROS generation [57].

3.2 Necroptosis in Cerebral Ischemia Disease

Necroptosis delayed mouse ischemic brain injury in the absence of apoptotic signaling [58]. In hippocampal neurons oxygen-glucose deprivation (OGD) models RIP3 mRNA and protein levels upregulation nevertheless caspase-8 mRNA downregulation. Similar to RIP3, RIP1 protein level was correlated with the activation of neuronal death. Consistent with the classical procedural necroptosis cellular pathways, ischemic injury upregulated RIP1-RIP3 expression and decreased the caspase-8 expression, which may be available afterwards for activation of necroptotic signaling [59].

Global brain ischemia and reperfusion (I/R) injury is another form of brain cell injury, which the hippocampal CA1 layer is especially vulnerable [60]. As a marker of necroptosis, RIP3 upregulated and transferred into nucleus after cerebral ischemia and reperfusion injury. RIP1–RIP3 complex is necessary for TNF induced necropoptosis in cell cytosol. ATP depletion is one of the results of the mitochondrial permeability transition pore (mPTP) leads to mitochondrial swelling. CypD as a gatekeeper of mPTP, alleviated the levels of RIP1 and RIP3, which mediated mPTP opening may contribute to not only apoptosis but also necroptotic cell death in cerebral I/R injury [61]. RIP3 was activated after I/R injury, and then interacts with AIF in the cytoplasm. The nuclear translocation of AIF and RIP3 is critical to neuronal necropoptosis, and the nuclear translocation of AIF may be RIP3-dependent [51]. AIF is the mediating molecule that links caspase-independent PCD with the necroptotic pathway.

It was observed that nerve cell necrosis occurred following focal middle carotid artery occlusion/reperfusion (MCAO/R) ischemic stroke model. TNFR1 and RIP3 were positively expressed and significantly increased following the volume of cerebral infarction post-reperfusion. Pre-administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3 [62]. In addition to phosphorylation modification, RIP3 S-nitrosylation in ischemia and reperfusion paralleled with elevated phosphorylation. It means RIP3 could be regulated by its S-nitrosylation triggered by NMDAR-dependent nNOS activation [63].

3.3 The Regulation of Necroptosis in Cerebral Ischemic Model

The classic inhibitor is a small molecule compound NSA, which did not block necrosis-induced RIP1 and RIP3 interactions, it blocks necroptosis downstream of RIP3 activation. In human glioblastoma cells, NSA switch from necrosis to apoptosis in edelfosine-treated [64].

In the field of cerebral ischemia, Necrostatin-1 (NEC-1) is another inhibitor of necroptosis has been shown to ameliorate tissue damage in ischemic brain injury animal models [58]. NEC-1 has a selective primary cellular target responsible for the death domain receptor-associated adaptor kinase RIP1 activity [65, 66]. It not only inhibited the expression of RIP1, prevented upregulation and nuclear translocation of RIP3, but also decrease cathepsin-B releasing in globe cerebral ischemic model. CA074-me and 3-methyladenine (3-MA), as autophagy inhibitors [67], were used to determine whether beneficial for global cerebral ischemia in the process of necroptosis signal pathways. The mechanism of 3-MA is inhibiting the nuclear translocation and co-localization of RIP3 and AIF. As the nuclear translocation of RIP3-AIF complex is critical to ischemic neuronal DNA degradation and necroptosis [51] (Fig. 5.2). Beside this, CA074-me almost completely hampered the loss of mitochondrial membrane depolarization, phosphatidylserine (PS) translocation, and plasma membrane rupture [68].

Fig. 5.2
figure 2

RIP1 and RIP3 are activated (phosphorylated) and combine with each other after CIRI. AIF is released from mitochondria and combines with RIP3 (perhaps phosphorylated RIP3) to form RIP3-AIF complexes. The RIP3-AIF complexes translocate into the nucleus resulting in chromatin condensation and DNA degradation, and then the neurons are triggered to undergo programmed necrosis. All of these changes after I/R injury are inhibited by pre-treatment with Nec-1 and 3-MA, except for the release of AIF from mitochondria in the 3-MA pre-treatment group. In neurons, the findings that caspase-8 expression was undetectable and caspase-3 was not activated indicate that caspase-dependent apoptosis is not involved in this process. Another necroptosis pathway in the cytoplasm induced by RIP1-RIP3-MLKL complexes, described by others, may also participate in this process

Full size image

4 Autophagic Cell Death in CIRI

Autophagy is the process by which a membrane engulfs organelles and cytosolic macromolecules to form an autophagosome, with the engulfed materials being delivered to the lysosome for degradation [69]. Briefly, autophagy proceeds through the capture of portions of cytoplasm containing target material inside expanding membranes, which finally enclose to form double-membrane vesicles called autophagosomes. Fully formed autophagosomes are shuttled along microtubules to lysosomes, whereupon fusion and degradation occur [70]. This removal and recycling serves as an emergency energy supply during starvation, but autophagy has also been linked to a diverse range of other protective roles [71, 72]. However, despite these pro-survival roles, autophagy has also been implicated as a mechanism of programmed cell death [73, 74]. Numerous studies have reported instances of dying cells displaying accumulated autophagosomes, which engulf large portions of the cell’s cytoplasm and which have been presumed to lead to excessive destruction of vital components [75, 76]. “Autophagic cell death” is morphologically defined as a type of cell death (type II) that occurs in the absence of chromatin condensation but accompanied by massive autophagic vacuolization of the cytoplasm [77].

4.1 Possible Autophagy Signaling Pathways in Cerebral Ischemia

The existence of autophagy in ischemic stroke has been found for many years; however, it is not sure whether autophagy plays a protective role in ischemic cerebral injury or not yet [78, 79]. Generally, in the neuronal system, moderate autophagy is thought to be neuroprotective because autophagy helps to clear aggregated-protein associated with neurodegeneration. Inadequate or defective autophagy may lead to neuronal cell death, while excess autophagy, often triggered by intensive stress, can also promote neuronal cell death.

Almost any signal can be a trigger for autophagy, some activating the pathway and some suppressing the pathway. By far, energy depletion and oxygen deficient environment are the most powerful triggers for stimulating autophagy, while the reverse environment factors, hormones, receptors with cytokine activities, receptors with tyrosine kinase activities and receptors that recognize pathogen ligands can also activate autophagy. Cerebral ischemia can activate multiple signaling pathways that subsequently feed into the autophagy pathway (Fig. 5.3).

Fig. 5.3
figure 3

Possible autophagy signaling pathways in CIRI

Full size image

The figure shows the many different signaling pathways involved in the activation of autophagy during cerebral ischemia. When activated, Akt and NF-κB activate mTOR to inhibit autophagy in cerebral ischemia. However, the activation of AMPK could inhibit the activity of mTOR and induce autophagy. Hypoxia caused by cerebral ischemia activates HIF-1α and induces autophagy through BNIP3 and p53. Excitotoxicity could induce autophagy by ER stress and block autophagic flux by glutamate in cerebral ischemia. Autophagy could also be induced through ROS and inhibited through PPAR-γ. PPAR-γ: Peroxisome proliferator-activated receptor-γ; AMP: Adenosine 5′-monophosphate; PI3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; HIF-1α: hypoxia inducible factor 1α; Bcl-2: B cell lymphoma/leukmia-2; Bcl-xL: B-cell lymphoma-extra large; AMPK: AMP-activated protein kinase; AMPK: AMP-activated protein kinase; Akt/PKB: protein kinase B; NF-κB: nuclear factor kappa B; ER: endoplasmic reticulum; BNIP3: Bcl-2 and adenovirus E1B 19 kDa interacting proteins 3; mTOR: mammalian target of rapamycin.

  1. 1.

    PI3K-Akt-mTORC1 mTOR is a 289 kDa serine/threonine protein kinase that regulates transcription, cytoskeleton organization, cell growth and cell survival. The mTOR is a high energy sensor, which on the other hand is a negative regulator of autophagy. By binding to different co-factors, mTOR can form two distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) [80]. mTORC1 is responsible for the inhibitory effect of rapamycin, more so than mTORC2. Recent studies suggest that the PI3K/Akt/mTOR pathway could regulate acute nervous system injury in cerebral hypoxia-ischemia [81]. PI3K consists of class I, class II and class III. Class I PI3K plays an important role in the PI3K-Akt-mTOR pathway. PI3K phosphorylates and activates Akt which in turn phosphorylates and inactivates tuberous sclerosis complex (TSC) 1/2. Inactivated TSC1/2 increases the activation of Rheb which is part of the Ras family GTP-binding protein, and mTOR is subsequently activated. Autophagy is inhibited by activating mTOR [82]. Beclin-1, a component of the class III PI3K, is essential for the initial steps of autophagy and could also induce autophagy via the interaction with other components of the class III PI3K pathway in cerebral ischemia [83]. Peroxisome prolif-erator-activated receptor-γ (PPAR-γ), a member of nuclear hormone receptor superfamily, is a ligand-activated transcription factor. PPAR-γ activation antagonizes beclin-1-mediated autophagy via upregulation of Bcl-2/Bcl-xl which interact with beclin-1 in cerebral ischemia/reperfusion [84].

  2. 2.

    AMPK-mTORC1 AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase and consists of three subunits: a catalytic α-subunit and regulatory β and γ-subunits. Each subunit appears to have distinct functions. The most studied is the catalytic α-subunit which contains a threonine phosphorylation site that when phosphorylated, activates AMPK. The status of nutrient and energy depletion is sensed and modulated by kinase B1 (LKB1), Ca2+/calmodulin-dependent kinase kinase beta (CaMKKβ) and transforming growth factor β activated kinase-1 (TAK1), resulting in phosphorylation of threonine residue at 172 position and activation of AMPK [85] and AMPK activation could subsequently inhibit the activity of mTOR to induce autophagy [86, 87].

  3. 3.

    Beclin 1-Bcl-2 complex.

    Beclin 1 was identified as a Bcl-2-interacting protein through its BH3 domain [88]. The binding of Bcl-2 to Beclin 1 disrupts the association of Beclin 1 with PI3K, hVps34 and p150, therefore inhibiting autophagy [89]. Intriguingly, only ER-localized, but not mitochondria-localized, Bcl-2 inhibits autophagy [89]. Under stress conditions, Beclin 1 is released and induces autophagy [90, 91]. As previously demonstrated, the expression of Beclin 1 in neurons is dramatically increased in neonatal HI or focal cerebral ischemia [92, 93]. Ischemia stimulates autophagy through the AMPK–mTOR pathway, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent pathway [94]. Although there are several different mechanisms to regulate the dissociation of Beclin 1 from Bcl-2 during autophagy in mammalian cells [95], the specific mechanism in cerebral ischemia is not yet established.

    Hypoxia-inducible factor 1 (HIF-1) is a key transcriptional factor that is activated in response to hypoxia during cerebral ischemia [96]. HIF-1 is composed of a constitutively expressed HIF-1β subunit and an inducibly expressed HIF-1α subunit. Since ubiquitination is inhibited under hypoxic conditions, HIF-1α can accumulate and dimerize with HIF-1β. This dimer activates transcription of a number of downstream hypoxia-responsive genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), glucose transporter 1, and glycolytic enzymes [97]. Bcl-2 and adenovirus E1B 19 kDa interacting proteins 3 (BNIP3) with a single Bcl-2 homology 3 (BH3) domain is a subfamily of Bcl-2 family proteins and also serves as an important target gene of HIF-1α [98]. BNIP3 can compete with beclin-1 for binding to Bcl-2 and beclin-1 is released to trigger autophagy [99]. BNIP3 also binds and inhibits Rheb, an upstream activator of mTOR, so it could activate autophagy by inhibiting mTOR activity. The induced p53 stabilization by up-regulation of HIF-1α also plays an important role in post-ischemic autophagy activation [97].

  4. 4.

    p53 The tumor suppressor and transcription factor p53 has been reported to be pivotal in neuronal apoptosis [100]. Crighton et al. demonstrated that p53 induced autophagy through the upregulation of damage-regulated autophagy modulator (DRAM), the p53 target gene encoding a lysosomal protein [101].Other study also demonstrated that the NF-κB-regulated p53 pathway contributes to excitotoxic neuronal death by activating the autophagic process [102]. Overstimulation of N-methyl-d-aspartate receptors (NMDARs) induces the upregulation of p53, its target gene DRAM, and other autophagic proteins including LC3 and Beclin 1. Moreover, the NF-κB inhibitor SN50 inhibits the excitotoxin-induced upregulation of p53, its target gene DRAM, and other autophagic proteins.

    Nuclear factor kappa B (NF-κB) is a transcription factor that regulates expression of multiple genes [103]. Recent experiments have demonstrated that the knockout of p50 (NF-κB1) enhanced autophagy by repression of mTOR in cerebral ischemic mice [104]. NF-κB-dependent p53 signal transduction pathway is also associated with autophagy and apoptosis in the rat hippocampus after cerebral ischemia/reperfusion insult [105]. Mitogen-activated protein kinases (MAPKs) include extracellular signal-related kinase (ERK), Jun NH2 terminal kinase (JNK) and p38 [106]. MAPK is one upstream regulator of mTORC1 and autophagy could also be induced via MAPK-mTOR signaling pathway in cerebral ischemia/reperfusion [107].

  5. 5.

    Others.

    Autophagic cell death is activated in the nervous system in response to oxidative stress [108]. Oxidative stress can occur in cerebral ischemia and could increase reactive oxygen species such as superoxide, hydroxyl radical and hydrogen peroxide. Recent studies have reported that selenium provides neuroprotection through preserving mitochondrial function, decreasing reactive oxygen species production and reducing autophagy [109]. Autophagy can also be induced under conditions of excitotoxicity which can also occur in cerebral ischemia [110]. Although excitotoxic glutamate blocks autophagic flux, it could also induce autophagy in hippocampal neurons [111]. Sustained elevations of Ca2+ in the mitochondrial matrix are a major feature of the intracellular cascade of lethal events during cerebral ischemia. Recently, it was reported that endoplasmic reticulum stress is one of the effects of excitotoxicity [1]. When endoplasmic reticula were exposed to toxic levels of excitatory neurotransmitters, Ca2+ was released via the activation of both ryanodine receptors and IP3R, leading to mitochondrial Ca2+ overload and activation of apoptosis. During endoplasmic reticulum stress, Ca2+ increase seems to be required for activating autophagy.

4.2 The Dual Roles of Autophagy in Cerebral Ischemia

Numerous data have demonstrated that autophagy is activated by ischemic insult in various models, and the elevated autophagic activity could be regulated by a wide range of interventions, mainly including pharmacological and genetic methods. There is no question that disrupting the autophagic process in brain is deleterious, particularly for the lifespan of the animal, resulting in the accumulation of dysfunctional or aging macromolecules and organelles [112, 113]. However, upon the acute cerebral ischemia stress, whether autophagy plays a beneficial or harmful role in the survival of neuronal cells is not an easy question. Adhami et al. [114] showed for the first time that many damaged neurons displayed features of autophagic/lysosomal cell death, and very few cells completed the apoptosis process in cerebral ischemic stress. This result suggested that the damaged neuronal cells can exhibit multiple forms of cell death morphological features, and autophagy is only one kind of cell death during ischemic injury. Alternatively, autophagy may protect neurons by degrading damaged organelles to abrogate apoptosis or generating energy to delay the onset of ionic imbalance and necrosis after cerebral ischemia–hypoxia. However, these early reports did not determine the exact role of autophagy. Dozens of later investigations pointed out the complex effects of autophagy in cerebral ischemia. The autophagy and the controversial impacts of autophagy on cerebral ischemic injury as a double-edged sword have been uncovered.

4.2.1 Detrimental Role of Autophagy in Ischemic Cerebral Injury

Mice deficient in Atg7, the gene essential for autophagy induction, showed nearly complete protection from both hypoxia-ischemia-induced caspase-3 activation and neuronal death, indicating autophagy is essential in triggering neuronal death after hypoxia-ischemia injury [115]. Wen et al. [116] confirmed autophagy was activated in a permanent middle cerebral artery occlusion (MCAO) model. In their paper, the infarct volume, brain edema and motor deficits could be significantly reduced by administration of 3-MA (an autophagy inhibitor). The neuroprotective effects of 3-MA were associated with an inhibition of ischemia-induced upregulation of LC3-II, a marker of active autophagosomes and autophagolysosomes. Moreover, it was observed that the inhibition of autophagy, either by direct inhibitor 3-MA or by indirect inhibitor 2ME2 (an inhibitor of hypoxia inducible factor-1α; HIF-1α) might prevent pyramidal neuron death after ischemia [97].

4.2.2 Beneficial Role of Autophagy in Cerebral Ischemic Injury

Carloni et al. [117] suggested that in neonatal hypoxia-ischemia, autophagy may be part of an integrated pro-survival signaling complex that includes PI3K-Akt-mTOR. When either autophagy or PI3K-Akt-mTOR pathways were interrupted, cells underwent necrotic cell death. Wang et al. [118] reported that neuronal survival was promoted during cerebral ischemia when autophagy was induced by nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), which is the rate-limiting enzyme in mammalian NAD+ biosynthesis and regulates the TSC2-mTOR-S6K1 signaling pathway. These studies suggest that autophagy may be a potential target for post-ischemic neuronal protection.

4.3 The Factors Determining the Role of the Autophagy in Cerebral Ischemia

4.3.1 The Degree of Autophagy Determines the Fate of Cells in Cerebral Ischemia

Kang and Avery [119] proposed that levels of autophagy were critical for the survival or death of cells: physiological levels of autophagy promote survival, whereas insufficient or excessive levels of autophagy promote death. This hypothesis was confirmed in an oxygen and glucose deprivation model that observed dual roles of the autophagy inhibitor 3-MA in different stages of re-oxygenation [75]. Twenty-four hours prior to reperfusion, 3-MA triggered a high rate of neuronal death. However, during 48–72 h of reperfusion, 3-MA significantly protected neurons from death. It is possible that prolonged oxygen and glucose deprivation/reperfusion triggers excessive autophagy, switching its role from protection to deterioration.

4.3.2 The Time at Which Autophagy Is Induced Determines Its Role

Autophagy could play a protective role in ischemic preconditioning but have a different effect once ischemia/reperfusion has occurred [120]. Infarct volume, brain edema and motor deficits induced by permanent focal ischemia were significantly reduced after ischemic preconditioning treatment. 3-MA suppressed neuroprotection induced by ischemic preconditioning, while rapamycin reduced infarct volume, brain edema and motor deficits induced by permanent focal ischemia [121]. This hypothesis was supported by a study by Yan et al. [122] in which 3-MA administrated through intracere-broventricular injection before hyperbaric oxygen preconditioning, attenuated the neuroprotection of hyperbaric oxygen preconditioning against cerebral ischemia. Moreover, 3-MA treatment before middle cerebral artery occlusion aggravated subsequent cerebral ischemic injury. In contrast, Carloni et al. [92, 117] found that when 3-MA and rapamycin were injected 20 min before hypoxia-ischemia, 3-MA inhibited autophagy, significantly reduced beclin-1 expression and caused neuronal death, while rapamycin increased autophagy and decreased brain injury. In addition, 3-MA administrated by intracerebroventricular injections strongly reduced the lesion volume (by 46%) even when given 4 h after the beginning of the ischemia [123]. Gao et al. [124] found that rapamycin applied at the onset of reperfusion might attenuate the neuroprotective effects of ischemic postconditioning. Conversely, 3-MA administered before reperfusion significantly reduced infarct size and abolished the increase of brain water content after ischemia. Targeting autophagy either pre- or post-treatment has different results and this may reflect the different effects of autophagy at early and late stages. The time of intervention could be related to the degree of autophagy at different stages of ischemia and further studies are necessary to confirm this.

4.3.3 Autophagy May Be Interrupted in Cerebral Ischemia

A common feature of many neurodegenerative diseases is the accumulation of an abnormally large number of autophagic vacuoles (autophagosomes and autolysosomes) or the frequent appearance of irregularly shaped autophagic vacuoles. Enhanced autophagosome formation seems to be reflected by increased density of autophagic vacuoles, but these increased autophagic vacuoles may also imply impaired autolysosomal degradation [125]. Rami et al. [93] also observed a dramatic up-regulation of Beclin-1 and LC3 in rats after cerebral ischemia. These results indicate that autophagy was activated in the brain following ischemia. Recently, however, it has been hypothesized that the increase in proteins may reflect a failure in lysosomal function leading to an accumulation of autophagosomes, or an improvement in the activity of autophagy [126]. Other studies found that accumulation of LC3-II was observed in sham-operated rats after treatment with lysosomal inhibitor-chloriquine, but the further change of LC3-II levels in post-ischemic brain tissues was not observed [127, 128]. The results indicated that accumulation of autophagy-associated protein following ischemia could be the result of failure of the autophagy pathway. Puyal and Clarke [129] found that lysosomal activity detected by LAMP-1 and cathepsin D was increased in neurons with punctate LC3 expression in neonatal focal cerebral ischemia model. The failure of autophagosome and lysosome fusion caused an increase of autophagosomes. The deficiency of acid phosphatase activity in the lysosome could lead to the increase of autophagosomes and autolysosomes. Further studies are required to verify whether the activity of autophagy is enhanced in cerebral ischemia.

5 Crosstalk Between Apoptosis, Autophagy and Necroptosis (Necrosis) After CIRI

PCD in vivo involves the complex interaction between apoptosis, autophagy, and necroptosis [130, 131] (Fig. 5.4). In some cases, a specific stimulus triggers only one type of programmed cell death, but in other situations, the same stimulus may initiate multiple cell death processes. Different types of mechanisms may co-exist and interact with each other within a cell, but ultimately, one mechanism dominates the others. The decision taken by a cell to undergo apoptosis, autophagy, or necroptosis is regulated by various factors, including the energy/ATP levels, the extent of damage or stress, and the presence of inhibitors of specific pathways (e.g., caspase inhibitors). ATP depletion activates autophagy. However, if autophagy fails to maintain the energy levels, necroptosis occurs [132]. Slight/moderate damage and low levels of death signaling typically induce apoptosis, whereas severe damage and high levels of the death signaling often result in necroptosis. Similarly, inhibition of caspase activity might change apoptosis to necrosis or autophagic cell death, whereas activation of calpain-mediated cleavage of autophagy-regulated protein, Atg-5, switches the mode of cell death from autophagy to apoptosis [133, 134]. Interestingly, although necroptosis, necrosis and secondary necrosis following apoptosis, represent different modes of cell death, all of them might eventually converge on similar cellular disintegration features, albeit with different kinetics [135]. Furthermore, apoptosis and autophagy differ from the necrosis by the feature of tissue inflammation [136]. Both apoptosis and autophagy do not exhibit tissue inflammation, while the latter does. Thus, learning more about the molecular mechanisms regulating various cell death modalities and their cross-talk is very important, since they play a critical role in CIRI.

Fig. 5.4
figure 4

Cross-talk between different modes of programmed cell death after CIRI. FADD Fas-associated death domain-containing protein, NSA necrosulfonamide, NEC-1 necrostatin-1

Full size image

Although death-receptor mediated apoptosis represents a canonical apoptotic pathway, stimulation of death receptors under apoptotic deficient conditions is now known to activate necroptosis [58]. The activation of death receptors by their respective ligands, such as FasL (CD95L) and TNF-α, respectively, leads to the formation of DISC (death-inducing signaling complex) that includes the adaptor protein FADD (Fasassociated death domain), caspase-8 and death domain-containing kinase RIP1. In apoptotic proficient condition, the recruitment of caspase-8 leads to its activation which in turn activates downstream caspases, such as caspase-3, and mitochondrial damage by cleaving Bid [137]. In apoptotic deficient cells when caspases cannot be activated, however, stimulation of death receptors leads to the activation of RIP1 kinase and necroptosis [54, 65]. Activation of AKT also appears to act as a switch, in addition to facilitating the necroptotic response, it also acts to inhibit apoptosis [138, 139]. These results clearly illustrate that the molecular pathways regulating death ligand-induced apoptosis and necroptossis are intimately intertwined. They also firmly establish the paradigm that inhibition of caspase-dependent apoptosis primes cells towards necroptosis.

Crosstalk between apoptosis and autophagy in CIRI is also complex. It has been acknowledged that appropriately controlled autophagy can induce neuroprotection and can rescue neurons from apoptotic cell death in the cerebral ischemia. For instance, clearance of damaged mitochondria via autophagy prevented neurons from caspase-dependent apoptosis [140]. However, autophagy may also act as a pro-apoptotic mechanism [99] and is causally connected with the subsequent onset of apoptotic cell death [141,142,143]. Cathepsin B, a protease which is normally confined inside the lysosomal-endosomal compartment, leaked from the lysosomes into the cytoplasm, initiating and promoting the execution of apoptosis [141]. It has been hypothesized that when the autophagic flux impairs, autolysosomes would extensively accumulate and the autophagic stress would be induced [126]. This would lead to autolysosomes and lysosomes membrane destabilization, which results in leakage of hydrolases, and subsequently provoke apoptosis [144]. As a result, the initial autophagy, as a defensive reaction, when over-activated, is converted into a damage response [75]. In this case, the inhibition of autophagy attenuates apoptotic cascades in ischemic injury. These evidences demonstrated that elucidating the interrelationships between autophagy and apoptosis will present novel opportunities for discovering targets in the therapy for cerebral ischemic injury.

So far, with the identification of several key molecules (e.g., ATG, Bcl-2 family members, Beclin 1, and p53) [105, 145,146,147,148,149,150], the mechanisms underlying the autophagy-apoptosis conversation are beginning to be uncovered. However, current researches seemingly only reveal a tip of the iceberg among the intricate interactions between autophagic and apoptotic cascades during the cerebral ischemic injury. Thus, more studies about the crosstalk between autophagy and apoptosis are warranted in the future.

The autophagy and necrosis can be activated in parallel or sequentially, and have either common or opposite objectives. The molecular underpinnings of this relationship remain largely elusive and somewhat controversial; autophagy has been shown to either promote [123] or suppress necroptosis (necrosis) [92, 151]. However, the ability of autophagy to suppress various forms of necrotic cell death is considered to be one of the most important pro-survival functions of autophagy that is achieved either by blocking apoptosis or suppressing necrotic cell death.

Therefore, because of the complex crosstalk between cell death pathways, much effort should be put on the finding of biomarkers that may predict the risk of a hypoxic-ischemic condition during the CIRI to initiate the treatment in an early stage, allowing the possibility of using the preconditioning effect of putative drugs. These early treatments may be followed by endovascular recanalization therapy (thrombolytic therapy or arterial embolectomy), that potentially reduces both apoptosis and necrosis. Of course, a better understanding of the mechanisms responsible for the switch among the different cell death phenotypes and the development of new and more selective molecules that can act upstream of these putative checkpoints will help to find new pharmacological strategies that could be associated to endovascular recanalization therapy.