2 Characters of the Ischemic Stroke
2.3 The Pathophysiology of Ischemic Stroke
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which including the regulation of blood flow, maintaining the integrity of BBB, affecting the synaptic function by the release of growth factors, reducing the gluta- mate amount and alleviating excessive inflammatory response [41].
As a key membrane protein of BBB, aquaporins 4 (AQP4) is crucial in astrocyte swelling and cerebral edema process [42]. AQP4 is relevant with the expression of Agrin that accumulates in brain microvessels at the time of BBB tightening and is responsible for BBB integrity [43]. The AQP4 deletion could reduce astrocyte swell- ing and brain edema, as well as improve the neurological outcome of mice after focal ischemia [44]. Because of interaction between IL-17 and its receptor on endothelial cells promotes the progress of BBB breakdown by disrupting tight junctions [45]. As a critical effector of cerebral ischemia tissue, IL-17 secreting T cells could accumu- late in the CNS and cross the BBB, further destroy BBB or directly damage neurons [46, 47]. In the middle cerebral artery occlusion (MCAO)-induced ischemic stroked mice, the brain tissue expression level of IL-17 elevated at 1 h after ischemia insult and peaked at day 6 [48]. However, the anti-inflammatory properties of IL-4 were reported that the ischemic damage were exacerbated in IL-4 knocked-out mice, which suggested its potential to attenuate ischemic injury and promote tissue repair [49]. Similarly, the regulatory T lymphocytes (Treg)-produced IL-10 is also regarded as the neuroprotective cytokine for inhibiting neurotoxic function of TNF-α and IFN-γ [50]. In addition, the essential immune roles of Toll-like receptors (TLR) including TLR2 and TLR4 were confirmed in the ischemic brain injury [51]. It was reported that TLR4 knock-out could protect the brain from high-mobility group pro- tein box-1 (HMGB1) mediated ischemic damage [52], as well as both TLR2 and TLR4 knock-out alleviated ischemic brain injuries [53]. The proinflammatory cytokines- mediated inflammatory response is more complicated, hence the identifi- cation of their roles in neuroprotection may provide effective immune therapeutic strategies for ischemic stroke.
2.3.3 Apoptosis
There are two general pathways of apoptosis activated after ischemia: the Caspase- 8- mediated extrinsic pathway and the Caspase-9 (mitochondria) or Caspase-12 (endoplasmic reticulum)-mediated intrinsic pathways. Intrinsic Caspase-9 pathway is initiated by the cytochrome C release of mitochondria, while the extrinsic path- way is due to the activation of cell surface death receptors, resulting in the stimula- tion of caspase-8 [54]. In the past decades, researchers focused on the apoptosis in the ischemic penumbra in order to find the potential target at the early stage of ischemic stroke onset. It suggests that mitochondria influence neuronal apoptosis primarily via the release of proapoptotic factors into the cytoplasm, including cyto- chrome C [55, 56]. Cytochrome C is involved to active Caspase-9, which is presum- ably an initiator of the cytochrome c-dependent Caspase cascade, then activates Caspase-3 [57]. Caspase-9 and Caspase-3 play the important roles in neuronal death after cerebral ischemia. The researches illustrated the cleaved Caspase-9 increased
2 Characters of Ischemic Stroke and Recanalization Arteries
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in brain tissue 12 h after ischemia and up-regulation of Caspase-3 mRNA in rat brain 1 h after the onset of focal ischemia [58, 59]. It has been also observed Caspase-3 was up-regulated in the human brain tissue after ischemia [60]. At the same time, some studies suggested that genetic deletion and pharmacological inhi- bition of Caspases have a strong neuroprotective effect in stroke animal models.
Some evidences showed that cerebral ischemia triggers the extrinsic apoptotic sig- naling cascade. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand, Fas and FasL expressions are up-regulated within 12 h after cerebral ischemia in the animal models and peaked between 24 and 48 h, which coincides with the time course of apoptotic death in neuronal cells [61, 62]. It was detected Caspase-3 and several other Caspase family members up-regulated in astrocytes and microglia after MCAO [63]. Although there are some studies discovering the mechanism of apoptosis after stroke, more detailed molecular process of apoptosis are needed to find the novel stroke therapies.
2.3.4 Autophagy
Programmed cell death includes type I (apoptosis), type II (autophagic cell death) and type III (necroptosis). The process of autophagy could degrade the damaged organelles and proteins through the autophagic vesicles [64]. More and more evi- dences indicate that autophagy may be involved in regulating neuronal death in isch- emic stroke, including global and focal ischemia [47, 65–68]. Among all the autophagy related proteins, the microtubule-associated protein 1 light chain 3 I (LC3-I) is noteworthy. It could be hydrolyzed to LC3-II during the process of autophagy starts. Therefore, ratio of LC3-II/LC3-I is commonly used to evaluate the level of autophagy [69]. Turnover of LC3 I to II was increased in the ipsilateral hemisphere 24–72 h after cerebral hypoxia–ischemia [70]. In 1999, Beclin-1 is the first identified mammalian gene with a role in autophagy initiation [71]. Beclin-1 protein level was observed to increase as early as 4 h, appeared to peak at 24–72 h after ischemia injury [72]. 3-methyladenine (3-MA) is used to inhibit autophagy as the inhibitor, which has the time-dependent protective effect on neuronal death [73].
Applying it before ischemia could be better in protecting the global I/R injury. The previous study shows that conventional protein kinase C (cPKC)γ activation has neuroprotective effect against ischemic injuries [74]. Recent study finds that cPKCγ deficit could increase Beclin-1 expression level and the ratio of LC3-II/LC3-I, at the same time, BafA1 is regarded as the autophagy inhibitor, which could aggravate or alleviate OGD-induced ischemic injuries in cPKCγ wild-type and cPKCγ knock- out neurons, respectively [75]. All in all, as an essential role in ischemia, autophagy remains worth further study to develop newly effective therapeutic strategies for ischemic stroke.
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2.3.5 Pyroptosis
Pyroptosis is an inflammasome-dependent programmed cell death. Inflammasomes activate Caspase-1 by the Caspase recruitment domain (CARD) or pyrin domain (PYD)-containing inflammasome, including nod-like receptor (NLRP1, 3, 6, 7, 12, NLRC4), absent in melanoma 2 (AIM2), or pyrin [76, 77]. And then Caspase-1 induces the activation and secretion of pro-IL-1b and pro-IL-18 [77]. It is the pri- mary cause of anthrax-lethal- toxin-induced lung injury that pyroptosis induced by the NLRP1B inflammasome or a gain-of-function mutation in Nlrp1a [78]. The newly research indicated that interdomain cleavage of gasdermin D (GSDMD) by caspase-1/4/5/11 determines pyroptosis [79]. AIM2 contains a HIN200 domain and is specific for cytosolic DNA [80–83]. In 2014, the research firstly reported embry- onic cortical neurons express a functional AIM2 inflammasome and identify pyrop- tosis as a cell death mechanism in neurons. It demonstrated pannexin1 as a cell death effector channel in pyroptosis and inhibition of pannexin1 by probenecid attenuates pyroptosis in neurons, which suggested that active Caspase-1 cleaves inflammatory cytokines, opens the pannexin1 pore, and induces cell death [84]. It is unclear of the pyroptosis mechanism in ischemic stroke. What is the role of pyrop- tosis and the relationship with apoptosis and autophagy? It is worthy to explore the role of pyroptosis deeply in the ischemic stroke models.