The biphasic function of microglia in ischemic stroke

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PRONEU 1417 1–25 Progress in Neurobiology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Progress in Neurobiology journal homepage: www.elsevier.com/locate/pneurobio 1 2 3 4 5 6 7

The biphasic function of microglia in ischemic stroke Q1 Yuanyuan

Ma a,b, Jixian Wang a,b, Yongting Wang b,*, Guo-Yuan Yang a,b,**

a

Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, China Neuroscience and Neuroengineering Research Center, Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2015 Received in revised form 22 December 2015 Accepted 10 January 2016 Available online xxx

Microglia are brain resident macrophages originated from primitive progenitor cells in the yolk sac. Microglia can be activated within hours and recruited to the lesion site. Traditionally, microglia activation is considered to play a deleterious role in ischemic stroke, as inhibition of microglia activation attenuates ischemia induced brain injury. However, increasing evidence show that microglia activation is critical for attenuating neuronal apoptosis, enhancing neurogenesis, and promoting functional recovery after cerebral ischemia. Differential polarization of microglia could likely explain the biphasic role of microglia in ischemia. We comprehensively reviewed the mechanisms involved in regulating microglia activation and polarization. The latest discoveries of microRNAs in modulating microglia function are discussed. In addition, the interaction between microglia and other cells including neurons, astrocytes, oligodendrocytes, and stem cells were also reviewed. Future therapies targeting microglia may not exclusively aim at suppressing microglia activation, but also at modulating microglia polarization at different stages of ischemic stroke. More work is needed to elucidate the cellular and molecular mechanisms of microglia polarization under ischemic environment. The roles of microRNAs and transplanted stem cells in mediating microglia activation and polarization during brain ischemia also need to be further studied. ß 2016 Elsevier Ltd. All rights reserved.

Keywords: Crosstalk Ischemic stroke Microglia microRNAs Polarization

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Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of microglia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microglia activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microglia activation in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Microglia activation during ischemic stroke . . . . . . . . . . . . . . . . . 3.2. Microglia activation in human ischemic stroke . . . . . . . 3.2.1. Microglia activation in experimental ischemic stroke . . 3.2.2. Receptors mediating microglia activation and function. 3.2.3. Roles of activated microglia in ischemic stroke (Table 1) . . . . . . . . . . . . Microglia and blood brain barrier permeability . . . . . . . . . . . . . . 4.1. The effect of microglia on neurogenesis . . . . . . . . . . . . . . . . . . . . 4.2.

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Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; BBB, blood brain barrier; CSF-1R, colony stimulating factor-1 receptor; EPCs, endothelia progenitor cells; FcRs, Fc receptors; HMGB1, high mobility group box1; IGF-1, insulin-like growth factor 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MCAO, middle cerebral artery occlusion; MerTK, Mer receptor tyrosine kinase; MFG-E8, milk fat globule-EGF factor-8 protein; MS, multiple sclerosis; MSCs, mesenchymal stem cells; NO, nitric oxide; NOX, NADPH oxidase; NSCs, neural stem cells; OPCs, oligodendrocyte progenitor cells; OGD, oxygen–glucose deprivation; PACAP, pituitary adenylate cyclaseactivating polypeptide; PD, Parkinson’s disease; RAGE, receptor for advanced glycation endproducts; ROS, reactive oxygen species; Runx1, runt-related transcription factor 1; SOCS-1, suppressor of cytokine signaling 1; TLRs, Toll-like receptors; TREM-2, triggering receptor expressed on myeloid cells-2; SVZ, subventricular zone; VZ, ventricular zone. * Corresponding author. Tel.: +86 21 62933186; fax: +00 86 21 62932302. Q2 ** Corresponding author at: Shanghai Jiaotong University, Med-X Reasearch Institute, 1954 Hua Shan Road, Shanghai 200030, China. Tel.: +86 21 62933186; fax: +00 86 21 62932302. E-mail addresses: [email protected] (Y. Wang), [email protected] (G.-Y. Yang). http://dx.doi.org/10.1016/j.pneurobio.2016.01.005 0301-0082/ß 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ma, Y., et al., The biphasic function of microglia in ischemic stroke. Prog. Neurobiol. (2016), http:// dx.doi.org/10.1016/j.pneurobio.2016.01.005

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4.2.1. Microglia influence neurogenesis under physiological condition . . . . Microglia influence neurogenesis during ischemic stroke . . . . . . . . . . 4.2.2. Interaction between activated microglia and NPCs . . . . . . . . . . . . . . . 4.2.3. Microglia polarization during ischemic stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microglia polarization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Ischemia induces microglia polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Substances involved in the modulation of microglia polarization . . . . . . . . . . . 5.3. Clinical drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. 5.3.2. Receptors and small molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . microRNAs and microglia (Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Microglia crosstalk with other cells during ischemic stroke . . . . . . . . . . . . . . . . . . . . . 6.1. Microglia and neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurons control microglia activation . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Neurons influence microglial function . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Microglia influence neuronal function . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. 6.2. Microglia and astrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cross-talk between microglia and astrocytes . . . . . . . . . . . . . . . . . . . . 6.2.1. Cross-talk between microglia and astrocytes during ischemic stroke. 6.2.2. Microglia and oligodendrocytes/oligodendrocyte progenitor cells . . . . . . . . . . . 6.3. Relationship between microglia and stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microglia as a potential candidate of cell based therapy for ischemic stroke. . . . . . . . Conclusion and prospect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10 Q3 1. Introduction 11 Q4 In the late 19th century, German anatomist Franz Nissl first 12 described microglia and named them as Staebchenzellen (rod 13 cells) for their rod-shaped nuclei. Nissl explained these cells as 14 reactive glia elements with the capacity of migration, phagocytosis 15 and proliferation (Kim and de Vellis, 2005). Later in 1913, Spanish 16 neuroscientist Santiago Ramo´ny Cajal renamed this type of cells as 17 a ‘‘third element’’. He pointed out that these cells were different 18 from neurons and astrocytes, and probably of mesodermal origin. 19 Further, this ‘‘third element’’ was divided into various cell types 20 based on morphological and functional differences (Ginhoux et al., 21 2013). The main population among the third elements was 22 oligodendrocytes, and the second population was microglia 23 (Cartier et al., 2014). Using a silver staining technique, the 24 presence of microglia in human brain was demonstrated. Microglia 25 were classified into three types based on their morphological 26 differences: amoeboid, ramified, and intermediate microglia (Kim 27 and de Vellis, 2005). Since then, the studies of microglia expanded 28 rapidly and widely (Nayak et al., 2014). 29 In the adult mouse brain, microglia constitute 5–12% of all glia 30 cells. The distribution of microglia is different in different regions 31 of the brain, ranging from 5% in the cerebral cortex and corpus 32 callosum to 12% in the substantia nigra. There are approximately 33 3.5  106 microglia in the adult mouse brain. More microglia are in 34 the gray matter than that in the white matter in the mouse brain 35 (Lawson et al., 1990). In normal human brain, the distribution of 36 microglia varies by up to one order of magnitude. The number of 37 microglia accounts for approximately 0.5% to 16.6% of all brain 38 cells. Different from the case in mouse brain, much more microglia 39 are located in the white matter than in the gray matter in human 40 brain. These findings provide valuable information to evaluate 41 microglial response under pathological conditions (Mittelbronn 42 et al., 2001). 43 Microglia are the resident macrophages and the first line of 44 defense against injury in the central nervous system (Hu et al., 45 2014; Prinz and Priller, 2014). After ischemic brain injury, 46 microglia rapidly migrate toward the lesion site and exacerbate 47 tissue injury by producing inflammatory cytokines and cytotoxic 48 substances. On the other hand, however, microglia also contribute 49 to tissue repair and remodeling by clearing up debris and

producing anti-inflammatory cytokines and growth factors (Shi and Pamer, 2011). Increasing evidence indicate that microglia could be beneficial for the functional recovery after cerebral ischemia (Neumann et al., 2009; Neumann et al., 2006). Microglia activation is a complicated process that can be affected by many substances and surrounding cells such as neurons, astrocytes, and oligodendrocytes. The dual role of microglia is associated with different polarization of microglia under different cellular context and pathological stages after brain injury (Hu et al., 2014; Kim et al., 2014). Elucidating the detailed mechanisms of microglia activation and polarization will help to drive microglia shift from a detrimental phenotype to a protective phenotype. Investigation of how microglia communicate with neurons, astrocytes, and oligodendrocytes will aid our understanding on the role of microglia during ischemic disease.

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2. Origin of microglia

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In the last decades, investigators were devoted to deciphering the origin of microglia since its discovery (Wieghofer et al., 2015). Although it was still under debate regarding what was the precise nature of microglia progenitors, the generally accepted concept is that microglia are of myeloid origin (Ginhoux et al., 2013). Cajal believed that microglia originated from mesodermal progenitors (Kettenmann et al., 2011). Meningeal macrophages could penetrate into the brain during embryonic development and transform into microglia (Cartier et al., 2014; Kim and de Vellis, 2005). Initially, circulating monocytes or precursor cells invaded into the developing brain, changed their amoeboid morphology, and formed resting microglia of ramified shape in the mature brain (Kim and de Vellis, 2005). Studies using bone marrow transplantation technique indicated that microglia derived from blood monocytes. In a model of primary demyelination induced by copper chelator cuprizone and a model of facial nerve axotomy in mice, blood derived Ly-6ChiCCR2+ monocytes could invade into the central nervous system and contribute to the repopulation of microglia after the brain was irradiated (Mildner et al., 2007; Varvel et al., 2012). However, further clarification was needed to identify these invading cells as true microglia (Nayak et al., 2014). Using a model of chimeras between chick embryo and quail yolk sac, a study showed that microglia progenitors originated

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from embryonic yolk sac (Cuadros et al., 1993). Myeloid cells originated from quail yolk sac invaded into the chick embryo during development, which was independent of blood supply. Another study supported this concept with the discovery of microglia precursors presented in the yolk sac and later in the brain rudiment at embryonic day eight (Alliot et al., 1999). Most microglia expressed proliferating cell nuclear antigen (PCNA), a marker of cell proliferation, suggesting that microglia could proliferate in situ. Recently, a study further confirmed that microglia were originated from primitive myeloid progenitors located in the yolk sac using fate mapping technique. Crossing mice expressing tamoxifen-inducible Cre recombinase under runt-related transcription factor 1 (Runx1) promoter with mice expressing loxP Rosa-YFP gene produced transgenic mice in which yolk-sacderived cells were YFP+. Because Runx1 was specifically expressed in yolk sac and embryonic liver hematopoietic progenitors, researchers were able to trace yolk-sac-derived cells (Cartier et al., 2014; Nayak et al., 2014). When tamoxifen was injected into pregnant mice before embryonic day 7.5, Runx1+ cells were restricted in yolk sac first and then migrated into the mouse brain from embryonic day 10 to 6 weeks after birth, in a circulation dependent manner (Samokhvalov et al., 2007). However, when tamoxifen was injected at embryonic day 8.5, when yolk sac hematopoiesis was already replaced by definitive hematopoiesis in the embryo, Runx1+ cells could not be found in the brain. These results collectively suggest that Runx1+ cells enter the brain during early development and give rise to microglia. In addition, microglia and yolk sac macrophages are markedly reduced in colony stimulating factor-1 receptor (CSF-1R) deficient mice while the amount of circulating monocytes is not affected, indicating that CSF-1R is essential for the development of microglia and macrophages in the embryo but not required by circulating monocytes. During embryonic development, definitive hematopoiesis is initiated in the aorta, gonads, and mesonephros (AGM) region, and then occur in the fetal liver, spleen, and bone marrow (Lichanska and Hume, 2000; Orkin and Zon, 2008). Tissue macrophages were thought to derive from bone marrow monocytes. Myb, a transcription factor, is required for the development of definitive hematopoiesis but not for the yolk sac myelopoiesis (Mucenski et al., 1991). It was noted that when PU.1, a transcription factor required for macrophage development, was deleted, all macrophages and microglia were absent (DeKoter et al., 1998; McKercher et al., 1996). These results revealed that yolk sac derived macrophages give rise to microglia in the embryo (Cartier et al., 2014). Another study demonstrated that microglia derive from primitive CD45c-kit+ erythromyeloid precursors in yolk sac. These precursors eventually mature into CD45+/c-kit/CX3CR1+ microglia and invade into the brain via specific matrix metalloproteinase. Transcription factors PU.1 and IRF8 are both essential for the development of microglia derived from yolk sac precursors. When PU.1 and IRF8 are deficient, the number of Iba1+ microglia decreased in mouse brain (Kierdorf et al., 2013). Collectively, all these studies suggested that microglia originate from primitive myeloid progenitors arising from yolk sac before embryonic day 8. In addition, microglia are a genetically distinct cell type in the mononuclear phagocyte system (Ginhoux et al., 2010; Schulz et al., 2012).

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3. Microglia activation

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3.1. Microglia activation in the brain

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Under physiological condition, microglia display classical ramified morphology with a small cell soma and fine processes, which is referred as ‘‘resting microglia’’ (Kreutzberg, 1996). Resting

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microglia are defined as having low expression of CD45, MHC II, CD80, CD86, and CD11c (Lynch, 2009; Ponomarev et al., 2005; Prinz et al., 2011). The processes of microglia are constantly in motion and survey the local microenvironment of the brain (Nimmerjahn et al., 2005). Disturbance of brain homeostasis could induce microglia activation (Ransohoff and Perry, 2009). Once activated, microglia undergo typical morphological changes, characterized by retracting and thickening of the processes, and hypertrophy of the cell body (Kreutzberg, 1996). A number of markers such as CD45, MHC II, and CD86 are up-regulated when microglia are activated (Ponomarev et al., 2013). In addition, cell surface markers such as Iba1, IB4, F4/80, and CD68 (ED-1) are commonly used to identify microglia activation. After focal brain ischemia, microglia display different expression patterns of markers depending on whether they are located in the peri-infarct region or in the infarct zone. In the peri-infarct region, microglia are positive for Iba1 but negative for CD68, while in the infarct zone microglia are both Iba1and CD68 positive and displayed enhanced CD11b expression (Ito et al., 2001; Morrison and Filosa, 2013). Activated microglia in the local infarct zone mainly express MHC I and act primarily as scavenger cells (Stoll et al., 1998). While activated microglia in regions far from infarct region mainly express MHC II and are thought to be associated with Wallerian (anterograde) degeneration (Block et al., 2005; Morioka et al., 1993). Therefore, microglia at different locations in respect to brain ischemia exert different function.

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3.2. Microglia activation during ischemic stroke

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Inflammation is recognized as a vital contributor to the 179 pathophysiology of ischemic stroke (Moskowitz et al., 2010). 180 Microglia activation is the first step of inflammatory response in 181 the brain, followed by infiltration of immune cells such as 182 neutrophils, macrophages/monocytes, natural killer cells, T cells, 183 and other neuronal cell activation (Iadecola and Anrather, 2011; Jin 184 et al., 2010). Microglia could be activated within minutes after 185 ischemic brain injury (Nakajima and Kohsaka, 2004). Activated 186 microglia can produce a variety of mediators including inducible 187 nitric oxide synthase (iNOS) (Iadecola and Ross, 1997), nitric oxide 188 (NO) (Gibson et al., 2005; Nakashima et al., 1995), pro- 189 inflammatory cytokines (such as TNF-a) (Lambertsen et al., 190 2009), anti-inflammatory cytokines (such as TGF-b) (Iadecola 191 and Anrather, 2011), growth and trophic factors such as insulin- 192 like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), 193 hepatocyte growth factor (HGF), platelet-derived growth factor 194 (PDGF) (Lai and Todd, 2006), nerve growth factor (NGF), brain- 195 derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), 196 neurotrophin-4/5 (NT-4/5), and plasminogen (PGn) (Elkabes 197 et al., 1996). Whether these mediators exacerbate or alleviate 198 tissue damage during brain ischemia needs to be further 199 investigated. For example, TNF-a, which is a key player in brain 200 inflammation, is up-regulated in microglia both in animals and 201 human after ischemic stroke (Clausen et al., 2008; Dziewulska and 202 Mossakowski, 2003; Gregersen et al., 2000). Administration of 203 recombinase TNF-aprotein before transient middle cerebral artery 204 occlusion (MCAO) exacerbated infarct volume, brain swelling, and 205 neurological deficit. These phenomenon could be reversed by pre- 206 treatment with TNF-a antibody (Yang et al., 1998). Blockage of 207 endogenous TNF-a resulted in a smaller infarct volume compared 208 to the control group (Barone et al., 1997). These results implied Q5209 that TNF-a plays a toxic role during focal cerebral ischemia. 210 However, a study using TNF-a, TNF-areceptor I (TNF-p55R), and 211 TNF-a receptor II knock out mice suggests they could function 212 differently. Mice lacking TNF-aand TNF-p55R develop larger 213 infarct volume and poorer neurological outcome compared to 214 the wild type mice and TNF-a receptor II knock out mice. 215

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Furthermore, TNF-ais mainly produced by activated microglia and could enhance the tolerance of cultured neurons and astrocytes against oxidative stress and ischemic injury (Ginis et al., 2002; Goodman and Mattson, 1996; Lambertsen et al., 2009). These results indicated that microglia could facilitate neuroprotective function against brain ischemia via TNF-p55R. The different conclusions mentioned above is due to that TNF-a exerts different function depending on the location of TNF-areceptors (Vexler et al., 2006). Further study is needed to elucidate the exact role and mechanism of mediators produced by microglia in ischemic stroke. The task would pave the road for developing intervention strategies targeting mediators produced by microglia.

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3.2.1. Microglia activation in human ischemic stroke With the rapid development of imaging methods such as positron emission tomography (PET) and MRI, it is feasible to study microglia activation in human. Clinical studies demonstrated that microglia activation is present in the brain at the acute phase (Krupinski et al., 1996; Tomimoto et al., 1996), the sub-acute phase (Price et al., 2006), and the recovery phase after ischemic stroke (Gulyas et al., 2012). Microglia activation can be detected in human brain within 24–48 h after brain ischemia, and mainly present in the ischemic core and extend to the peri-infarct region over time (Krupinski et al., 1996; Tomimoto et al., 1996). The time course of microglia activation in the infarct zone or the peri-infarct region is different in stroke patients (Thiel et al., 2010). Combining PET using radiolabeled ligand 11C-(R)-PK11195 with structural brain imaging by MRI, microglia activation was dynamically evaluated in vivo. Activated microglia were observed in 6 patients from 3 to 150 days after ischemic stroke. Imaging results showed that microglia activation could be observed as early as day 3. One patient was examined at 28 and 150 days after brain ischemia. Microglia accumulated at the primary infarct site at 28 days, and spread to the connected region in the ipsilateral and contralateral thalamus at 150 days (Gerhard et al., 2005). Another study examined 4 patients with ischemic stroke showed that activated microglia increased in the first week both in the infarct zone and peri-infarct region, but decreased over time. Microglia activation could persist for up to 14 weeks in the peri-infarct region, which is longer than that in the infarct zone, indicating a longer neuro-inflammatory activity in the peri-infarct region (Gulyas et al., 2012). A clinical study of 18 patients who experienced a first-ever sub-cortical stroke indicated that activated microglia in the infarct zone or in the area far from infarct zone display different association with anterograde pyramidal tract damage (Thiel et al., 2010). The extent of microglia activation in the remote area is related to the amount of anterograde tract damage in the first week of ischemic stroke, while activated microglia in the infarct zone is related to anterograde tract damage 6 months after brain ischemia. Activated microglia in the infarct zone has a negative correlation with clinical outcome, but in the remote area, activated microglia has a positive correlation with clinical outcome (Thiel et al., 2010). The difference of dynamic changes of microglia activation in the infarct zone and in the peri-infarct area observed in different studies mentioned above might be due to different characteristics of patients recruited. More patients should be recruited to obtain more longitudinal data and information on the spatiotemporal evolution of microglial response (Thiel and Heiss, 2011). Whether microglia activation in the infarct zone or in the peri-infarct region is beneficial or detrimental for the recovery of ischemic stroke is controversial (Lai and Todd, 2006). On one hand, activated microglia in the infarct zone could exacerbate delayed neuronal death via producing toxic substances. On the other hand, activated microglia contribute to neuronal regeneration via producing growth factors such as BDNF (Madinier et al., 2009). Microglia could also protect the brain via removing debris from the infarct

zone (Stoll et al., 1998; Thiel et al., 2010). These findings suggested that activated microglia in the extended period after ischemic stroke might be an intervention target to limit late neuronal damage and improve outcome (Price et al., 2006; Thiel and Heiss, 2011). However, the exact role of microglia activation in the infarct zone and in the peri-infarct area in stroke recovery needs to be further studied.

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3.2.2. Microglia activation in experimental ischemic stroke

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3.2.2.1. Diversity of microglia morphology in the brain ischemic region. Microglia activation has been extensively studied in animal models of ischemic stroke. Microglia activation appears as an early, sensitive, and reliable signal for neuronal damage (Gehrmann et al., 1992). During the different courses of reperfusion after brain ischemia (from 1 to 14 days), the number and maximum length of microglia processes change in multiple ways (Korematsu et al., 1994; Korzhevskii et al., 2012; Morrison and Filosa, 2013). The diversity of activated microglia morphology is correlated to microglia function and could reflect the severity of ischemia in certain circumstances. For example, in a mouse model of focal cerebral ischemia, a study observed morphological changes of microglia during the course of brain ischemia. Classical resting microglia with fine ramified processes were found in the normal cerebral cortex. At 1 to 3 days after ischemia, ‘‘stellate’’ microglia with intense and shortened processes were mainly found in the peri-infarct region. At 6 days after MCAO, ‘‘amoeboid’’ microglia with a feature of large and round cell body but no processes predominated the region close to the infarct zone (Schroeter et al., 1997). Consistent with these observations, another study showed that ramified microglia and ‘‘amoeboid’’ microglia were found surrounding intact and dying neurons after ischemic stroke, respectively (Perego et al., 2011; Zhang et al., 1997).

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3.2.2.2. Activation periods of brain resident microglia and blood immune cells. After ischemic brain injury, neuro-inflammation rapidly increases and contributes to irreversible neuronal damage (Tobin et al., 2014). Both brain resident microglia and blood immune cells are involved in mediating post-stroke neuroinflammation (Aktas et al., 2007). However, there are different activated periods of brain resident microglia and blood immune cells after brain ischemia. Studies demonstrated that microglia are the dominating cell type in post-stroke neuro-inflammation in the first week after ischemic stroke (Schilling et al., 2003; Schroeter et al., 1997; Thiel and Heiss, 2011). Using a mouse model of focal cerebral ischemia induced by photo thrombosis, a study showed that brain resident microglia played a major role in phagocytosis within three days, while bone marrow derived macrophages participated in the removal of necrotic tissue at 6 days after cerebral ischemia (Schroeter et al., 1997). Using a bone marrow transplantation approach to distinguish brain resident microglia from bone marrow derived macrophages, a study revealed that brain resident microglia, defined as F4/80+ and GFP, could be activated and proliferate as early as 24 h after ischemic attack. Microglia activation peaks at 4 days and then gradually decreases to baseline level until 28 days. Whereas F4/80+ and GFP+ macrophages begin to invade into the brain starting at 4 days and peaked at 7 days. The majority of macrophages in the infarct zone are GFP, suggesting that brain resident microglia carry out predominant defense function during brain ischemia other than blood derived macrophages (Schilling et al., 2003). Microglia and macrophages can be distinguished from each other based on the different expression levels of CD45 using flow cytometry (Campanella et al., 2002; Sedgwick et al., 1998). Cells identified as CD45low CD11b+, CD45intermediate CD11b+, or CD45high CD11b+ represents resting microglia, activated microglia, or macrophages,

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respectively (Gelderblom et al., 2009). Both microglia and macrophages are present in the ischemic hemisphere 3 days after MCAO (Gelderblom et al., 2009). However, another study using similar method showed different results, in which both microglia (CD45dim/CD11b+) and macrophages (CD45high/CD11b+/CD11c) persistently increase at 7, 14, and 28 days after MCAO (Stubbe et al., 2013). The different results regarding the peak time of microglia activation and infiltration of blood immune cells could be due to differences in detection methods, ischemia time, and experimental models. For example, permanent cerebral ischemia causes more pronounced microglia activation at 1 and 5 days after ischemia than transient focal cerebral ischemia. Moreover, blood immune cells infiltration occur early in permanent cerebral ischemia (Chu et al., 2014; Zhou et al., 2013). It is essential to develop novel and sensitive detection methods to explore the role of microglia activation in comparable models.

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3.2.3. Receptors mediating microglia activation and function As mentioned above, different pathological conditions could affect microglia activation. The question is what controls and mediates microglia activation? In the past few years, scientists found that many surface receptors are involved in mediating microglia activation and its functions including inflammation, migration, phagocytosis, motility, and survival during ischemic stroke. Toll-like receptors (TLRs) and purinergic receptors are two widely studied receptors essential for microglia-mediated inflammation. Stimulation of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) activated microglia, mediated pro-inflammatory cytokine production, and caused brain damage after focal cerebral ischemia (Caso et al., 2007; Kilic et al., 2008; Lehnardt et al., 2003, 2007; Rosenberger et al., 2014; Yenari et al., 2010). Deficiency of TLR2 or TLR4 reduced TNF-a, iNOS, and COX2 production, contributing to reduced brain infarct volume and improved neurological outcome (Pradillo et al., 2009; Tang et al., 2007). Recent study indicated that inhibition of TLR2 and TLR4 could be an effective method to reduce brain damage within 6 h after ischemic stroke (Wang et al., 2014). IL-17, which is mainly secreted by Th17 cells, gd T cells, and innate lymphoid cells group 3, is a major contributor to the delayed inflammatory response of ischemia stroke (Roy et al., 2005). Microglia could secrete IL-17 upon activation of TLR2/IL-23/IL-17 signal pathway after brain ischemia (Lv et al., 2011). Microglia upregulate IL-17 specific receptor IL-17RA when TLR is activated and produce more IL-17 in an autocrine manner, contributing to neuronal damage (Kawanokuchi et al., 2008; Liu et al., 2014a; Sonobe et al., 2008; Wang et al., 2009). Blocking IL-17 reduced brain infarct volume and neurological deficiency (Gelderblom et al., 2012), possibly due to the fact that IL-17 can impair neural stem cells (NSCs) proliferation and differentiation (Li et al., 2013). Therefore, TLR2/IL-23/IL-17 pathway could be a potential target for dampening delayed inflammatory response and improving neurological outcome after brain ischemia. Purinergic receptors are constituted of P1 adenosine receptors and P2 ATP receptors (Hu et al., 2014). Among P1 adenosine receptors, A2A receptor is up-regulated in microglia after focal cerebral ischemia and is involved in controlling microglia proliferation and BDNF release induced by LPS stimulation (Gomes et al., 2013; Trincavelli et al., 2008). A2A receptor antagonists or genetic depletion of A2A receptor attenuated ischemia induced brain damage (Rivera-Oliver and Diaz-Rios, 2014; Yang et al., 2013). P2X receptors are composed of seven distinct subunit subtypes (P2X1 to P2X7) (Abbracchio et al., 2009; Burnstock, 2008). P2X4 and P2X7 receptors are predominantly expressed on microglia and mediate microglia activation after cerebral ischemia, hypoxia or hypoxia-ischemia (Burnstock, 2007; Cavaliere et al., 2003; de Rivero Vaccari et al., 2012; Del Puerto

5

et al., 2013; Li et al., 2011; Wixey et al., 2009). P2X7 receptor activation is responsible for inducing microglia to release proinflammatory cytokines such as TNF-a, IL-1b, NO, CXCL2, and CCL (Gendron et al., 2003; Hide et al., 2000; Kataoka et al., 2009; Shiratori et al., 2010). Blocking P2X7 receptor reduced proinflammatory cytokines IL-1b, TNF-a and IL-6 release, neuronal death, and neurological deficit after global cerebral ischemia (Chu et al., 2012). P2X7 receptor antagonist treatment reduced microglia death under Oxygen–Glucose Deprivation (OGD) (Eyo et al., 2013). In contrast, the exact role of P2X4 receptor in cerebral ischemia is less well studied. P2Y12 is another type of purinergic receptors expressed on microglia. P2Y12 is down-regulated after microglia activation. Deficiency of P2Y12 in mice impaired microglia migration, polarization, and its ability to extend their processes toward the lesion site, where nucleotides are released by damaged brain tissue (Haynes et al., 2006). Recently, P2Y12 was found to mediate microglia neurotoxicity. P2Y12 knockout reduced microglia accumulation in the peri-infarct region and reduced neuronal death after cerebral ischemia in mice (Webster et al., 2013). Collectively, these studies suggested that TLRs and the purinergic receptors could be potential targets for controlling microglia activation and reducing post-stroke inflammation mediated by microglia. The chemokine receptor CCR2 is highly expressed in nearly all immune cells and inflammatory monocytes (Ajami et al., 2011). However, CCR2 is expressed at a low level in the brain microglia under normal condition (Hu et al., 2014; Savarin-Vuaillat and Ransohoff, 2007). After cerebral ischemia, CCR2 and its ligand monocyte chemoattractant protein-1 (MCP-1) are up-regulated in microglia (Inose et al., 2014). Activation of CCR2 is responsible for enhancing cerebral inflammation and brain infarct volume (Dimitrijevic et al., 2007). In a model of hypoxia in neonatal rats, administration of MCP-1 induced microglia migration from adjacent area toward the injection site in peri-ventricular white matter (Deng et al., 2009). However, absence of CCR2 in mice mainly reduced the recruitment of blood immune cells but did not affect brain resident microglia activation after transient MCAO (Schilling et al., 2009). Thus, CCR2 seems to be critical for blood immune cells recruitment but not essential for microglia activation after focal brain ischemia. Triggering receptor expressed on myeloid cells-2 (TREM-2), which is a member of triggering receptors expressed on myeloid cells (TREM) family, is expressed on microglia (Schmid et al., 2002). TREM-2 activation promotes microglia migration and phagocytosis of apoptotic neurons without inducing inflammatory cytokines expression (Takahashi et al., 2005). After cerebral ischemia, TREM-2 is up-regulated in microglia (Heldmann et al., 2011; Sugimoto et al., 2014). However, up to now, the role of TREM-2 in cerebral ischemia is largely unknown. Fc receptors (FcRs) are also involved in regulating microglia phagocytosis. In a mouse model of Alzheimer’s disease (AD), microglia were found capable of phagocytose extracellular amyloid plaques via FcRs (Bard et al., 2000). In addition, FcRs expressed on microglia also mediate inflammatory cascade and produce superoxide and NO (Le et al., 2001; Ravetch, 1994; Sylvestre and Ravetch, 1994). FcRs knockout in a transient MCAO model inhibited microglia activation and iNOS production, contributing to reduced infarct volume and mouse mortality at 3 days after MCAO (Komine-Kobayashi et al., 2004). This study suggested that interventions targeting FcRs in the acute phase of brain ischemia might be beneficial for attenuating ischemia induced brain injury. However, whether FcRs knockout exerts beneficial function during the recovery phase of brain ischemia is still unknown and needs further study. The receptor for advanced glycation end products (RAGE) is another receptor which mediates microglia activation and inflammatory response in many diseases (Fang et al., 2010; Lue

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et al., 2001; Ramasamy et al., 2009; Yamagishi and Matsui, 2010; Zhai et al., 2008). In ischemic stroke patients, RAGE is up-regulated in the brain and plasma (Menini et al., 2014; Zhai et al., 2008). The interaction between RAGE and its ligand high mobility group box1 (HMGB1) is involved in exacerbating ischemia induced brain damage, since anti-HMGB1 or RAGE depletion treatment reduced brain infarct volume (Muhammad et al., 2008). In vitro study showed that HMGB1-RAGE interaction is essential for microglia activation induced neuronal death (Muhammad et al., 2008). Recent studies indicated that cysteinyl leukotriene receptor 2 is also involved in microglia mediated inflammatory response and neuronal death after brain ischemia. Selective inhibition of cysteinyl leukotriene receptor 2 using HAMI 3379 via intraventricular injection before ischemia reduced microglia activation and brain injury within 3 days after MCAO (Shi et al., 2012, 2015; Zhao et al., 2011). Thus, HAMI 3379 treatment could be a protective strategy for attenuating microglia mediated inflammatory response at the acute and subacute phase of ischemic stroke. Both Galectin-3 and CD36 belong to pattern recognition receptors (PRR) expressed on microglia. Galectin-3 is required for microglia proliferation, while CD36 is involved in phagocytosis of dying neuronal cells and mediates inflammatory response. Lack of Galectin-3 reduced microglia proliferation, which is associated with enhanced infarct volume and neuronal apoptosis after MCAO (Lalancette-Hebert et al., 2012). Similarly, lack of CD36 in mice caused poorer neurological outcome at 1 day after MCAO, as a result of exacerbated inflammatory response and reduced removal of neuronal debris (Woo et al., 2012). These studies suggested that Galectin-3 and CD36 play a positive role in acute stroke. Sema4D, another molecule found to mediate microglia activation via interacting with its receptors Plexin B1 and CD72 expressed on microglia (Okuno et al., 2010). Sema4D is up-regulated in the periinfarct region of the ischemic brain (Taniguchi et al., 2009). Sema4D deficiency caused an increase in the number of oligodendrocytes in healthy and injured mouse brain. In Sema4D/ mice, the number of amoeboid microglia and iNOS production in the peri-infarct area of the cortex were both reduced after permanent cerebral ischemia, accompanied by better neurological outcome (Sawano et al., 2015). These studies demonstrated that a large number of receptors expressed on microglia contribute to regulating microglia activation under ischemic environment. Many studies investigated the potential of targeting these receptors to treat neurological diseases (Binder et al., 2011; Clark et al., 2007; Tsiperson et al., 2010; Yenari et al., 2010). However, whether treatment strategies targeting these receptors could be developed into effective treatments for brain ischemic disease awaits further study because other cell expressing the identical receptors would also be affected. Furthermore, when and how long should these receptors be blocked or activated also need to be considered. Inhibition of microglia activation for a long term might cause dysfunction of innate immune response in the brain (Hu et al., 2014). Further understanding of the activity of receptors expressed on microglia would facilitate the development of novel and effective therapeutic strategies for the treatment of ischemic stroke.

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4. Roles of activated microglia in ischemic stroke (Table 1)

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4.1. Microglia and blood brain barrier permeability

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Blood brain barrier (BBB) disruption plays a critical role in the pathogenesis of ischemic stroke (da Fonseca et al., 2014; Yang and Rosenberg, 2011). BBB disruption leads to cerebral vasogenic edema, hemorrhagic transformation, leukocytes infiltration, and the leakage of toxic molecules into the brain. Early phase BBB disruption occurs at 12–24 h after ischemia, and the delayed

secondary phase occurs at 48–72 h. During this period, microglia are activated and affect BBB disruption (da Fonseca et al., 2014). A recent study dynamically observed microglia activation within 72 h after MCAO using time lapse two-photon imaging (Jolivel et al., 2015). Microglia in the penumbra rapidly expanded cellular protrusions toward blood vessels, with morphological changed over time. Increased number of microglia was accompanied by decreased blood vessels. Immunostaining results showed that Iba1+ microglia colocalize with CD31+/Gul+/caveolin-1+/claudin5+/podocalyxin+ blood vessels, indicating that activated microglia phagocytosed endothelial cells. This phenomenon is associated with BBB disruption, confirmed by high levels of IgG and Evans blue extravasation, and high matrix metalloproteinase-9 expression. CX3CR1 knockout mice, in which microglia function is impaired, were used to confirm the role of microglia in BBB disruption after focal cerebral ischemia. CX3CR1 knockout mice displayed a reduced infarct size and contrast agent extravasation (observed by MRI). Inhibition of microglia activation by minocycline treatment after MCAO reduced infarct volume, hemorrhagic transformation, and BBB permeability, partly via preventing the degradation of collagen IV and laminin (Machado et al., 2009; Tikka et al., 2001; Yenari et al., 2006). Moreover, inhibiting microglia activation increased blood vessel integrity (Jolivel et al., 2015). Collectively, these studies suggested that microglia activation could induce BBB disruption after brain ischemia through disintegration of blood vessels in the penumbra. Tables 1 and 2. Several mechanisms and molecules are associated with microglia mediated BBB disruption. Oxidative stress, generally caused by excess production of reactive oxygen species (ROS), is considered a key contributor to the early phase of BBB disruption (Obermeier et al., 2013). Microglia can be activated by ROS and subsequently produce more ROS. The high amount of ROS could in turn amplify microglia activation, injure endothelial cells, and exacerbate BBB disruption (Kacimi et al., 2011). Hypoxia-inducible factor-1 and NF-kB are up-regulated in microglia after cerebral ischemia. The up-regulation of Hypoxia-inducible factor-1 and NFkB could stimulate microglia to produce matrix metalloproteinases, which are key mediators in the delayed secondary phase of BBB disruption (da Fonseca et al., 2014). Pro-inflammatory cytokines such as IL-1b, IL-6, and TNF-a, are released by microglia and up-regulate after ischemic stroke (Lee et al., 2014; Zhou et al., 2013). Recently, our laboratory showed that microglia activation could up-regulate aquaporin-4 expression in astrocytes by releasing IL-1b, IL-6, and TNF-a, causing enhanced astrocyte apoptosis and BBB disruption. P38 signaling pathway was found to be involved in microglia mediated BBB disruption. P38 inhibitor treatment immediately after ischemic reperfusion reduced aquaporin-4 expression in the mouse brain. MSCs transplantation inhibits the microglia mediated aquaporin-4 up-regulation and astrocyte apoptosis, thus contributing to maintaining the integrity of BBB (Tang et al., 2014). Up to now, there are only limited studies focusing on the interaction between microglia activation and BBB disruption. Some studies showed that blood protein fibrinogen could deposit in the area of activated microglia after BBB disruption and cause microglia activation through interacting with Mac-1 on microglia (Adams et al., 2007; Gay et al., 1997), Thus, BBB disruption could cause microglia activation in reverse. However, microglia possibly play a protective role on attenuating BBB disruption via secreting progranulin, which is involved in reducing BBB disruption and brain edema after ischemic stroke (Egashira et al., 2013; Jackman et al., 2013; Kanazawa et al., 2015). Therefore, the causal relationship between microglia activation and BBB disruption is still elusive. The effect of microglia activation on BBB disruption remains to be further explored.

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Model

Methods or treatment

Injection time

marker

Observe location

Observe time

outcome

Beneficial/ detrimental

Reference

Rats PGRN/ mice

tMCAO 1.5 h

100 mg of PGRN was injected via inguinal vein

4 h after MCAO

Iba1

Peri-infarct zone

4,10,18,24,72 h

Beneficial

Kanazawa et al. (2015)

Rats

tMCAO 2 h

4 h after MCAO

Iba1

Peri-infarct zone of cortex

0.5,1,3,7 d

Detrimental

van Minnen et al.

Sema4D/ mice

pMCAO

Ginsenoside Rd treatment via intralventrical injection Depletion of Sema4D in mice

Detrimental

Sawano et al. (2015)

Rats

tMCAO 1 h

CYSLT2R antagonist administraion

1, 3 d before MCAO Or 0.5 h before MCAO

ED1 Iba1

Ischemic core peri-infart zone in the cortex

1,3 d

Detrimental

Haan et al. (2015)

Mice

20 min BCCAO

Minocycline treatment via intralventrical injection

Immediately after reperfusion

OX-42

Dentate gyrus

1,3,7,14,21,28 d

PGRN knock out do not affect the number of microglia, but reduce brain edema Reduced microglia is accompanied by enhanced neurological function Reduce iNOS production and neurological function deficency Reduce microglia activation, neurological deficit, and infarct volume Reduce neurogenesis

Beneficial

Choi et al. (2015)

18 month-old rats

tMCAO 50 min

ED1 Iba1

peri-infarct area

3,14&28 d

Detrimental

Titova et al. (2014)

Postnatal rats

Normal

Beneficial

ShigemotoMogami et al. (2014)

Mice

tMCAO 1.5 h

Detrimental

Li et al. (2014)

MKP-1 knockout mice

tMCAO 1 h

Beneficial

Li et al. (2014)

Rats CX3CR1-GFP mice Nrf2/ mice and LysMCreHmox 1D/D mice Galectin-3 knockout mice

pMCAO

Detrimental

Gelosa et al. (2014) Parada et al. (2013)

Q11

Photothrombotic stroke

Peri-infarct zone of cortex

Minocycline (30 mg/ kg) treatment via intraperitoneal injection Adjudin (50 mg/kg) treatment via intraperitoneal injection Depletion of MKP-1 in microglia

Begin on postnatal day 2 (P2).for 3 d

CD11b CD68

SVZ

P1,P4,P10,P14,P30

Immediately,5&24 h after reperfusion

CD11b

Cortex striatum

3d

Ticagrelor 3 mg/kg treatment per os a7 nAChR agonist PNU282987 treatment

10 min, 22&36 h after MCAO 1h postphotothrombosis

tMCAO 1 h

Depletion of Galectin3

Rats

Endothelin-1 induced cerebral ischemia

Seven-day-old rats

tMCAO 1.5 h

BMMCs transplantation or compared with minocycline via caudal vein, CB2R and CB1R agonist WIN 55,212-2 (WIN) treatment via subcutaneous injection

Iba1

ED1/Iba1

3d

Peri-infarct area infarct core

Iba1

2, 24, 48 & 72 h 24 h

Reduced microglia is accompanied by enhanced neovascularization Microglia inhibition reduce neurogenesis and oligodendrogenesis Reduced microglia activation is associated with reduced BBB disruption Depletion of MKP-1 in microglia enhance infarct size and neurological deficit Enhance neurological deficit Reduce infarct size and improve motor skills

Beneficial

Mac-2 Iba1

Infarct region

1&3 d

Reduce infarct size and improve motor skills

Beneficial

5  106 cells, 24 h postischemia

ED1

Cortex

7,14&21 d

Reduce infarct size and improve functional recovery

Detrimental

Immediately,4 h after reperfusion twice daily

Iba1

Cortex

24&72 h

Reduce infarct size

Detrimental

LalancetteHebert et al. (2012) Franco et al. (2012)

FernandezLopez et al. (2012)

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Animal

Y. Ma et al. / Progress in Neurobiology xxx (2016) xxx–xxx

Please cite this article in press as: Ma, Y., et al., The biphasic function of microglia in ischemic stroke. Prog. Neurobiol. (2016), http:// dx.doi.org/10.1016/j.pneurobio.2016.01.005

Table 1 Biphasic function of microglia in different models and periods of ischemic stroke.

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Methods or treatment

Injection time

marker

Observe location

Observe time

outcome

Beneficial/ detrimental

Reference

Seven-day-old rats

tMCAO 3 h

2 d before MCAO

Iba1 IB4 ED1

Ischemic core penumbra

8,24&72 h

Enhance infarct size

Benificial

Faustino et al. (2011)

Rats mice

tMCAO 2 h tMCAO 0.5 h

Immediately after reperfusion; immediately& 1 week later

Mac-1 Iba1 ED1 TREM-2

SVZ striatum

7,14&28 d

Do not affect neurogenesis

No effect

Heldmann et al. (2011)

Hv1 knockout mice CX3CR1-GFP mice Rats

pMCAO tMCAO 2 h

Peri-infarct area

24&72 h

Reduce infarct size and neurological scores

Detrimental

Lozano et al. (2012)

tMCAO 1.5 h

3,5,7&14 d

Reduce infarct size and apoptotic cell

Beneficial

Narantuya et al. (2010a,b)

Rats

tMCAO 2 h

Rats

tMCAO 1.5 h

Rats

Endothelia-1 induced cerebral ischemia tMCAO 1 h

Depletion of microglia via intracerebral injection of liposomeencapsulated clodronate Mac-1-saporin treatment via inntraventricular injection Depletion of voltagegated proton channel Hv1 Microglia transplantation via jugular vein injection Minocycline (90 mg/ kg) treatment via intraperitoneal injection P2X7 receptor agonist BzATP or antagonist OxATP treatment via inntraventricular injection Two-photon microscopy imaging Depletion of CX3CR1

Q12

CX3CR1-GFP CX3CR1 knockout mice Mice

Mongolian gerbil

tMCAO 0.5 h tMCAO 1 h 5 min BCCAO

CD11b-TKmt-30 mice

tMCAO 2 h

Rats

10 min BCCAO

Mice

tMCAO 2 h

Chimeric mice

tMCAO 0.5 h

Rats

tMCAO 2 h

Rats

tMCAO 2 h

3  106 cells,48 h after stroke

ED1

Immediately after reperfusion

Ox6

SVZ peri-infarct zone

1,4&7 d

Reduce neurogenesis

Beneficial

Kim et al. (2009)

0.5 h before reperfusion

CD11b

Cortex striatum

1&3 d

Reduce neurological deficiency

Beneficial

Yanagisawa et al. (2008)

24&48 h after OGD

Reduce neuronal death

Beneficial

Reduce infarct size and apoptotic cells, accelerate recovery Reduced microglia is accompanied by smaller infarct size Increase neuronal survival

Detrimental

Neumann et al. (2008) Denes et al. (2008)

IB4

Magnetic Resonance Imaging

Iba1

Cortex striatum hippocampus

4,24,&72 h

Iba1 lectin

Cortex, striatum

4,24,48&72 h

IB4

Hippocampus

7d

Microglia transplantation via subclavian artery Ganciclovir treatment via intraperitoneal, injection

1  106 cells 24 h before,24 h or 24 h after ischemia, 48 h before&24 to 72 h after stroke

Mac-2 Iba1

Microglia transplantation via subclavian artery injection 45 mg/kg minocycline treatment via intraperitoneal injection

2  106 cells, 4 to 7 days before ischemia

Iba1 Ox42/ CD11b

Hippocampus

2&4 d

0.5&12 h after reperfusion

IB4

Peri-infarct area

24 h

Iba1

Cortex striatum

1,2,4,7,10&14 d

3 d before MCAO, daily

CD11b ED-1

Cortex, striatum

7,14&28 d

5  104 cells, 1 h after reperfusion

Iba1 CD11b

Striatum substantia nigra

8d

Indomethacin (2.5 mg/ kg) treatment via intraperitoneal injection Microglia transplantation via introcerebroventrical injection

3d

Beneficial

Denes et al. (2007)

Beneficial

Imai et al. (2007)

Microglia depletion enhance infarct size and neuronal apoptosis Reduce neuronal death and protect synapse transmission

Beneficial

LalancetteHebert et al. (2007)

Beneficial

Hayashi et al. (2006)

Reduce infarct size, BBB disruption, and neurological deficiency Phagocytosis of neuronal cell debris Enhance neurogenesis

Detrimental

Yenari et al. (2006)

Beneficial

Schilling et al. (2005) Hoehn et al. (2005)

Improve motor function recovery

Beneficial

Detrimental

Kitamura et al. (2005)

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Model

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Animal

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Please cite this article in press as: Ma, Y., et al., The biphasic function of microglia in ischemic stroke. Prog. Neurobiol. (2016), http:// dx.doi.org/10.1016/j.pneurobio.2016.01.005

Table 1 (Continued )

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Abbreviations: a7 nAChR, alpha-7 nicotinic acetylcholine receptor; BCCAO, bilateral common carotid artery occlusion; BMNCs, Bone marrow mononuclear cells; BzATP, 20 -30 -O-(4-benzoylbenzoyl)adenosine 50 -triphosphate; CYSLT2R, cysteinyl leukotriene receptor 2; OGD, Oxygen-Glucose Deprivation; OxATP, adenosine 50 -triphosphate-20 ,30 -dialdehyde; PGRN, progranulin; SVZ, sub ventricular zone; tMACO, transient middle cerebral artery occlusion.

Monje et al. (2003) Detrimental 2 months ED1

Dentate gyrus

Microglia activation is accompanied by reduced neurogenesis

Kitamura et al. (2004) Enhance neural survival 3d Infarct core border zone CD11b 4  103 or 4  104 cells, 1 h after reperfusion Microglia transplantation via introcerebroventrical injection

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tMCAO 1 h

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

Rats

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Beneficial

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4.2. The effect of microglia on neurogenesis 4.2.1. Microglia influence neurogenesis under physiological condition Neurogenesis exists in the adult brain, both in human and animals (Ernst et al., 2014; Shors et al., 2001; Tobin et al., 2014). Neurogenesis increases in rodents and primate brain after ischemic stroke (Arvidsson et al., 2002; Jin et al., 2001; Tonchev, 2011; Tsai et al., 2011; Tureyen et al., 2004). Microglia are located in the neurogenic niche and have a complex link with neurogenesis in both healthy and injured brain (Ekdahl, 2012; ShigemotoMogami et al., 2014). Microglia are involved in excise induced neurogenesis in adult mice. The number of brain microglia increased in aged mice and removal of microglia reduced neural progenitor cells (NPCs) activity (Adachi et al., 2010). In postnatal mice, minocycline treatment inhibited microglia activation and reduced neurogenesis in the sub ventricular zone (SVZ) (Shigemoto-Mogami et al., 2014; Vukovic et al., 2012). It is noted that microglia maintain the homeostasis of baseline neurogenic cascade via phagocytosing the new born cells in the sub granular zone of the dentate gyrus in young normal brain. In aged or LPSstimulated rat brain, inhibiting microglia enhanced the number of new born cells and promoted neurogenesis in the sub granular zone (Sierra et al., 2010). These findings indicated that microglia exert different functions on neurogenesis under different physiological conditions. 4.2.2. Microglia influence neurogenesis during ischemic stroke Under pathological conditions including ischemic stroke, microglia exhibit biphasic functions in neurogenesis (Ekdahl et al., 2009). Minocycline treatment for 4 weeks reduced microglia activation, enhanced neurogenesis in dentate gyrus, and improved neurological function after MCAO (Liu et al., 2007). Indomethacin treatment before focal brain ischemia enhanced neurogenesis, which is associated with the inhibition of microglia activation by indomethacin (Hoehn et al., 2005). These findings suggested that microglia activation play a detrimental role in neurogenesis. However, in a rat model of transient cerebral ischemia, minocycline treatment reduced the number of DCX+/BrdU+ cells in the SVZ at 4 days and NeuN+/BrdU+ cells at 7 days after ischemia, though minocycline treatment inhibits microglia activation (Kim et al., 2009). Another study found that microglia accumulating in the peri-infarct striatum peaked at 2 weeks and maintained to 16 weeks after MCAO. IGF-1 mRNA was up-regulated in the SVZ and interestingly only microglia expressed IGF-1 protein in the SVZ. These phenomenon were accompanied by enhanced neurogenesis in the SVZ at 2, 6, and 16 weeks after ischemia, suggesting that microglia are supportive of neurogenesis after ischemic brain injury (Thored et al., 2009). In contrast, a later study showed that microglia depletion did not affect neurogenesis in the SVZ and striatum at 2 weeks after MCAO. DCX+/BrdU+ neuroblasts were examined after the removal of Mac-1+ microglia via Mac-1-saporin intraventricular injection. There was no difference in the number of DCX+/BrdU+ neuroblasts in the SVZ and striatum between Mac1-saporin treated group and control group (Heldmann et al., 2011). These controversial results could be due to the different observation time of neurogenesis, since the later study only focused on two weeks after MCAO, while the former observed up to 16 weeks. A study with longer observation time with multiple time points is needed to clarify the effect of microglia on neurogenesis after ischemic stroke. In addition, different stimulation methods of microglia activation may contribute to these opposite effects on neurogenesis. For example, microglia activation induced by bacterial endotoxin, lipopolysaccharide (LPS), or cranial irradiation all cause severe inflammatory response and inhibit neurogenesis in hippocampus or striatum (Ekdahl et al., 2003). While IL-4 or IFN-g induced microglia activation support neurogenesis or oligoden-

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Table 2 miRNAs associated with microglia activation in different pathological conditions. miRNA

Target

Function

Treatment

Model

Reference

miR-155

SOCS-1

Butovsky et al. (2015)

–

Knock out of miR-155 in mice Ab stimulation

ALS

miR-155

AD

Guedes et al. (2014)

miR-155

c-Maf

Knock out of miR-155 in mice

MCAO

Bsibsi et al. (2014)

miR-155

–

LPS stimulation

Primary microglia

Freilich et al. (2013)

miR-155

SOCS-1

LPS stimulation

N9 cells

Cardoso et al. (2012)

miR-124

–

miR-124 deficiency

zebrafish

Svahn et al. (2015)

miR-Let7A

–

Knock out of miR-Let7A

BV2 cells

Choi et al. (2015)

miR-Let7c-5p

caspase 3

miR-Let7c-5p overexpression

MCAO

miR-204

SIRT1

Inhibition of miR-204 under LPS stimulation

N9 and BV2 cells

Albiero et al. (2015)

miR-203

MyD88

Promote microglia activation Promote IL-6 and IFN-b production Promote proinflammatory cytokines production Mediate proinflammatory pathways Promote iNOS and proinflammatory cytokines production Reduce motility and phagocytosis capacity of microglia Modulate autophagy activity Inhibit microglia activation and reduce brain injury Promote inflammatory response and proliferation of microglia Attenuate brain injury

MCAO

Haan et al. (2015)

miR-Let-7a

-

miR-181c

TLR4

miR-124

p65 TRAF6

inhibit iNOS and IL-6 expression, enhance BDNF expression reduce TNF-a, IL-1b, and iNOS production reduce TNF-a, IL-1b, and IL-6 production

Overexpression of miR203 Overexpression of Let-7a under LPS stimulation

miR-99a, 125-5p, and 342-3p miR-9

–

–

–

Human microglia

Qiu et al. (2015) See comment in PubMed Commons below Butovsky et al. (2014)

MCPIP1

Overexpression of miR-9

Primary microglia

Li et al. (2014)

miR-17

Jadhav et al. (2014b)

Traumatic brain injury

Jadhav et al. (2014a)

miR-124

–

HIV-1 Tat C protein treatment Overexpression of miR200b IL-4 stimulation

HMC3

miR-200b

NOX2 and NOX4 c-Jun

Mediate inflammatory response Reduce ROS production

Primary microglia

Freilich et al. (2013)

miR-29b

TNFAIP3

Japanese encephalitis virus treatment

Primary microglia And BV2 cells

Thounaojam et al. (2014)

miR-29a,b

–

Aged humans microglia and primary microglia MCAO

Fenn et al. (2013)

miR-424

IGF-1 and CX3CL1 –

miR-181c

TNF-a

miR-21

FasL

miR-Let7f

IGF-1

miR-146a

–

Reduce microglia activation, Reduce brain infarct volume –

miR-124

–

Reduce microglia activity

miR-146a

MCP-2

Reduce MCP-2 expression

Reduce iNOS and NO production Mediate antiinflammatory pathways Inhibit iNOS, COX-2, and pro-inflammatory cytokine production Inhibit microglia activation Reduce microglia activation, brain infarct volume, and neuronal apoptosis Reduce TNF-a production

OGD Morphine treatment

Overexpression of miR424

OGD OGD Anti-miR-Let7f treatment Ab42 peptide and TNF-a stimulation Knock out or overexpression of miR124 HIV infection

BV2 cells

Primary microglia and BV2 cells Primary microglia and BV2 cells

van Minnen et al.

Zhao et al. (2013)

Primary microglia and BV2 cells Primary microglia and BV2 cells MCAO

Zhang et al. (2012a)

Selvamani et al. (2012)

HMG

Ajami et al. (2011)

EAE

Ponomarev et al. (2011)

Primary human fetal microglia

Felger et al. (2010)

Zhang et al. (2012b)

Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; COX-2, cyclooxygenase-2; EAE, Experimental Autoimmune Encephalomyelitis; HMC3, human microglia clone 3 cell lines; HMG, Human microglial cells; FasL, Fas ligand; MCP-2, monocyte chemoattractant protein 2; MCAO, middle cerebral artery occlusion; MCPIP1, monocyte chemotactic protein-induced protein 1; OGD, Oxygen-Glucose Deprivation; SIRT1, Sirtuin1; SOCS-1, suppressor of cytokine signaling 1; TLR4, Toll-like receptor 4; TNFAIP3, tumor necrosis factor alpha-induced protein 3; TRAF6, TNFR-associated factor 6.

668 669 670 671

drogenesis from adult NPCs. Co-culturing NPCs with microglia stimulated with IL-4 or IFN-g promoted NPCs differentiation into Tuj1+ neurons and NG2+ oligodendrocytes. It was suggested that IL-4 stimulated microglia promoted NPCs differentiation into

oligodendrocytes partly via secreting IGF-1, because anti-IGF-1 treatment effectively blocked NPCs differentiation (Butovsky et al., 2006). These studies indicated that different activating stages of microglia may have different effects on neurogenesis.

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In conclusion, it is increasingly apparent that microglia activation plays a biphasic role in neurogenesis. Elucidating when and how microglia influence neurogenesis will be helpful for advancing our understanding of brain injury and repair after ischemic stroke (Tobin et al., 2014).

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4.2.3. Interaction between activated microglia and NPCs As mentioned above, microglia exert positive or negative effect on neurogenesis in vivo. A biphasic role regarding the influence of microglia on neurogenesis was also found in vitro. Conditioned medium from primary microglia or co-culturing with microglia enhanced the capacity of NPCs to generate neuroblasts and differentiate into neurons (Aarum et al., 2003; Walton et al., 2006). These studies suggested that microglia could be beneficial and supportive for NPCs differentiation via releasing neurotrophic factors. Another study further supported the paracrine function of microglia on NPCs. Depletion of microglia from cultured hippocampal slice reduced NPCs survival and proliferation while cocultured with microglia or conditioned medium from microglia reversed the phenomenon. In addition, the study found that microglia directly targeted NPCs via releasing IL-4 (Nunan et al., 2014). However, microglia could inhibit the proliferation of NPCs via releasing NO. NPCs co-cultured with microglia from iNOS knockout mice (iNOS/) displayed enhanced proliferation compared with that co-cultured with microglia from wild type mice (iNOS+/+) (Carreira et al., 2014). Interestingly, increasing numbers of studies indicated that microglia activation could be regulated by NPCs. Using GFAPCre/bCateninEx3 transgenic mice, in which CXCL12 expressing progenitor cells were abolished, a study found that microglia were parallelly reduced in the ventricular zone (VZ)/SVZ of developing cerebral cortex. The apoptosis of NPCs in transgenic mice controlled by suicide gene Thymidine Kinase (TK) and Ganciclovir (GCV) treatment induced microglia migration into the VZ/SVZ and exerted phagocytosis function. Moreover, using transgenic mice lacking CSF-1R to deplete microglia inhibited NPCs migration into the cerebral cortex, indicating that NPCs activity could drive microglia migration into the VZ/SVZ in the developing brain (Arno et al., 2014). In turn, microglia are essential for NPCs development. Another study explored the regulation effect of NPCs on microglia activation (Mosher et al., 2012). Microglia and NPCs displayed a close correlation in their positions in the brain. The number of microglia in the SVZ and dentate gyrus, which are origins of NPCs (Kokaia et al., 2012), were higher than that in the cerebral cortex in mice, suggesting a functional relationship between microglia and NPCs. Co-culturing primary microglia or BV2 cells with NPCs enhanced microglia proliferation, migration, and phagocytosis. Further experiments showed that NPCs regulate microglia activation via releasing VEGF. Intrastraital injection of conditioned medium from NPCs or VEGF alone promoted microglia proliferation, which could be blocked by knocking down VEGF in NPCs via lentiviral shRNA. These studies suggested that microglia and NPCs cross-regulate each other, but the regulation mechanisms need to be further explored.

729

5. Microglia polarization during ischemic stroke

730

5.1. Microglia polarization

731 732 733 734 735 736 737

Macrophages could adopt different phenotypes depending on the stimulus, the period, and the environment (Biswas and Mantovani, 2010; Gordon and Taylor, 2005). The process is called polarization. Similar to macrophages, microglia could also polarize to many phenotypes, which was demonstrated in vitro and in vivo (Girard et al., 2013). Based on the stimulation of pro-inflammatory or anti-inflammatory cytokines, microglia could generally polarize

11

into inflammatory and anti-inflammatory phenotypes (Gordon, 2003). Inflammatory phenotype is designated as classically activated microglia and could be induced by IFN-g and LPS stimulation (Nakagawa and Chiba, 2014). Inflammatory phenotype, also known as M1 phenotype, is characterized by the production of a variety of pro-inflammatory cytokines (IL-1b, IL-6, TNF-a, CCL2, and CXCL10), ROS, NO, and proteolytic enzymes matrix metalloproteinase-9 and matrix metalloproteinase-3 (Kettenmann et al., 2011; MacMicking et al., 1997; Saijo and Glass, 2011; Varnum and Ikezu, 2012; Yenari et al., 2010). The functions of classically activated microglia are to present antigens and kill intracellular pathogens to maintain environmental homeostasis (Cherry et al., 2014). However, in vitro studies showed that LPS stimulation induced classically activated microglia (an inflammatory phenotype) exacerbated neuronal injury under OGD condition and inhibited neurogenesis in the hippocampus (Hu et al., 2012; Walter et al., 2011). Anti-inflammatory phenotype, known as M2 phenotype, is the alternatively activated microglia. Based on different stimulation and function, M2 phenotype is further divided into three sub-class including M2a, M2b, and M2c (Chhor et al., 2013; Latta et al., 2015; Sudduth et al., 2013). M2a microglia could be induced by IL-4 or IL13 stimulation and are mainly considered to suppress inflammation. M2a microglia display enhanced expression of Arginase-1, Ym1, IGF-1, CD206, chitinase 3-like 3 and Fizz1 (Latta et al., 2015; Mecha et al., 2015). Intracerebral injection of IL-4 or IL-13 in the brain enhanced Arginase-1, Ym1, CD206 expression (Girard et al., 2013), supporting that IL-4 and IL-13 could promote microglia polarization into M2a phenotype. Unlike M2a phenotype, M2b phenotype does not express Arginase-1, Ym1, and Fizz1. M2b phenotype could be induced by immune complexes and TLRs agonists, and IL-1R ligands and shows increased expression of IL1RA, CD86, and SOCS3 (Chhor et al., 2013; Mecha et al., 2015). However, the role of M2b phenotype is less understood compared to M2a phenotype. The last sub-class is M2c phenotype and could be induced by TGF-b, IL-10, and glucocorticoids. M2c phenotype is also called as ‘‘deactivated microglia’’ (Chhor et al., 2013; Mecha et al., 2015). Instead of having no function, M2c phenotype is involved in tissue regeneration when inflammatory response become weakened (Mantovani et al., 2004). Anti-inflammatory phenotype is capable of producing antiinflammatory cytokines IL-10, TGF-b, IL-4, IL-13, and IGF-1, as well as expressing scavenge receptors, contributing to inhibiting inflammation and promoting tissue repair (Cherry et al., 2014; Ponomarev et al., 2013). Generally, anti-inflammatory phenotype is considered as having a protective function. A study showed that treatment with M2 conditioned media enhanced neuronal survival and process extension after spinal cord injury (Kigerl et al., 2009). Conditioned medium from M2 phenotype reduced neuronal death in brain slices under OGD condition. These results suggested that promoting M2 phenotype polarization is helpful for facilitating tissue regeneration after injury.

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5.2. Ischemia induces microglia polarization

790

Microglia polarization could either exacerbate damage or promote tissue repair, which is partially dependent on different pathophysiological conditions (David and Kroner, 2011; O’Neill and Hardie, 2013). Furthermore, microglia polarization could be affected by the time and severity of injury, aging, and microenvironment (Hu et al., 2015; Norden and Godbout, 2013; Perego et al., 2011). After ischemic stroke, there is dynamic microglia polarization in the peri-infarct region (cortex and striatum). CD206+ M2 microglia were observed in the peri-infarct region as early as day 1, with a transient elevation within 7 days after MCAO, and then gradually decreased. In contrast, CD16/32+ M1 microglia appeared

791 792 793 794 795 796 797 798 799 800 801

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at 3 days and gradually increased until 14 days after MCAO. The mRNA expression of M1 microglia markers iNOS, CD11b, CD16, CD32, and CD86 and M2 microglia markers CD206, Arginase-1, CCL22, Ym1/2, IL-10, and TGF-b were consistent with the immunostaining results mentioned above (Hu et al., 2012). In vitro study showed that neurons injured by ischemia could induce microglia to polarize toward M1 phenotype. Conditioned medium from M1 microglia enhanced neuronal death while M2 microglia protected neurons against OGD (Hu et al., 2012). Different phenotypes of microglia could differentially modulate cell death after brain injury (Girard et al., 2013). Adding M1 or M2 microglia into brain slices after OGD treatment, researchers found that M1 microglia was detrimental for the survival of neurons, while M2 microglia was protective in reducing neuronal death in the CA3 and CA1 region of the hippocampus after OGD treatment. In a transient MCAO model, there was no apparent microglia polarization within 24 h after ischemia (Girard et al., 2013). These results suggested that microglia polarization might exert different functions during the pathology of ischemic stroke. Inhibiting microglia activation as a therapeutic strategy should be thought over, considering it would compromise the beneficial role of microglia for the recovery and tissue repair of ischemia induced brain injury. Future studies are required to explore the function of microglia polarization to guide the development of therapeutic strategies for ischemic stroke.

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5.3. Substances involved in the modulation of microglia polarization

828 829 830

Many substances could modulate microglia polarization, contributing to improved functional recovery or exacerbated brain injury.

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5.3.1. Clinical drugs Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a peptide with neuroprotective function. A recent study used stem cell transplantation to deliver PACAP into mice brain and found that PACAP delivered at 3 days after MCAO could reduce brain infarct size, improve functional recovery, and induce microglia polarization toward M2 phenotype at 7 and 14 days after ischemia (Brifault et al., 2015). This study provided evidence that we could prevent delayed tissue damage through modulating microglia polarization. Metformin, widely used to treat type 2 diabetes, has been shown to be able to reduce the incidence of stroke and promote M2 polarization of macrophages in vivo and in vitro (Jin et al., 2014; Nath et al., 2009; Tsoyi et al., 2011; Zhou et al., 2001). Metformin treatment daily at 24 h after MCAO enhanced M2 polarization related gene expression, promoted angiogenesis and neurogenesis, and improved functional recovery. In vitro study performed by the same group showed that M2 polarization of microglia was essential for Metformin induced angiogenesis (Jin et al., 2014). Two clinical trials showed that Ginsenoside treatment could improve neurological outcome in patients with acute ischemic stroke. The beneficial role of Ginsenoside treatment was associated with inhibiting microglia activation and reducing M1 phenotype related pro-inflammatory cytokines expression (Zhang et al., 2015). Minocycline, which is widely used to inhibit microglia activation, has been used to treat ischemic stroke in clinical trials (Fagan et al., 2010; Lampl et al., 2007). Recent studies showed that minocycline treatment could exert a long-term protective function via attenuating BBB permeability and promoting microglia polarization toward M2 phenotype after focal cerebral ischemia (Yang et al., 2015). Minocycline was also shown to modulate microglia polarization during the early pathogenesis phase of amyotrophic lateral sclerosis (ALS), in which microglia are significantly activated (Kobayashi et al., 2013; Liao et al., 2012). These results suggested that minocycline could be a promising

therapeutic drug for inhibiting microglia activation mediated inflammatory response and tissue injury.

865 866

5.3.2. Receptors and small molecules Recent studies showed that Class A scavenger receptor (SR-A) and cannabinoid type 2 receptor (CB2R) are involved in modulating microglia polarization after ischemic stroke. In SR-A knockout mice, the numbers of F4/80+CD11b+ macrophages and CD16/32+ M1 microglia were reduced in the ischemic brain compared with control mice, while the number of F4/80+CD206+ M2 microglia was increased. Absence of SR-A reduced ischemia induced inflammation and infarct volume, accompanied by lower expression of M1 microglia related genes TNF-a, iNOS, MCP-1, and IL-1b and higher expression of M2 microglia related genes IL-10 (Xu et al., 2012). In CB2R knockout mice, brain infarct volume was increased and neurological impairment was worse. Intraperitoneal injection of CB2R agonist JWH-133 at 10 min or 3 h after MCAO reversed the phenomenon. JWH-133 treatment also reduced Iba1+ microglia activation, concomitant with lower expression of both M1 and M2 microglia markers (Zarruk et al., 2012). Culture supernatants derived from Th1 cells promoted microglia to up-regulate M1 phenotype related genes such as MCP-1 and CCL-2 expression, indicating that Th1 could affect microglia polarization in a paracrine manner. However, the molecular mechanism of the interaction between Th1 cells and microglia in ischemic brain is rarely investigated (Prajeeth et al., 2014). A study in IL-10 knockout mice showed that IL-10 absence exacerbated brain ischemic injury and reduced M2 microglia marker expression, suggesting that IL-10 is critical in modulating microglia polarization toward M2 phenotype (Perez-de Puig et al., 2013). In a model of experimental autoimmune encephalomyelitis (EAE), lentivirus mediated IL-25 gene delivery promoted microglia polarization to M2 phenotype, with high expression of M2 markers Arginase-1, CD206, and Ym1 (Maiorino et al., 2013). Studies showed that TGF-b could induce microglia polarization to M2 phenotype (Blobe et al., 2000; Hanisch and Kettenmann, 2007). On the other hand, M2 polarization of microglia could produce TGFb(Hu et al., 2015). Recent studies found that TGF-b involved in promoting IL-4 mediated M2 polarization of microglia and enhancing the sensitivity of microglia response to IL-4 (Zhou et al., 2012). In summary, microglia polarization is a complicate process and could be modulated by many molecules. Understanding how these molecules modulate microglia polarization under ischemia environment could help us drive microglia toward a protective phenotype, thus protecting brain against ischemic attack.

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5.3.3. microRNAs and microglia (Table 2) Increasing evidence revealed that microRNAs play an important role in the pathophysiology of CNS diseases, including AD, Parkinson’s disease (PD), EAE, multiple sclerosis (MS), ALS, and stroke (Butovsky et al., 2012; Junn and Mouradian, 2012; Moore et al., 2013; Parisi et al., 2013; Ponomarev et al., 2011; Sun et al., 2013; Zhao et al., 2013). Several microRNAs were found to be associated with microglia activation or microglia polarization in CNS diseases. The differential expression of microRNAs could affect microglia polarization and impact the function of microglia in brain injury or tissue repair (Butovsky et al., 2015; Hu et al., 2015; Ponomarev et al., 2011; Su et al., 2015). MiR-124 is unique among microRNAs, with selective expression in the CNS and the highest expression in neurons (Kim et al., 2004; Landgraf et al., 2007; Weng et al., 2011). In a zebrafish model, miR-124 was specifically over expressed in microglia. MiR124 overexpression did not cause morphological changes of microglia, but reduce microglia activity and the capacity of clearing apoptotic cells. Conversely, depletion of miR-124 in

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zebrafish enhanced microglia motility and phagocytosis. These results suggested that stable expression of miR-124 is important for maturity of microglia (Svahn et al., 2015). In a transient cerebral ischemia model in rat, plasma miR-124 was increased at 6 h and continued to elevate at 48 h after ischemia onset, however, there was no correlation between the level of plasma miR-124 and brain infarct volume (Weng et al., 2011). A clinical study of 65 patients with cardiac arrest found that the up-regulation of blood miR-124 at 24 h and 48 h after cardiac arrest was associated with poorer neurological outcome at 6 weeks after cardiac arrest, suggesting that miR-124 could be a biomarker for the prognosis of ischemic stroke and cardiac arrest (Gilje et al., 2014). It remains controversial whether miR-124 exerts beneficial or detrimental role in ischemic brain injury (Doeppner et al., 2013; Liu et al., 2013; Sun et al., 2013; Zhu et al., 2014). One previous study showed that miR-124 delivery could protect cultured neurons against OGD condition. MiR-124 delivery enhanced angiogenesis and neurogenesis at 8 weeks after MCAO, with a mechanism of Usp14-dependent REST degradation in vivo. This concept was supported by the evidence that miR-124 delivery reduced Usp14 and REST expression after MCAO, and REST was down-regulated after Usp14 inhibitor IU-1 treatment (Doeppner et al., 2013). However, another study showed that knocking out miR-124 reduced ischemia induced cell death and improved neurological function. Treatment with miR-124 antagomir enhanced Ku70 expression (a DNA repair protein) in the ischemic region, suggesting that miR-124 promoted ischemia induced brain injury via regulating Ku70 expression (Zhu et al., 2014). Under normal physiological condition, non-activated microglia express high level of miR-124 and mainly express M2 phenotype markers such as Fizz1, Ym1, IL-10 and IL-4 (Ponomarev et al., 2007; Ponomarev et al., 2011). Under pathological condition, with proinflammatory cytokines stimulation or in a model of EAE, activated microglia displayed up-regulated M1 phenotype markers (e.g. MHC II), and down-regulated miR-124. Transfection of miR-124 in mice at different stages of EAE reduced microglia activation and leukocyte infiltration, ameliorated clinical symptoms, and promoted neuronal function recovery. Conversely, repression of miR124 using miR-124 antisense oligonucleotides led to microglia activation. The study indicated that miR-124 is essential for maintaining the quiescent phenotype of microglia and contribute to M2 phenotype in normal condition (Ponomarev et al., 2011; Ponomarev et al., 2013). MiR-124 inhibition reduced inflammation and delayed the course of EAE, suggesting that we might use miR124 as a target for the treatment of inflammation or microglia activation associated diseases including ischemic stroke. MiR-155 is widely accepted as a pro-inflammatory microRNA and has a direct relationship with M1 phenotype (Guedes et al., 2013). MiR-155 expression is increased in microglia after being stimulated by pro-inflammatory cytokines such as LPS, IFN-??, and TNF-?? (Bala et al., 2011; Cardoso et al., 2012; Wang et al., 2010). Knocking down miR-155 by anti-miRNA oligonucleotides enhanced suppressor of cytokine signaling 1 (SOCS-1) protein expression, which is a target of miR-155 and critical for inhibiting inflammation. Knocking down miR-155 also reduced the production of NO as well as the expressions of inflammatory cytokines and iNOS. Conditioned medium collected from microglia, in which miR-155 was inhibited, protected neurons from inflammation mediated injury. This study provided evidence that miR-155 promotes microglia toward pro-inflammatory phenotype (M1 phenotype) via inhibiting SOCS-1 expression (Cardoso et al., 2012). LPS stimulated microglia could inhibit neural stem cells (NSCs) differentiation, which could be reversed via depletion of miR-155 in microglia. When miR-155 was depleted under LPS induced inflammatory environment, NSCs proliferation was restored and the number of amoeboid morphological microglia enhanced in

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dentate gyrus of mice. Thus, miR-155 in microglia plays an important role in inflammation induced neurogenic deficit (Woodbury et al., 2015). In a mouse model of ALS, microglia were abnormal because many molecules essential for microglia survival were lost. However, the miR-155 expression was increased. The expression of pro-inflammatory genes in microglia was associated with the severity of ALS in mice. MiR-155 ablation suppressed microglia polarization to M1 phenotype and corrected with the abnormality of microglia. In addition, anti-miR-155 treatment delayed the course of ALS and prolonged survival in SOD1 mice, a model used for ALS (Butovsky et al., 2015). Similarly, another study found that miR-155 was highly expressed in cultured M1 phenotype microglia from MS patients. MiR-155 transfection enhanced TNF-a and IL-6 secretion and M1 phenotype marker expression including CD80, CD86, and CCR7 while miR-155 inhibition reduced pro-inflammatory cytokines secretion via inhibiting SOCS-1 expression (Moore et al., 2013). These studies further supported that miR-155 is crucial for M1 polarization of microglia. Therefore, miR-155 inhibition could be neuroprotective for the treatment of neurological diseases where inflammation is excessive and detrimental. Although miR-155 is critical for microglia activation mediated inflammatory response under many pathological conditions, few studies has investigated the role and mechanisms of miR-155 in microglia activation or polarization in ischemic stroke. In a model of permanent MCAO in mice, miR-155 expression was reduced in the brain at 24 h after ischemia, but whether low expression of miR-155 was associated with inflammatory response and microglia activation was not investigated (Liu et al., 2010). Another study indicated that miR-155 in microglia was increased after transient MCAO and induced proinflammatory response in microglia (Su et al., 2014). Combination with these results, whether miR-155 inhibition could restore inflammatory response, modulate microglia polarization, and benefit tissue repair and functional recovery after ischemic stroke needs to be explored in the future. Other microRNAs were also found to have a connection with microglia polarization and activation. MiR-Let7ais involved in modulating microglia polarization toward M2 phenotype. Overexpression of miR-Let7a in BV2 cells, a widely accepted cell line used for investigation of microglia activation, reduced M1 related cytokines including ROS, iNOS, and IL-6 expression, but enhanced M2 related cytokines including IL-10, IL-4, and BNDF expression. MiR-Let7a was found to mediate autophagy activity in microglia under LPS induced inflammation environment (Cho et al., 2015; Song et al., 2015). MiR-Let-7c-5p, a highly conserved miRNA in stroke, is down-regulated in the plasma of patients with ischemic stroke. In mice model of MCAO, miR-Let-7c-5p was demonstrated to be down-regulated in the brain. MiR-Let-7c-5p overexpression in the brain could inhibit microglia activation and promote neurological function recovery. Further experiment showed that ischemia and inflammation could induce miR-Let-7c-5p upregulation in microglia and miR-Let-7c-5p overexpression inhibited microglia activation via suppressing caspase 3 pathway. The study suggested that miR-Let-7c-5p is a promising target for inhibiting microglia activation after cerebral ischemia (Butovsky et al., 2014; Ni et al., 2015; Zhao et al., 2013). MiR-181c is another potential inhibitor of microglia activation. MiR-181c decreased in microglia after OGD treatment. Conditioned medium from miR181c transfected microglia reduced TNF-a production and protected neurons against OGD condition. TNF-a is a direct target of miR-181c, which was elucidated by a luciferase reporter assay. Overexpression of TNF-a compromised the protective effect of miR-181c on neurons under OGD condition. Thus, miR-181c could inhibit microglial production of TNF-a and attenuate ischemiahypoxia induced neuron apoptosis (Zhang et al., 2012a). MiR-9, which is specifically expressed in rodents brain, was used to induce

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transgene expression in brain resident microglia (Akerblom et al., 2013; Deo et al., 2006). MiR-9 was shown to promote microglia activation through down-regulating the expression of miR-9 downstream protein monocyte chemotactic protein-induced protein 1 (MCPIP1) (Yao et al., 2014). MiR-125b-5p, miR-99a, miR-342-3p are highly expressed both in human and mouse brain microglia, but it needs further study to identify the role of these miRNAs in modulating microglia activation or polarization (Butovsky et al., 2014). Collectively, these results indicated that microglia polarization is complicated and could be affected by many substances. Whether inhibition of microglia activation is beneficial or detrimental for the recovery of ischemia induced brain injury might depend on when and how microglia activation is inhibited, and whether microglia polarization is affected. Promoting the switch of microglia toward a neuroprotective M2 phenotype might be a promising approach for ischemic disease therapy.

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6. Microglia crosstalk with other cells during ischemic stroke

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6.1. Microglia and neurons

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6.1.1. Neurons control microglia activation Neurons could control microglia activation via ‘On’ and ‘Off’ signals. ‘On’ signal is mainly found under pathological condition and included chemokines, purines, glutamate, and matrix metalloproteinase-3. ‘Off’ signal mainly occurs in the healthy brain and is mediated by release of CX3CL1, CD22, neurotransmitters and neurotrophins from neurons, which could bind with receptors on microglia and help microglia exert physiological function (Biber et al., 2007). The interaction between neurons and microglia is complicated and involves a variety of ligands and receptors (Correa et al., 2013). CD200/CD200R and CX3CL1/ CX3CR1 are the two most studied ligands and receptors pairs involved in controlling microglia activation. CD200 is constitutively expressed on neurons and CD200R is expressed mainly on microglia in the brain. Deficiency of CD200 or blockade of CD200R in mice led to enhanced microglia activation and worsened symptoms in the mouse model of EAE, facial nerve transection, retinal inflammation, and encephalitis (Broderick et al., 2002; Deckert et al., 2006; Hoek et al., 2000). These results suggested that CD200/CD200R contributes to maintaining microglia activation at an appropriate level. Similar to the function of CD200/ CD200R, CX3CL1/CX3CR1 could also inhibit microglia activation. CX3CL1 is expressed on neurons and CX3CR1 is only expressed on microglia in the brain. Absence of CX3CR1 in mice enhanced microglia neurotoxicity, which caused more neuronal death in models of PD and ALS (Cardona et al., 2006). In a retinal transplantation study, time-lapse confocal imaging was used to evaluate the influence of absence of CX3CR1 on microglia processes dynamism and mobility. It was observed that the absence of CX3CR1 did not affect microglia processes dynamism including extension and retraction of processes, formation of new processes, and elimination of existing processes, but reduced the rate of movement toward injury induced by laser. Administration of CX3CL1 induced the transformation of microglial spindle processes toward a more branched and shorter counterparts in CX3CR1 heterozygote mice. However, no microglia morphological changes could be detected after the administration of CX3CL1 in CX3CR1 knockout mice, suggesting that CX3CL1/CX3CR1 plays an important role in the interaction between neurons and microglia (Liang et al., 2009).

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cells release damage-associated ligands and excitotoxic glutamate, exacerbating neuronal damage (Neher et al., 2013). However, a recent study showed that injured neurons could promote microglia polarization toward protective phenotype via releasing lipocalin-2. Lipocalin-2 is highly expressed in cultured neurons from human or mice brain after ischemia. Lipocalin-2 treatment induced microglia to secrete IL-10, change from ramified shape to less branches and long-rod shape, and enhance microglia capacity of phagocytosis. Conditioned medium from lipocalin-2 treated microglia enhancedsynaptophysin and post-synaptic density 95 (PSD95) expression and prevented neuronal death from OGD stress (Xing et al., 2014). Damaged neurons induced by ischemia could release IL-4, which could enhance IL-4 receptor expression on microglia and promote microglia polarization to M2 phenotype. IL-4 activated peroxisome proliferator activated receptor g (PPARg) on microglia and enhanced microglia capacity of phagocytosis of apoptotic neurons (Zhao et al., 2015). Glutamate stressed neurons could enhance the capacity of microglia to clear neuronal debris via secreting soluble fractalkine (sFKN) (Noda et al., 2011). These studies implied that injured neurons could promote microglia to exert protective function to help neuron survive under ischemic condition.

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6.1.3. Microglia influence neuronal function Under pathological condition, microglia are traditionally recognized with a function in immunological surveillance (Eyo and Wu, 2013). They could affect disease progression via clearing debris, secreting pro-inflammatory or anti-inflammatory cytokines, and producing growth factors. Microglia are essential for the development and maintenance of healthy brain homeostasis including modulating neural circuit via specifically interacting with neuronal synapse (Eyo and Wu, 2013; Hirasawa et al., 2005; Wake et al., 2013). Two-photon microscopy allows the monitoring of the interaction between neurons and fluorescent-labeled microglia in transgenic mice in vivo (Wake et al., 2009). Microglia directly contacted neuronal synapse through extending processes every 5 min for 1 h. It took microglia in the peri-infarct region longer to connect with neurons, up to approximately 1 h after ischemia. The prolonged connection time after ischemia stimulation caused disappearance of a few presynaptic boutons, suggesting that microglia could monitor the functional state of synapses (Wake et al., 2009). However, the mechanisms driving microglia to make direct contact with neuronal synapse and the exact role of prolonged connection with neuronal synapse after brain ischemia are still unclear. Microglia are professional phagocytes in the brain and could engulf neurons within hours (Neher et al., 2011). Microglial CX3CR1 knockout prevented neuronal loss in a mouse model of AD (Fuhrmann et al., 2010). Microglia play a beneficial role for tissue remodeling and regeneration after ischemic stroke via clearing dead or dying neurons (Brown and Neher, 2014; Neumann et al., 2009). Insufficient clearance of cell debris by microglia could cause failure of tissue repair after focal cerebral ischemia (Stoll et al., 2004). Chimeric mice in which brain resident microglia were GFP, and infiltration of macrophages were GFP+ were used to explore the phagocytosis of brain resident microglia. It was found that brain resident microglia could engulf neuronal debris as early as day 1 and peaked at 2 days, while infiltrated macrophages started to clear neuronal debris at 4 days after MCAO. The study indicated that brain resident microglia are more sensitive and important on the defense of ischemia via clearing dead or dying neurons (Schilling et al., 2009). However, the exact benefit of removing dead or dying neurons by microglia is not clear, maybe partly because it help reduce inflammation (Sierra et al., 2013). In a rat focal cerebral ischemia model induced by endothelin-1, a study

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observed that microglia played anti-inflammatory role through phagocytosing polymorph nuclear granulocytes at 24 h after ischemic injury, and reduced neuronal death (Neumann et al., 2008). Depletion of Mac-2+ resident microglia in the brain caused enhanced neuronal apoptosis and high expression of proinflammatory cytokines at 72 h after transient cerebral ischemia (Lambertsen et al., 2009). These studies suggested that microglia are critical for the neuron survival within the acute phase of ischemic stroke (Lalancette-Hebert et al., 2007). The detrimental role of microglia in neurological diseases had been widely debated over the past several decades (Block et al., 2007; Hanisch and Kettenmann, 2007). Several studies showed that microglia activation is associated with ischemia induced neuronal death (Denes et al., 2008; Franco et al., 2012; Neher et al., 2013; Wu et al., 2012). Inhibition of microglia activation by a number of drugs reduced brain injury (Lai and Todd, 2006). A recent study showed that microglia mediated delayed neuronal death through phagocytosis activity after ischemia. Microglia expressed phagocytosis related proteins Milk fat globule EGF-like factor-8 (MFG-E8) and Mer receptor tyrosine kinase (MerTK) at 3 to 7 days after focal brain ischemia. Blocking microglia phagocytosis via depleting MFG-E8 or MerTK in mice reduced neuronal loss and brain atrophy. This study further demonstrated that MFG-E8 or MerTK mediated phagocytosis of microglia specifically affect the living and viable neurons. In vivo experiment showed that there was no difference in the accumulation of Fluorojade C positive dead neurons between MFG-E8 or MerTK deficient mice and control wild type mice after ischemic injury. Addition of cultured microglia from wild type, MFG-E8, or MerTK deficient mice showed no difference in the loss of untreated neurons, but reduced the loss of viable neurons stressed by glutamate. Moreover, MFG-E8 or MerTK deficiency displayed lower microglial capacity of phagocytosing neurons, suggesting that the interaction between microglia and neurons is a complicated process, and the different functional stages of neurons could influence microglia function differently. Therefore, further study aims at elucidating the role of microglia in different stages of ischemic brain injury would help pave the way in developing microglia targeted treatments for ischemic stroke (Neher et al., 2013). NADPH oxidase (NOX) and NOX induced ROS are common mediators of neuronal damage caused by microglia activation after ischemia (Dringen, 2005). Knocking out NADPH oxidase type 4 (NOX4) reduced BBB leakage and neuronal apoptosis in ischemic brain in mice (Kleinschnitz et al., 2010). Knocking out NADPH oxidase type 2 (NOX2) promoted tissue repair through angiogenesis following ischemic reperfusion in mice (McCann et al., 2014). A recent study found that microglia expressed voltage-gated proton channel Hv1 protein, which is responsible for ischemia induced neuronal death. Microglia cultured from Hv1 knockout mice could not produce NOX and ROS. Treatment of cultured microglia with NOX inhibitor diphenyleneiodonium (DPI, 30 mM) and apocynin (300 mM) or the Hv1 inhibitor Zn2+ suppressed NOX activation and ROS production. In Hv1 knockout mice, brain infarct volume and neurological scores were smaller than wild type mice at 24 and 72 h after MCAO. The number of caspase3+ or TUNEL+ apoptotic neurons decreased in the peri-infarct region. These results suggested that Hv1 contributes to neuronal death via NOX activation and ROS production. In vitro study further supported the hypothesis. Co-culture of neurons with microglia from Hv1 knockout mice reduced neuronal death after 30 min OGD, comparing with neurons co-cultured with microglia from wild type mice. Pre-treatment with NOX inhibitor or Hv1 knockout in microglia protected neurons against OGD. Thus, the study provided a new target protein Hv1 for ischemic stroke therapy (Wu et al., 2012).

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6.2.1. Cross-talk between microglia and astrocytes Astrocytes are the most abundant cell type in the brain and are thought to serve and protect neurons (Nedergaard and Dirnagl, 2005; Tsai et al., 2012). Both microglia and astrocytes are major components of the innate immune system in the brain. They are able to sense the changes of brain environment and play an important role in various pathological conditions including ischemic stroke (Hanisch and Kettenmann, 2007; Ransohoff and Brown, 2012; Seifert et al., 2006; Tremblay et al., 2011). Based on time-lapse recording from two-photo microscopy imaging, a study observed that microglia directly contacted astrocytes via extending their processes toward astrocytes (Nimmerjahn et al., 2005). Microglia display a comparable density to astrocytes in the brain and express membrane receptors for all known neurotransmitters, which allow microglia to modulate neuronal activity and receive information from astrocytes (Jinno et al., 2007; Pocock and Kettenmann, 2007). Indeed, emerging evidence indicated that there exists a functional communication between microglia and astrocytes, which is crucial for the innate immune response in the brain (Ransohoff and Brown, 2012). The modulators contribute to the communication between microglia and astrocytes including IL1b, TNF-a, TGF-b, adenosine, ATP, and glutamate (Boison et al., 2010; Burnstock et al., 2011; Herrera-Molina and von Bernhardi, 2005; Pascual et al., 2012; Rouach et al., 2002a,b). Recently, several new discoveries further advanced our understanding about the molecular mechanisms regarding the communication between microglia and astrocytes. For example, LPS stimulated microglia could release ATP, and promote astrocytes to release glutamate, which modulated neuronal activity via metabotropic glutamate receptors (mGluRs) (Pascual et al., 2012). LPS stimulation induced a transient spontaneous excitatory postsynaptic currents (EPSCs) in cultured brain slices, which could be blocked by depletion of microglia in brain slices (Pascual et al., 2012). Blocking purinergic receptors via broad spectrum antagonists decreased the frequency of EPSCs, while activating purinergic receptor P2Y1R, which is exclusively expressed on astrocytes in cultured brain slices, reversed the phenomenon. These results collectively suggested that microglia are essential for astrocytes mediated neuronal activity via releasing ATP, which could bind to P2Y1R located on astrocytes. Further experiments found that inhibition of mGluR5 receptor on neurons decreased the effect of LPS stimulation on neuronal activity, implicating that activated microglia induced astrocyte modulated neuronal activity through mGluR5 (Pascual et al., 2012). In conclusion, studies supported that microglia could modulate neuronal activity via communicating with astrocytes in a ATP-P2Y1R-mGluR5-mGluR5 receptor dependent manner. Microglia could trigger astrocytes mediated neuroprotection via an ATPP2Y1R-P38-MAPK-IL-6 dependent signal pathway. Microglia sensed the low concentration of methylmercury (MeHglow), which induced neuronal death, and released ATP. ATP bound to the P2Y1R on astrocytes, which in turn activated P38-MAPKs and promoted astrocytes to produce IL-6. ATP release and IL-6 production could be blocked via inhibition of vesicular nucleotide transporter (VNUT) or P2Y1R. Moreover, additional IL-6 in cultured brain slices attenuated MeHglow induced neuronal death (Shinozaki et al., 2014). These discoveries further implicated that astrocytes are required for microglia mediated neuroprotective function on neurons. Therefore, astrocytes could be a potential choice for regulating microglia function in neurological diseases.

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6.2.2. Cross-talk between microglia and astrocytes during ischemic stroke CX3CL1-CX3CR1 signal in microglia contributes to the regulation of neuronal viability under ischemia condition (Cipriani et al.,

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2011; Denes et al., 2008; Soriano et al., 2002). In a mouse model of permanent MCAO, CX3CL1 and CXCL16 acted synergistically on the crosstalk between neurons, microglia, and astrocytes, and prevented ischemia or glutamate induced neuronal death. Administration of CX3CL1 or CXCL16 at 30 min before MCAO in mice reduced brain infarct volume at 24 h after ischemia. Deficiency of CXCL16 receptor in mice eliminated the protective effect of CX3CL1 or CXCL16. CX3CL1 stimulation promoted cultured astrocytes to release CCL2 and reduced glutamate-excitotoxicity induced neuronal death, which was reversed by depletion of CCL2 receptor A3R or CCL2 antibody treatment. These results suggested that CX3CL1-CXCL16-CCL2 signaling contributes to mitigating ischemia induced brain damage via mediating the interaction between neurons, microglia, and astrocytes (Rosito et al., 2014). Pro-inflammatory mediator lysophosphatidylcholine (LPC) produced by neurons and astrocytes after ischemic stroke stimulated microglia to up-regulate mRNA of MCP-1 and CCR2, which involves in mediating inflammatory response after cerebral ischemia (Inose et al., 2014). Therefore, there are complicated communications between microglia and astrocytes. The understanding of molecular mechanisms of this communication is still limited and calls for the further investigation.

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Oligodendrocytes form the myelin sheath in the CNS and are highly vulnerable to ischemia (Back et al., 2002; Dewar et al., 2003). After 30 min of an artery occlusion, swelling of oligodendrocytes occurs. Most of oligodendrocytes are dead at 3 h after ischemia, which appears earlier than neuronal death in the ischemic region (Pantoni et al., 1996). Oligodendrocyte progenitor cells (OPCs) derived from NPCs in the SVZ could proliferate and differentiate into oligodendrocytes in ischemic brain (Li et al., 2010; Zhang et al., 2001, 2012b). Oligodendrocytes are major components of white matter. Damage of oligodendrocytes and white matter is associated with function impairment after ischemia induced brain injury (Matute et al., 2013). Several studies demonstrated that microglia activation causes the impairment of oligodendrocytes/OPCs via producing pro-inflammatory cytokines such as TNF-aand IL-1b (Deng et al., 2008; Haynes et al., 2005; Moxon-Emre and Schlichter, 2010). For example, in a rat model of chronic cerebral ischemia, a study observed that increased loss of oligodendrocytes was coupled with increased microglia activation from 2 to 4 weeks after surgery. Interestingly, TNF-a was highly expressed at 2 weeks after surgery (Masumura et al., 2001). Another study using the same model found that activated microglia expressing TNF-a appeared at the white matter lesion, where oligodendrocytes died in a caspase3 dependent apoptotic death manner (Jalal et al., 2012). Minocycline treatment inhibited microglia activation, reduced pro-inflammatory cytokines expression, and attenuated oligodendrocytes/OPCs damage under hyperoxia and ischemia-hypoxia conditions in neonatal rats (Cai et al., 2006; Schmitz et al., 2014). These studies suggested that there is a link between microglia activation mediated neuro-inflammation and hyperoxia/ischemia induced oligodendrocytes loss, but the detailed molecular mechanism connecting the link is unclear. Recently, emerging evidence indicated that microglia exert beneficial function on oligodendrocytes/OPCs, and it is mainly M2 polarization of microglia that plays the protective role. In vitro, conditioned media from LPS induced M1 microglia or untreated microglia enhanced oligodendrocytes death after OGD treatment for 24 h, while conditioned media from IL-4 induced M2 microglia reduced oligodendrocytes apoptosis (Wang et al., 2013). Ina mouse model of MS, depletion of M2 microglia in mice impaired OPCs differentiation. Conditioned medium from M2 microglia promoted

cultured OPCs differentiation. Inhibiting activin-A released by M2 microglia suppressed OPCs differentiation after demyelination (Miron et al., 2013). These two studies shed new light on the potential role of microglia polarization in different pathological conditions. M2 microglia derived activin-A could be a target for the treatment of neurological diseases. Another study found that there is a temporal and spatial activation of microglia in the SVZ in neonatal rats. Inhibiting microglia activation via minocycline treatment reduced NPCs and OPCs proliferation in the SVZ, as well as the expression of pro-inflammatory cytokines IL-1b, IL-6, TNFa, and IFN-g. Co-culture of neurosphere with microglia enhanced NSCs differentiation into OPCs, which could be blocked by inhibiting microglia activation or blocking production of IL-1b, IL-6, TNF-a, and IFN-g. This study further supported the beneficial effect of microglia activation on oligodendrocytes/OPCs (Shigemoto-Mogami et al., 2014). Thus, it is imprecise to simply declare that microglia activation is detrimental or beneficial for oligodendrocytes/OPCs, as well as the role of pro-inflammatory cytokines released by microglia. Different polarization of microglia and different pathological conditions could influence the function of microglia. Understanding when and how microglia activation exert detrimental or beneficial role on oligodendrocytes/OPCs will be helpful for the regeneration of white matter injury under different pathological conditions including ischemic stroke.

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7. Relationship between microglia and stem cells

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Stem cell transplantation is a promising strategy for the treatment of various neurological diseases including AD, PD, ALS, MS, spinal cord injury, and ischemic stroke (Abe et al., 2012; Boulis et al., 2011; Gogel et al., 2011; Liu et al., 2014b; Martino et al., 2010; Sahni and Kessler, 2010). The interaction of grafted cells with its surrounding environment and host cells are important for the neuroprotective effects (Darsalia et al., 2011; Gogel et al., 2011). Emerging evidence indicated that transplanted stem cells could interact with and influence brain resident microglia activation after ischemia. In a rat model of transient cerebral ischemia, transplantation of human NSCs, CTX0E03, promoted microglia proliferation in the striatum from 1 to 4 weeks after MCAO. CTX0E03 transplantation slowed the rate of decline of DCX+/Ki67+ neuroblasts after 4 weeks of ischemia (Hassani et al., 2012). These results suggested that transplanted NSCs could facilitate neuroblast proliferation, which is associated with enhanced microglia activation. Further studies to examine the phenotype of these activated microglia could help elucidate the mechanism of how transplanted NSCs affect microglia activation. Compared with NSCs, transplanted mesenchymal stem cells (MSCs) exert beneficial function on neurological recovery after ischemic brain injury partly via anti-inflammatory response, which is mainly mediated by brain resident microglia, macrophages, and lymphocytes (mainly T and B cells) (Kokaia et al., 2012; Sheikh et al., 2011; Tang et al., 2014). MSCs transplantation inhibited microglia activation following3 and 7 days of transient MCAO in rats. Fractalkine and IL-5 secreted by MSCs could reduce microglia response to LPS stimulation (Sheikh et al., 2011). However, another study showed that MSCs transplantation via tail vein improved motor behavior recovery after MCAO accompanied by enhanced microglia activation (Yang et al., 2010). The controversial results of transplanted MSCs on microglia activation might partly due to the timing and dose of MSCs transplantation, as well as the observation time during experiments. In a model of hypoxic-ischemia (HI) in nine-day-old neonatal mice, transplantation of MSCs reduced microglia response and induced microglia polarization to M2 phenotype, contributing to enhanced regeneration and improved neurological outcome at 18 or 21 days after ischemic brain injury (Donega et al., 2014; van Velthoven et al.,

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2010). In vitro studies showed that MSCs could promote M2 polarization of microglia and affect microglia activity via releasing NO (Hegyi et al., 2014; Neubrand et al., 2014). Collectively, transplanted stem cells could either inhibit or promote microglia activation and modulate microglia polarization after ischemia. However, there is still no strong evidence to support that microglia activation could influence transplanted stem cells survival, migration, and differentiation after ischemic stroke. Endothelia progenitor cells (EPCs) is another effective stem cell type used for the treatment of ischemic disease. The number of EPCs in circulation is considered as a predictor for acute stroke (Chu et al., 2008). There is a transient elevation of circulating EPCs

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after stroke (Zhou et al., 2009). The level of circulating EPCs negatively correlates with the severity of ischemia (Bogoslovsky et al., 2010). Recent studies demonstrated that EPCs transplantation reduces brain infarct volume and improves neurological outcome (Chen et al., 2012; Fan et al., 2010; Iskander et al., 2013; Zhang et al., 2002). Our previous study demonstrated that there is an interaction between microglia and EPCs. LPS activated microglia could release HMGB1, which promoted cultured EPCs to release IL8. Conditioned medium from EPCs enhanced the viability of human umbilical cord vein cells (HUVEC) under OGD condition and promoted tube formation. Administration of HMGB1 inhibitor glycyrrhizin or knocking down IL-8 in EPCs reversed the beneficial

Fig. 1. Microglia polarization after ischemic stroke. The figure illustrates the role of M1 or M2 microglia in ischemic brain injury. Ischemia causes neural death and tissue damage. Injured tissue release a variety of damage associated molecular pattern molecules, pro-inflammatory cytokines, and reactive oxygen species (ROS), causing microglia to polarize toward M1 or M2 phenotype. M1 microglia could exacerbate neural death, astrocyte apoptosis, and BBB disruption via releasing IL-6, TNF-a, IL-1b, MMPs, and iNOS. Conversely, M2 microglia exert a protective role after ischemia through releasing neurotrophic factors including BDGF, IGF-1, IL-4, and IL-10. M2 microglia could maintain BBB integrity, promote the proliferation and differentiation of neural stem cells (NSCs) and oligodendrocyte progenitor cells (OPCs), and facilitate myelin regeneration and tissue repair. However, the exact molecular mechanism mediating microglia polarization toward M1 or M2 phenotype after ischemic brain injury needs further investigation.

Please cite this article in press as: Ma, Y., et al., The biphasic function of microglia in ischemic stroke. Prog. Neurobiol. (2016), http:// dx.doi.org/10.1016/j.pneurobio.2016.01.005

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effect of EPCs on HUVEC. Intravenous EPCs transplantation enhanced angiogenesis, reduced brain atrophy volume, and improved neurological behavior after transient focal cerebral ischemia. While administration of HMGB1 inhibitor glycyrrhizin compromised the therapeutic effects of EPCs transplantation. Therefore, microglia could modulate EPCs paracrine function via releasing HMGB1, contributing to enhanced therapeutic function of EPCs transplantation after ischemic stroke (Chen et al., 2014). Although microglia display a positive regulation on transplanted EPCs after ischemia, whether transplanted EPCs affect microglia activation and function is unknown. Studies exploring the interaction between microglia activation and EPCs transplantation are also scarce.

vehicle for gene therapy for glioma (Ribot et al., 2007). Collectively, these studies suggested that microglia transplantation manifests a potential for the treatment of neurological diseases including ischemic stroke. However, several questions remain to be addressed before microglia can be considered as an alternative choice of cell based therapy for ischemic diseases. What is the exact mechanism of microglia transplantation in influencing the neurological outcome after tissue injury? What is the advantages of microglia transplantation? More studies are needed to further the understanding of the role of transplanted microglia in neurological diseases.

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9. Conclusion and prospect

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1483 8. Microglia as a potential candidate of cell based therapy for 1484 ischemic stroke

Microglia are resident macrophages in the brain. Unlike other neural cells such as neurons, astrocytes, and oligodendrocytes, which are of neuroectodermal origin, microglia are originated from primitive progenitor cells located in yolk sac (Ginhoux et al., 2010; Kierdorf et al., 2013). Microglia could sense even small imbalances of environmental homeostasis and are rapidly activated with a characterization of dynamic morphology and polarization (Prinz and Priller, 2014). The temporal and spatial course of microglia activation are detected both in human being and experimental animals (Darsalia et al., 2011; Gulyas et al., 2012; Perego et al., 2011; Price et al., 2006; Tomimoto et al., 1996; Zhang et al., 1997). Traditionally, microglia activation are considered to play a deleterious role during ischemic stroke, as inhibition of microglia activation attenuates ischemia induced brain injury (Franco et al., 2012; Machado et al., 2009; Yenari et al., 2006). Activated microglia could mediate BBB disruption and induce neuronal cell death via releasing a variety of pro-inflammatory cytokines and toxic substances (da Fonseca et al., 2014; Jolivel et al., 2015; Lai and Todd, 2006). However, increasing evidence showed that microglia activation could also be beneficial through attenuating neuronal apoptosis, enhancing neurogenesis, and promoting functional recovery after cerebral ischemia (Faustino et al., 2011; LalancetteHebert et al., 2007; Neumann et al., 2008; Thored et al., 2009). Different polarization of microglia could likely explain the biphasic role of microglia in different pathological conditions (Cherry et al., 2014; Hu et al., 2015) (Fig. 1). Many substances are involved in modulating microglia polarization, among which microRNAs show

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As microglia are scavenger cells in the brain and could produce various neurotrophic factors to promote neurological recovery after ischemic brain injury, several studies directly transplanted microglia into the brain to explore whether microglia could be protective for ischemic stroke (Hanisch and Kettenmann, 2007; Kreutzberg, 1996). In a mouse model of transient MCAO, intracerebroventricular injection of microglia at 1 h after brain ischemia enhanced neuronal survival, reduced neurodegeneration, and improved neurological behaviors (Kitamura et al., 2004; Kitamura et al., 2005). In a rat model of transient MCAO, intravenous injection of microglia at 48 h after brain ischemia contributed to reducing neuronal apoptosis, increasing neurotrophic factors including GDNF, BDNF, VEGF, and BMP7 and antiinflammatory cytokinesIL-4 and IL-15 expression, and promoting neurological function recovery (Narantuya et al., 2010a). However, in a rat model of permanent MCAO, microglia transplantation via tail vein did not affect neurological outcome (Jiang et al., 2013). The controversial results are likely due to the different experimental animals, different time, routes, and dose of microglia transplantation. Besides ischemic stroke, microglia transplantation was also conducted in chronic ischemia model, spinal cord injury, AD, and PD, and showed a beneficial effect on tissue repair and functional recovery (Danielyan et al., 2014; Narantuya et al., 2010b; Takata et al., 2007; Yu et al., 2009). In addition, microglia were used as a

Fig. 2. Possible therapeutic strategies targeting microglia activation to reduce inflammatory response and promote tissue repair. Microglia could be either beneficial or detrimental for tissue repair after brain injury, which is depending on how microglia are activated. Clinical trial and basic research indicated that we could possibly modulate microglia activation via suppressing microglia to secrete pro-inflammatory cytokines or promoting microglia polarization to M2 phenotype. Receptors (e.g. TLRs, FcRs, and RAGE) located on the left microglia body mainly mediate inflammatory response and could be inhibited via knocking out these receptors or using antagonist and anti-inflammatory response drugs, for example Minocycline and Ginsenoside. Similarly, receptors (e.g. TREM-2 and CB2R) located on the right microglia body mainly mediate microglia protective function. Applying receptor agonist or neuroprotective drugs, for example Metformin, could dampen microglia mediated inflammatory response and promote microglia to secrete neuroprotective factors, such as IGF-1, TGF-b and IL-4. Overexpression of anti-inflammatory miRNAs or suppression of pro-inflammatory miRNAs in microglia could also reduce inflammatory cytokines release and promote microglia polarization to M2 phenotype. Therefore, these receptors and miRNAs in microglia could be potential therapeutic targets for modulating microglia activation and polarization, and eventually promote tissue repair.

Please cite this article in press as: Ma, Y., et al., The biphasic function of microglia in ischemic stroke. Prog. Neurobiol. (2016), http:// dx.doi.org/10.1016/j.pneurobio.2016.01.005

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a potential for microglia-targeted therapy (Ponomarev et al., 2013). More and more current studies demonstrated that there are biphasic communications between microglia and other neuronal cells including neurons, astrocytes, and oligodendrocytes/OPCs (Eyo et al., 2013; Inose et al., 2014; Ransohoff and Brown, 2012; Rosito et al., 2014; Schmitz et al., 2014; Wang et al., 2013; Xing et al., 2014). Exclusively suppressing microglia activation might compromise the protective effects of microglia and therefore not a suitable therapeutic strategy for ischemic brain injury. There is an urgent need to establish the best strategy to modulate microglia activation and drive microglia polarization to a protective phenotype at different stages of ischemic stroke (Fig. 2). Stem cell transplantation shows a promising future for cell-based therapy. Many studies showed that transplanted stem cells modulate microglia activation and contribute to reducing brain injury (Donega et al., 2014; Hassani et al., 2012; Sheikh et al., 2011; Tang et al., 2014). Whether microglia activation could in turn affect transplanted stem cell survival, migration, and differentiation is largely unknown. Our study indicated that microglia enhance EPC therapeutic effects after focal brain ischemia (Chen et al., 2014). Understanding the detailed mechanism of interaction between microglia and transplanted stem cells would facilitate clinical translation of stem cell therapy for ischemic stroke.

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Conflict of interest statement

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None. Acknowledgements

1574 Q6 This work was supported by China 973 Program 2011CB504405 1575 Q7 (GYY, WY), NSFC 81070939 (GYY) and U1232205 (GYY). 1576

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