Introduction

Stroke ranks as one of the top causes of death and disability globally.1 Over the past thirty years, there has been a 70% rise in the global incidence of stroke, a 43% increase in its mortality rate, and at the same time, a 32% increase in disability-adjusted life years.2 It is predicted that 12 million people will die from stroke by 2030.3 Stroke is divided into two types. Most strokes are essentially ischemic strokes due to blood clots or lumps blocking blood vessels, while a few strokes are hemorrhagic strokes caused by blood vessel rupture.4 The latter includes intracerebral hemorrhage and subarachnoid hemorrhage.5 Stroke has a significant impact on both physical and mental health, disrupting regular daily activities and leading to economic hardship for families and society. Numerous factors have been identified as controllable risk factors for stroke, including hyperlipidemia, alcohol and drug abuse, smoking, unhealthy diet, and diabetes.6

Even though ischemic stroke has a high incidence rate, the treatment options remain limited. Commonly used treatments include thrombolysis and surgical treatments. Thrombolysis includes intravenous thrombolysis (IVT),7 arterial thrombolysis (IAT),8 and bridging therapy (IVT combined with IAT).9 When it comes to thrombolytic drugs, the FDA only approves thrombolysis with tissue plasminogen activator. The surgical treatment for stroke is mechanical thrombectomy.10 Further research is being conducted on treatments such as antiplatelet therapy, neuroprotective therapy, stem cell therapy, and rehabilitation therapy, yet their therapeutic effects remain limited.6 The significance of inflammation in stroke has been emphasized more and more in recent studies. Moreover, the promotion of effective neurogenesis is increasingly being recognized as a crucial therapeutic approach for stroke treatment. The response of microglia post-stroke is intricate and closely linked to the development and prognosis of the stroke. Microglia, functioning as resident immune cells, rapidly activate after an ischemic injury, displaying both pro-inflammatory and anti-inflammatory characteristics and regulating neuroinflammation through polarization and secretion of various chemokines.11 Microglia-targeted therapy for ischemic stroke has significant potential due to their ability to clear away debris and secrete deleterious factors and trophic cytokines to support tissue regeneration and remodeling.12 Examining the basic roles of microglia in both physiological and ischemic brains, along with their communication with neurons and other glial cells, this review summarizes potential therapeutic targets for ischemic stroke that have been linked to them in the last decade. The article highlights potential therapeutic targets for ischemic stroke associated with microglia over the past ten years.

Physiological Functions of Microglia

Microglia, accounting for up to 10% of the cells in the brain,13 play a crucial role in protecting the central nervous system (CNS) from damage.14 Controversy remains regarding the exact origin of microglial progenitors, which are usually thought to originate from primitive yolk sac macrophages and infiltrate the CNS in the early stages of development.15 Most human microglia are situated in the gray matter, and they typically have a morphology characterized by cells with numerous short, fine processes.16 Microglia also express a number of specific and non-specific molecular markers that manifest different functions in different physio-pathological processes, respectively (Table 1).17 Microglia are typically referred to as “resting” in physiological conditions, whereas their reactive morphology under pathological conditions is referred to as “activated”. However, this terminology has been pointed out to be imprecise, since microglia are constantly responding to changes in their CNS environment (the adjacent tissue cells and the various factors they secrete) in different ways (by continually retracting and extending their ramified processes), even in normal physiological circumstances.18 Microglia also play a unique “sentinel” surveillance role,13 monitoring the quantity of synapses, which are neuronal structures that rapidly transmit information between neurons and transform this information during transmission.19 They also promote synapse formation, prune synapses, phagocytose cellular synapses, control neuronal firings, and tidy away debris, thereby preserving homeostasis in the CNS.20,21 So there is no sudden shift from “resting” to “activated” in response to disease, injury, or other pathological conditions. Therefore, many scholars prefer to rename resting microglia as “homeostatic” and to use “reactive to” or “responding to” (ie, the context) to describe microglial activation.22 Despite the variety of names, the traditional terms “resting” and “activated” are still widely used.

Table 1 Major Markers for Specific Identification of Microglia

Microglia and Phagocytosis

The primary role of microglia is the destruction of entire cells or cellular substructures, particularly synapses.44 When brain tissue is infected or brain cells perish, the parenchyma is left with dead cells and aggregated proteins. Failure to remove these substances can lead to tissue damage as they release reactive oxygen species (ROS) and inflammatory mediators.45 In cases of brain tissue infection or cell death, the parenchyma may accumulate dead cells and aggregated proteins. Therefore, under physiological conditions, microglial phagocytosis is the main way of getting rid of unwanted materials in the parenchyma.46 During development, nucleotides (adenosine triphosphate (ATP), adenosine diphosphate (ADP), or uridine triphosphate (UTP)) released by neurons are involved in synaptic pruning through activation of P2Y12 receptors on microglia. These receptors are upregulated, particularly during ischemic brain injury.47,48 During pathological conditions, neurons also release nucleotides that act as “find-me” signals, directing microglia to the site of damage and promoting their activation.46 Phosphatidylserine (PS), the primary phospholipid in the inner leaflet of the neuronal plasma membrane,49 acts as a crucial “eat-me” signal. Nevertheless, it remains unclear whether the presence of PS alone is adequate to trigger phagocytosis. During cell death/dying, the irreversible translocation of PS to the cell surface can be caused by ATP depletion, intracellular calcium accumulation, DNA damage, and oxidative stress.50,51 This is recognized either directly or indirectly (via bridging proteins that interact with PS) by microglial receptors, which then induce its phagocytosis.52 Several receptors directly recognize PS, including brain angiogenesis inhibitor I, Stabilin2,53 members of the T-cell immunoglobulin and mucin family,52,53 and the phosphatidylserine receptor. Bridging proteins, such as growth arrest-specific gene 6 and milk fat globule-EGF factor 8, bind to PS to facilitate indirect recognition.54 Interacting with low-density lipoprotein receptor-related protein 1 on microglia, cell surface calreticulin serves as an “eat-me” signal to initiate phagocytosis.50,55 Microglia are possibly the sole cells in the brain that express various crucial complement components, including complement component C1q (C1q), complement receptor 3 (CR3), and CR5.56 By binding to cell debris or cells that are meant to be phagocytosed, complement can enhance phagocytosis. C1q, for instance, induces phagocytosis by binding to calreticulin or cell-surface PS to enhance signaling recognition, or directly to the functional phagocytosis CR3 on the surface of microglia.57

The Role of Microglia in Ischemic Brain

Ischemic strokes are typically divided into three stages: the acute phase (within 3 days), the subacute phase (7–10 days), and the chronic phase (weeks to months). The ischemic-damaged brain tissue is comprised of two main regions: the ischemic core and the penumbra. With minimal blood flow in the ischemic core leading to instant neuronal death, the penumbra, accounting for half of the lesion volume, is a hypoperfused area on the periphery of the core.58 Over time, this area gradually transforms into irreversibly damaged tissue, becoming the ischemic core. However, with rapid reperfusion, the penumbra can be salvaged and normal function can be restored.59 We have summarized the manifestations of microglia in the ischemic brain into three main areas: activation, polarization, and crosstalk with other cells (Figure 1).

Figure 1 Microglial behavior after ischemia and their crosstalk with other cells. After ischemic stroke, complement factors are released at the site of the pathology, directing microglia to the lesioned or damaged area. Damage-associated molecular pattern molecules (DAMPs) are released from damaged or dying cells, which activate microglia. In addition to morphological changes, activated microglia polarize in response to different factors towards an anti-inflammatory phenotype that secretes neurotrophic factors and acts as a neuroprotectant, and a pro-inflammatory phenotype that releases deleterious factors that not only injure oligodendrocytes, but also induce A1-responsive astrocytes. At the same time, signals from these cells can, in turn, modulate microglial responses.

Abbreviations: TNF, tumor necrosis factor; IL, interleukin; NO, nitric oxide; ROS, reactive oxygen species; MMPs, matrix metalloproteinases; IFN, interferon; iNOS, inducible nitric oxide synthase; TGF, transforming growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor; VEGF, vascular endothelial growth factor; GLT, glutamate transporter; GDNF, glial-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; BDNF, brain-derived neurotrophic factor; NT, neurotrophins; C, complement; HMGB1, damage-associated molecular pattern molecules; ATP, adenosine triphosphate; PRX, peroxiredoxin; LPS, lipopolysaccharide; IRF, interferon regulatory factor; Glu, glutamate; UTP, uridine triphosphate; HSPs, heat shock proteins.

Microglia and Activation

When cerebral ischemia occurs, neurons in the vicinity of the affected arteries tend to perish rapidly, accompanied by cerebral oedema, blood-brain barrier destruction, and neuroinflammation.60 When cells are under stress and are dying, they begin to emit molecules of danger that have an immediate effect on microglia including ATP, UTP, and damage-associated molecular pattern molecules (DAMPs).61 Since complement factors such as C5a are released at pathological sites, the motility of microglia can be enhanced and directed to the diseased or damaged area through G-protein-dependent pathways and rearrangement of the actin cytoskeleton.62 Purinergic receptors P2X7 and P2Y12 are expressed in microglia and act as key sensors of brain injury, triggering microglia activation upon stimulation.63 They can both be activated by ATP, with activation of the P2X7 receptor triggering microglial proliferation,64 superoxide production,65 and release of interleukin 1β (IL-1β)66 and tumor necrosis factor (TNF)-α.67 Upon activation of the P2Y12 receptor, microglia undergo process extension and then migrate towards the stimulus by interacting with integrin-β1.25,68 Microglia possess Toll-like receptors (TLRs), a group of transmembrane proteins that are essential for detecting and defending against microbials. They can be stimulated by endogenous ligands and agonists, such as high-mobility group box 1 (HMGB1) protein,69 fibronectin, heparan sulfate, galectin-3,70 peroxiredoxin (PRX) family proteins,71 heat shock proteins (HSPs),72 and hyaluronic acid. This triggers the activation of microglia, which increases the expression of several pro-inflammatory genes.73 Prostaglandin E2, an inflammatory mediator, interacts with the G protein-coupled receptor EP2 expressed on microglia. Activation of the EP2 receptor exacerbates the induction of inflammatory mediators such as cyclooxygenase-2, IL-6, TNF-α, and IL-1β.74 The triggering receptor expressed on myeloid cell 2 (TREM2) is an immune-related receptor on the surface of microglia. One of its ligands, HSP60, stimulates TREM2 to activate microglial phagocytosis, and the absence of TREM2 promotes inflammation.75,76 In addition, zinc released from neurons after injury has been shown to trigger microglial activation by sequentially activating nicotinamide adenine dinucleotide phosphate oxidase, poly (ADP-ribose) polymerase-1 (PARP-1), and nuclear factor kappa B (NF-κB).77 However, when microglia become over-activated, it can result in neuronal dysfunction, oxidative stress, and neuroinflammation. This is caused by the excessive production of various cytotoxic factors including ROS, IL-1β, TNF-α, and nitric oxide (NO).78,79 Microglial activation is the initial stage of the inflammatory response triggered by ischemic brain injury, followed by infiltration of various immune cells, such as T-cells, natural killer cells, and neutrophils, all of which exacerbate the inflammatory process.80,81

There is a connection between the morphology and function of microglia as well. Ramified microglia are associated with a homeostatic state, while activated microglia have rounded cell bodies and usually have fewer and shorter processes.22 After stroke, microglia undergo four distinct morphological stages that signify a rising level of activation: ramified, intermediate, amoeboid, and round.82 In the uninjured area, microglia show a ramified pattern, while in the peri-infarct region, intermediate and amoeboid cells can be seen.83 When microglia take on a rounded form, it is a sign of a highly activated state, usually found in the core of an infarct.84 In the acute phase of ischemic stroke, neurons in the ischemic core region experience a lack of glucose and hypoxia, causing rapid activation of microglia within a few minutes.85 At 24 hours, activated microglia can be found in the ischemic core and the boundary region, with their presence increasing in the ischemic core after 72 hours.86 Simultaneously, activated microglia release various inflammatory factors that play a role in a robust inflammatory response.87 Microglial activation reaches its peak on Day 2 or 3 and lasts for several weeks thereafter.60 In addition to the infarct site, activated microglia can be observed in areas further away during the chronic phase.86 In the ischemic core, microglia are primarily activated due to excitotoxic cascade signals,88 while in the peri-infarct regions, their activation is initiated by DAMPs.89 Activated microglia remain in and around the area of the residual infarct for a period of 30 days.90

Microglia and Polarization

Microglia not only undergo morphological changes, but also exhibit different patterns of gene expression in different contexts, species, sex, time, and space, as well as genetic transcriptional profiles, leading to differentiation of cells into different phenotypic groups (including motility, morphology, and ultrastructure).22 Many current studies have used integrative analyses of gene and protein expression, single-cell technologies, and multi-omics to propose new nomenclatures based on the different microglia states observed in various species and models. For example, the disease-associated microglia (DAMs) in Alzheimer’s disease (AD) pathology models;23 microglial neurodegenerative phenotype (MGnD) in several disease models;91 activated response microglia (ARMs) and interferon-responsive microglia (IRMs) in an AD pathology mouse model;92 human AD microglia (HAMs).93 For more information on microglia nomenclature, we refer the reader to the distinguished review “Microglia States and Nomenclature: A Field at Its Crossroads”. The application of this nomenclature is somewhat controversial, and there is insufficient evidence to suggest that “DAM” applies to microglia in all cases of injury. Previous studies have classified microglia into anti-inflammatory and pro-inflammatory phenotypes based primarily on their responses, including pro- and anti-inflammatory characteristics of individual cells and small populations in the vicinity.94 Upon stimulation of TLR or ATP receptors by DAMPs, lipopolysaccharide (LPS) and interferon (IFN)-γ can induce activated microglia to the pro-inflammatory phenotype, resulting in the secretion of numerous pro-inflammatory mediators.95,96 In contrast, fractalkine 16 (CXCL16), IL-4, and IL-13 can trigger a shift in microglia towards an anti-inflammatory phenotype, leading to the secretion of anti-inflammatory cytokines that inhibit neuroinflammation mediated by the pro-inflammatory phenotype of microglia.96–98 The molecular processes underlying microglial polarization are still unclear, though.99 Early in ischemic stroke, microglia and recruited macrophages exhibit an anti-inflammatory phenotype that protects neurons from oxygen-glucose deprivation (OGD).100,101 However, as the stroke progresses, these cells gradually change into a pro-inflammatory phenotype in the peri-infarct regions, which exacerbates the damage that caused by OGD to neurons.100 Hence, it is important to consider the equilibrium between proinflammatory and anti-inflammatory reactions in order to predict the outcome of a stroke.102

Microglia and Secretion

It is thought that microglia, primarily of the pro-inflammatory phenotype, contribute to the advancement or escalation of neuronal degeneration and inflammation in numerous brain diseases. This is a result of their generation of damaging elements such as inflammatory cytokines, NO, and superoxide anions.80 First, in response to an inflammatory stimulus, pro-inflammatory phenotype microglia secrete inflammatory molecules like inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, and IL-6 to trigger a robust inflammatory response.103–105 Nonetheless, data is accumulating that microglial cells, predominantly of the anti-inflammatory phenotype, are able to protect nerve tissue from neuroinflammation by releasing substances that have anti-inflammatory properties.106 For example, CD11c+ microglia appear after behavioral pain hypersensitivity following nerve injury and express insulin-like growth factor 1 (IGF-1), which contributes to pain recovery.107 Two anti-inflammatory molecules, brain-derived neurotrophic factor (BDNF) and amylase 1, can be released by microglia when they are activated by astrocytes during tissue healing.108 In the normal brain, microglial subtypes express neurotrophins from the nerve growth factor (NGF) gene family, which play a role in promoting the development and normal function of neurons and glia. Additionally, they express neurotrophin 3 (NT-3), which encourages phagocytic function and microglial growth.109 Extracellular vesicles (EVs) secreted by microglia in the ischemic cerebral environment are enriched with transforming growth factor β1 (TGF-β1). This molecule stimulates the polarization of microglia into anti-inflammatory phenotypes, thereby aiding in the anti-inflammatory response.110 Furthermore, in response to inflammatory stimuli, microglia express higher levels of neuregulin-1, glial cell-derived neurotrophic factor (GDNF), and their receptors.111

Microglial Crosstalk with Other Cells

Microglia are essential for interactions with different types of cells in the CNS and are in charge of a number of developmental and functional activities, such as the removal of dead neurons and synaptic pruning.112 Acute damage and recovery after stroke can be impacted by the dynamic interactions that occur between microglia and neurons, as well as between other glial cells.113

Microglia and Neurons

Following ischemic stroke, neurons are severely damaged by the effects of ischemia and hypoxia.114 When neurons run out of energy, lactic acid builds up and causes acidosis. By raising H+ concentrations and speeding up the conversion of superoxide anion (O2−) to hydrogen peroxide (H2O2) or the more reactive hydroperoxyl radical (HO2), an acidic environment encourages a pro-oxidant effect.115 ROS are naturally occurring byproducts of oxygen metabolism, including peroxides, free radicals, and oxygen ions.116 One of the main causes of neuronal malfunction and death is excess ROS, which also causes oxidative stress by oxidizing proteins, DNA, and RNA. Lipid peroxidation is another impact of excess ROS.117 As the primary excitatory transmitter in neuron-to-neuron communication, glutamate quickly rises in the ischemic brain as soon as ischemia occurs, with activated microglia being a source.118,119 Ionotropic glutamate receptor overactivation causes excitotoxicity, resulting in the death of neurons.120 By releasing “On” and “Off” signals, neurons can regulate microglial activity based on the normal or pathological conditions.121 Interactions between neurons and microglia involve a range of ligands and receptors.122 For example, CX3CL1 is a neuronally expressed chemokine, and its G-protein-coupled receptor, CX3CR, is predominantly expressed on microglia.123,124 In reaction to pro-inflammatory stimuli, CX3CL1 is secreted and its levels are increased, contributing to neuron-microglia communication. Mice that lack CX3CL1 show smaller infarct sizes and experience better functional outcomes.125 The pyrin domain-containing 3 (NLRP3), which has recently attracted the attention of many researchers, has the potential to induce inflammatory responses and trigger a number of inflammatory pathways.126 Research indicates that CX3CR1 adversely regulates NLRP3 signaling.127 Neurons have the capacity to affect microglial functioning, and a recent study revealed that lipocalin-2 secretion from damaged neurons can cause microglia to shift towards an anti-inflammatory phenotype. In addition, Lipocalin-2 stimulates BBB breakdown, white matter damage, neuronal death, and neutrophil infiltration. It also intensifies inflammation following a stroke.128 Furthermore, ischemia-induced damaged neurons can trigger the release of IL-4, which may then induce microglia to express the IL-4 receptor more strongly. Subsequently, this triggers the transformation of microglia into an anti-inflammatory phenotype.129 Moreover, the role of IGF-1 derived from microglia in maintaining neuronal life has been demonstrated. By encouraging angiogenesis and enhancing cerebrovascular function in ischemic regions, the IGF-1/insulin-like growth factor 1 receptor (IGF1R) axis mitigates ischemia-induced harm by supporting cerebral angiogenesis and neurogenesis.130

Microglia and Astrocytes

Within the CNS, astrocytes are not only the most abundant cell type, but also an integral part of the brain’s innate immune system.131 It has been shown that activated astrocytes exhibit two polarization states: a neurotoxic or pro-inflammatory phenotype (A1) and a neuroprotective or anti-inflammatory phenotype (A2).132 (Similar to the microglia paradigm) A1 astrocytes are considered deleterious due to the up-regulation of synaptically detrimental genes and secretion of toxins, whereas A2 astrocytes up-regulate neurotrophic or anti-inflammatory genes and promote reparative functions, suggesting that they are protective.132,133 Recently, astrocyte subtypes with different expression patterns have been identified using single-nuclei and single-cell studies in different species, diseases and injury models, such as Alzheimer’s disease,134 amyotrophic lateral sclerosis,135 HIV infection,136 and spinal cord injury.137 The emergence of new typing patterns further illustrates the complexity of astrocyte expression in disease development. Microglia are the first responders to injury, and they can trigger astrocyte activation. It has been established that microglial activation is able to induce A1 reactive astrocytes both in vitro and in vivo conditions. This is due to the release of three cytokines: IL-1α, TNF-α, and C1q.132 A1 astrocytes lose their phagocytic function and fail to support neurite growth and synaptogenesis. Instead, they trigger the death of neurons and oligodendrocytes. In contrast, microglia induce the A2 phenotype in astrocytes by downregulating the astrocytic P2Y1 purinergic receptors and forming an astrocytic scar for neuroprotective and repair functions.138 By inducing intracellular signal transduction in astrocytes, activated microglia secrete various pro-inflammatory mediators that either directly initiate or promote astrocytic responses. These mediators include NF-κB, signal transducer and activator of transcription (STATs), and mitogen-activated protein kinase pathways.139 These responses include the formation of scars to exert a protective effect or the release of cytokines to exacerbate inflammatory responses.140 Further activation of distant microglia by astrocytes results in heightened secretion of specific inflammatory cytokines.141 Furthermore, astrocyte-derived IL-33 signals primarily to microglia in physiological circumstances and encourages them to engulf synapses.142 Pathologically, by increasing the production of heme-oxygenase (HO)-1 and lowering intracellular ROS levels in microglia, astrocytes can further regulate the inflammatory response of microglia, thereby preventing excessive inflammatory responses in the brain.143 The extracellular release of C3 is initiated by the activation of the NF-κB pathway, an inflammatory pathway in astrocytes. C3 then interacts with microglial C3aR to facilitate microglial phagocytosis.144

Microglia and Oligodendrocytes

Oligodendrocytes, which form the myelin sheath in the CNS, are particularly susceptible to ischemia.145 After merely 30 minutes of ischemia, there was obvious swelling of oligodendrocytes. By the time 3 hours had passed, a large number of oligodendrocytes had been fatally damaged, occurring before the neurons in the ischemic region met their demise.146 Various mechanisms have been identified as potentially causing harm to oligodendrocytes in ischemic conditions, including a deficiency of trophic factors, oxidative stress, and activation of apoptotic pathways.147 Following hypoxic injury, microglia produce inflammatory cytokines such as TNF-α and IL-1β via the mitogen-activated protein kinase signaling pathway, leading to oligodendrocyte loss.148 In addition, the expression of N-methyl D-aspartate receptor subunits on microglia after hypoxia can lead to oligodendrocyte death by mediating nitric oxide production through the NF-κB signaling pathway.149 Microglia-generated ROS can be damaging to both neurons and oligodendrocytes.150 However, evidence has come to light that microglia have a beneficial effect on oligodendrocytes/oligodendrocyte pro-genitor cells, with the anti-inflammatory phenotype polarization of microglia being the primary source of its protective function. This can drive the differentiation of oligodendrocytes and promote myelin regeneration during myelin regeneration after stroke.151 The pro-inflammatory phenotype of microglia exacerbates oxygen glucose deprivation-induced oligodendrocyte death.152

Treatment Targets for Microglia in Ischemic Stroke

A wave of studies on microglia has been conducted in the last ten years, revealing possible targets for treatment. Overall, we divide them into three categories: adjusting the polarization of microglia to promote anti-inflammatory effects, inhibiting their activation, and controlling how other cells interact with microglia (Table 2).

Table 2 Therapeutic Targets for Ischemic Stroke Associated with Microglia

Promoting Anti-Inflammatory Microglial Polarization

Since microglia show a dynamic response to ischemic injury,100 it has been proposed that inhibiting the pro-inflammatory phenotype may be a plausible therapeutic strategy for cerebral ischemia. Different inducers have been successfully used in in vitro experiments to analyze and regulate the polarization of microglia. Tanshinol borneol ester (DBZ) is a newly developed synthetic compound that has anti-inflammatory and anti-atherosclerotic characteristics. Research has demonstrated that in both LPS-stimulated BV2 cells and mouse primary microglia cells, NF-κB activity is dramatically inhibited by DBZ, which also reduces the production of pro-inflammatory molecules and increases the expression of mediators of anti-inflammatory phenotypes.167 A series of studies on mice with permanent middle cerebral artery occlusion (pMCAO) by Zhang et al discovered that atorvastatin had a positive effect on the defects in sensorimotor function, as well as reducing microglia-induced neuroinflammation through the suppression of proinflammatory polarization of microglia in the peri-infarct cortex.168 What’s more, through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway, ginkgetin and recombinant human fibroblast growth factor 21 (rhFGF21) therapies have been shown to enhance the anti-inflammatory polarization of microglia, which ultimately leads to the suppression of neuroinflammation and the enhancement of neurological function recovery in individuals suffering from ischemic stroke.160,170 In a mouse model of acute ischemic stroke (AIS), Chuanzhitongluo (CZTL), an ancient Chinese medicine mixture, was found to attenuate the inflammatory response by blocking the activation of the NLRP3 inflammasome and promoting a shift of microglia from a pro-inflammatory phenotype to an anti-inflammatory one.171 Research utilizing the transient middle cerebral artery occlusion mouse model (tMCAO) has revealed that IL-13 can enhance the long-term prognosis of ischemic stroke by inducing microglia/macrophages to adopt an anti-inflammatory phenotype, possibly through the inhibition of STAT3 phosphorylation.157,158 The process of microglia/macrophage polarization towards the anti-inflammatory phenotype in cerebral ischemia/reperfusion (I/R) damage has also been demonstrated to be regulated by the janus-activated kinase (JAK2)/STAT3 signaling pathway.158 The study by Qin et al identified Fingolimod (FTY720) as an immune modulator that reduces microglia-mediated neuroinflammation after ischemia and uses the STAT3 pathway to polarize microglia into an anti-inflammatory state, stimulating the development of oligodendrocytes.159

In response to ischemic injury, the body spontaneously generates other anti-inflammatory and pro-inflammatory responses associated with microglia. These responses include the regulation of gene transcription and the production of various functional proteins and molecules. Our goal is to identify these regulators and guide them in a favorable direction to enhance endogenous brain repair. The interferon regulatory factor (IRF) family, particularly the IRF5-IRF4 regulatory axis, has recently been found to be significantly associated with neuroinflammation and the polarization of microglia following cerebral ischemia. Abdullah et al found that IRF4 suppresses inflammation and encourages the anti-inflammatory polarization of microglia, in contrast to IRF5, which induces pro-inflammatory polarization.172 An increasing amount of research has demonstrated that the impact of microRNAs (miRs) on the polarization or activation of microglia in CNS disorders is growing. For example, miR-124 and miR-Let7a have roles in regulating microglia polarization towards the anti-inflammatory phenotype.173,174 MiR-155 has been identified as a pro-inflammatory microRNA. The absence of miR-155 diminishes microglial polarization to the pro-inflammatory phenotype.175 Moreover, exosomes (Ex), which are released by macrophages, have been demonstrated in many studies to regulate inflammation by transitioning gene transcription.176 In contrast, exosomes produced by the LPS-stimulated macrophage RAW264.7 cell line (LPS-Ex) contained higher levels of anti-inflammatory factors and genes (miR-212, miR-21-3p, or miR-21#, and miR-126-5p),177–179 which promoted the polarization of microglial cells from a pro-inflammatory to an anti-inflammatory phenotype and exerted a more potent and neuroprotective effect.154

Knockout models or inhibitors of different inflammatory proteins, signaling receptors, and cytokines generated from microglia may be fully neuroprotective in ischemic brain injury.102 In the brain, innate immune cells (astrocytes, microglia, and oligodendrocytes) are the main sources of colony-stimulating factor 1 (CSF-1). Many CSF-1R inhibitors have been created and are widely used to deplete microglia, such as Dasatinib, PLX3397, and PLX5622.180 In an experiment using tMCAO (30 min) mice as models, investigators assessed the immunomodulatory effects of long-term administration of PLX5622 at different time periods after ischemia through in vivo multimodal imaging. They found that CSF-1R inhibition transiently reduced neuroinflammation within the infarct.181 Recently, continuous CSF-1R inhibition before and after injury has been found in other disease models (tMCAO and pMCAO rodent models, TBI mouse model) to instead exacerbate inflammation and neurological deficits during the first few days of the injury, highlighting the anti-inflammatory role of microglia in the early stage of brain damage.138,182,183 It is not yet clear when inhibition of microglia is most effective, and research into the optimal therapeutic window may become critical for this targeted therapy. In the wounded CNS, microglia selectively express the calcium-activated potassium channel K(Ca)3.1. This channel’s activity is linked to the activation of microglia, which promotes inflammation. By using Senicapoc, a K(Ca)3.1 inhibitor, it could lead to modulation of stroke outcome by interfering with the inflammatory cascade that occurs after I/R.184 Following middle cerebral artery occlusion (MCAO), the stimulator of interferon genes (STING) is activated by mitochondrial DNA (mtDNA) and mediates microglia polarization towards a pro-inflammatory phenotype through IRF3/NF-κB signaling. In a recent study, I/R-induced neuronal injury, edema, and cerebral infarction were reduced by intraperitoneal injection of the specific STING inhibitor C-176 into a mouse model of MCAO.166 Furthermore, after ischemia, there is an increase in TLR4 expression on microglial membranes, which activates NF-κB and leads to a pro-inflammatory response in microglia.185 The neuroprotective effects of TLR4 knock-out mice in focal cerebral ischemia further support the possibility that we may be able to offer a potential treatment strategy for ischemic stroke by altering microglial phenotypes through the TLR4/NF-κB signaling pathway.161,162 In addition, Vx-765, a small-molecule caspase-1 inhibitor, safeguards against MCAO injury and reduces microglia-induced neuroinflammation mainly by altering microglia polarization from a pro-inflammatory phenotype to an anti-inflammatory phenotype.155

There are a number of other manipulations in experiments that have also been suggested to be connected to the anti-inflammatory effects of microglia, and thus, may hold therapeutic promise. In a study, investigators implanted stem cells that produced pituitary adenylate cyclase-activating polypeptide (PACAP) into the brains of mice. After seven days of ischemia, the data demonstrated that this caused microglia to polarize towards an anti-inflammatory phenotype.156 What’s more, it has recently been discovered that cerebral I/R injury-induced neuronal pyroptosis and locomotor impairments can be lessened by the non-invasive neuromodulatory method of repetitive transcranial magnetic stimulation (rTMS). This is achieved by restraining the pro-inflammatory activation and promoting the anti-inflammatory activation of microglia in the peri-infarcted region.163

Inhibition of Microglia Activation

Impeding the activation of microglia after stroke can reduce neuroinflammation. When human umbilical cord blood mononuclear cells are administered systemically after a MCAO in rats, it leads to a reduction in activated microglia and results in a decrease in the infarct size.186 Blocking the cellular P2Y12 receptors could impede microglial activation, resulting in a decrease in proinflammatory cytokine levels. This suggests that P2Y12 inhibitors have additional neuroprotective and anti-inflammatory benefits against ischemic stroke.187 According to Ying et al’s study, photothrombotic (PT) stroke mice have significantly elevated levels of PARP14 (poly (ADP-ribose) polymerase family, member 14) in the proximity of the infarct zone. Overexpression of PARP14 inhibits the transcription of the lysophosphatidic acid receptor 5 (Lpar5) gene, thus inhibiting microglial activation post-stroke.165 Other inhibitors of microglial activation, such as minocycline, ginsenoside Rd, and calycosin, have shown possible beneficial effects.188–190 Researchers combined the histone deacetylase inhibitor MS-275 and resveratrol in the pMCAO mouse model and found that it had a primary direct effect on inhibiting microglia and macrophage activation.191 MiRs can also affect microglial activation, in addition to their involvement in microglial polarization in neurological diseases. For instance, overexpression of let-7c-5p, a highly conserved miRNA, was observed to inhibit microglial activation in mice with experimental stroke, correlating with a decrease in infarction volume and improved neurological outcomes.192 As numerous signaling events are linked to microglial activity during ischemia, it is difficult to fully deactivate microglial activation.141

Regulating the Interaction Between Microglia and Other Cells

Controlling the exchange of information between microglia and other cells more precisely could open up possibilities for new treatments for stroke. Microglia express CX3CR1 as described previously. A deficiency in CX3CR1 has been shown to dysregulate microglial responses in three distinct in vivo models, leading to neurotoxicity. Consequently, enhancing CX3CR1 signaling could serve to protect against microglial neurotoxicity.193 Furthermore, the collaboration between microglia and astrocytes is thought to be a part of the process of forming a glial scar after stroke. In studies conducted in vivo and in vitro, treatment with anti-inflammatory microglial small EVs was observed to inhibit astrocyte proliferation, leading to a decrease in glial scar formation and an improvement in recovery after stroke.110,194 Yun et al reported that NLY01, a potent glucagon-like peptide-1 receptor agonist, can protect neurons by directly blocking microglia-mediated astrocyte transition into the neurotoxic A1 phenotype.195 What’s more, it has been demonstrated that cottonseed oil (CSO) guards against peripheral tissue damage, such as inflammatory bowel disease, through its anti-inflammatory effects.196 To investigate the anti-inflammatory effects of CSOs in stroke, it has recently been discovered that the use of CSO can reduce the severity of ischemic stroke damage by preventing microglial and astrocyte activation and inflammation. This is associated with a decreased activation of the neurotoxic A1 phenotype astrocytes and an inhibition of the TLR4/NF-κB pathway.164 In addition, Wnt-3a, a newly discovered Wnt protein, has been found to specifically enhance the Wnt/β-catenin signaling pathway, influencing cell proliferation and apoptosis. Results from research conducted using the tMCAO mouse model revealed that intranasal Wnt-3a could reduce the toxic effects of microglia/macrophages and astrocytes in ischemic brain injury.169

Conclusion

With the prevalence of ischemic stroke across the world, researchers are dedicating more resources to investigating treatments for stroke, as the major available treatments remain limited. This review gives a quick rundown of the physiological roles played by microglia and how they appear in the ischemic brain, including phagocytosis by microglia, microglial activation and polarization processes, and crosstalk with other glial cells. We summarize the therapeutic targets of microglia to prevent deterioration of the affected area and attenuate the inflammatory response by intervening at different stages of stroke, ultimately improving the prognosis and providing neuroprotection against stroke. With the increasing identification of substances and signaling pathways implicated in the intricate process of microglial activation and polarization following a stroke, it highlights the potential of microglial-targeted therapy. However, the modulation of microglial function following a stroke remains a topic of debate, and many therapies targeting microglia, such as immune regulatory strategies and anti-inflammatory treatments, have only been successful in animal models, such as mice. Because of the many differences in the physiological systems of rodents and humans, and the controversy over whether the microglial cell state found in mice exists in humans, it is essential to study the differences in the types and functions of microglia in humans and animal models. Because of the many differences in the physiological systems of rodents and humans, and the controversy over whether the microglial cell state found in mice exists in humans, further investigation is required before attempting to utilize microglia for clinical applications.

Funding

This study was funded by the College Students’ Innovative Entrepreneurial Training Plan Program (2022183).

Disclosure

The authors report no conflicts of interest in this work.

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