2.1 Phenotype of Microglia After IS

In 1919, with the breakthrough in specific silver staining techniques, Río-Hortega precisely described the morphological features of the third largest cell population in the brain, named this population microglia. Río-Hortega carefully noted that microglia experience notable changes in shape following brain damage, establishing a crucial basis for future studies [28].

In the initial phase of acute IS, microglia display unique diversity. Single-cell RNA sequencing analysis revealed three homeostatic groups, as well as five reactive groups which predominantly appeared post-IS. The Mic_M1L1 and Mic_M1L2 groups presented characteristics of M1-like polarization due to increased inflammatory gene expression following IS. Three distinct cell clusters, Mic_np1, Mic_np2, and Mic_np3, exhibit low levels of inflammation and are distinguished by elevated levels of Arhgap45, Rgs10, and Pkm expression, respectively [29]. The phenotypic changes in microglia are not confined to only the M1/M2 polarization state; rather, they can exhibit more complex and diverse phenotypes under different physiological and pathological conditions. Additional investigations are needed to explore the mechanisms underlying microglial phenotypic alterations to develop successful treatment strategies for neurological disorders.

Microglia, the primary immune cells in the CNS, are activated in the context of cerebral ischemia/reperfusion injury (CIRI), which leads to the production of numerous proinflammatory mediators that contribute to secondary damage [30]. On the contrary, microglia can provide further neuroprotection by secreting a range of anti-inflammatory substances and growth factors that are crucial for sustained neuroprotection and recovery from ischemic events [31]. Research indicates that microglial polarization is influenced by various factors, including nuclear factor kappa-B (NF-κB) [32-36]. Typically, NF-κB, which is present in the cytoplasm and bound to the inhibitory protein IκB, is suppressed and forms a dimeric structure. The nuclear localization signal of NF-κB is obscured by IκB, which renders it inactive. In the context of cerebral ischemic damage, cells are activated, leading to the degradation of IκB proteins through phosphorylation. Through exposure to a nuclear localization signal, NF-κB is activated and translocates to the nucleus, where it regulates transcription to increase the synthesis and expression of inflammatory mediators, ultimately worsening brain injury [37, 38]. CIRI is decreased by mesencephalic astrocyte-derived neurotrophic factor (MANF), which regulates the anti-inflammatory phenotype polarization of microglia via the A20/NF-κB pathway [32]. Monocyte locomotion inhibitory factor (MLIF) induces the transition of microglia to the anti-inflammatory phenotype. The NF-κB pathway plays a role in the effects of MLIF by targeting eukaryotic elongation factor 1A1e (EF1A1) [36]. Erythropoietin-producing human hepatocellular receptor A4 (EphA4) upregulation exacerbates brain damage by promoting proinflammatory phenotype polarization of microglia through NF-κB signaling after cerebral ischemia [34]. NF-κB-regulated chemokine-like factor 1 (CKLF1) upregulation in neurons is a key mechanism in promoting proinflammatory phenotype of microglia in the ischemic penumbra [35]. Salvianolic acid D, identified as a polyphenol within the plant Salvia miltiorrhiza Bunge, alleviates CIRI by suppressing the cytoplasmic translocation and release of high mobility group box 1 (HMGB1)-triggered NF-κB activation to inhibit inflammatory response (Table 1); [39]. Additionally, a variety of natural substances, including triptolide, β-patchoulene, ginkgetin, tanshinone IIA, breviscapine, diosgenin, icariin, and berberine, have been shown to protect the brain from ischemic injury. This protective effect is mediated by their ability to inhibit the NF-κB signaling pathway [40-44].

Inhibiting proinflammatory phenotype polarization and promoting anti-inflammatory phenotype polarization of microglia is a suggested therapeutic strategy to reduce neuroinflammation and enhance neuroprotection following CIRI. Microglia proinflammatory phenotype polarization leads to poor prognosis in IS patients and is associated with systemic inflammation. The activation of solute carrier 15A3 (SLC15A3) by p65 and hypoxia-inducible factor 1 alpha (HIF1α) leads to microglial polarization towards the proinflammatory phenotype [45]. By inducing microglia to become proinflammatory phenotype polarized via the AMP-activated protein kinase (AMPK) signaling pathway, long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) suppresses the angiogenic activity of cerebral artery endothelial cells [46]. Patients who experience acute ischemic stroke (AIS) have increased numbers of cerebral microglia that express high levels of Netrin-1 and Unc-5 netrin receptor A (UNC5a) [47]. In vitro, Netrin-1 facilitates anti-inflammatory phenotype polarization of microglia via UNC5a. Swell1 may also be involved in the regulation of the WNK1-SPAK/OSR1-NKCC1 signaling pathway, which helps promote microglial anti-inflammatory phenotype polarization [48]. During CIRI, the JAK2/STAT3 signaling pathway is involved in regulating microglia and macrophage polarization towards the anti-inflammatory phenotype [49]. Protein arginine methyltransferase 8 (PRMT8) promotes the anti-inflammatory phenotype polarization of microglia and reduces neuronal apoptosis to improve CIRI by increasing Lin28a expression [50]. Ac2-26, an ANXA1 mimetic peptide, protects against CIRI by shifting microglial/macrophage polarization from the proinflammatory phenotype to the anti-inflammatory phenotype, possibly by interacting with the FPR2/ALX receptor and activating the AMPK–mTOR pathway [51]. Triggering receptor expressed on myeloid cells 2 (TREM2) is involved in regulating the shift of microglia towards the anti-inflammatory phenotype via a range of signaling pathways including the TGF-β/Smad2/3 pathway [52], the JAK/STAT pathway [53], and the PI3K/Akt pathway [54]. Additionally, lncRNA SNHG8 [55] and Wip1 [56] can regulate microglial activation in CNS disorders (Figure 1).

2.2 Interactions of Microglia With Neurons

After IS, neurons are the primary cells affected and are also engaged in multiple regulatory functions that are closely associated with microglia. In summary, in both pathological and physiological states, neurons have the ability to regulate the activation of microglia through signaling mechanisms known as “On” and “Off” [57]. Following ischemia, the activation of microglia is first initiated by neuronal death [58]. Furthermore, microglia play various roles in the regulation of neurons. One example is the ingestion of nerve cells [59]. Following a cerebral ischemic event, activated microglia engulf dead or deteriorating neurons, which aids in the recovery process [60]. Recent research has indicated that in the penumbra area, the complement pathway directs the phagocytosis of stressed but recoverable neurons by microglia, which suggests promising targets for future therapeutic studies.

2.3 Interactions of Microglia With Other Glial Cells

3.1 Glycometabolism of Microglia

The glycometabolism of microglia primarily involves the transformation between oxidative phosphorylation (OXPHOS) and glycolysis (Figure 2). In the acute phase of IS, due to the sharp decline in cerebral blood flow, along with the resulting ischemia and hypoxia in brain tissue, the glycometabolism of microglia transitions into the glycolysis pathway to meet the energy required for the synthesis of various proinflammatory factors under hypoxic conditions [70-74]. The pentose phosphate pathway is another main pathway of glycometabolism and is interchangeable with glycolysis, shares the G-6-P step, and presents a competitive inhibitory relationship. Its main product is a reduced coenzyme, which dominates the reduction reaction in the process of biosynthesis [75]. Excessive glycolysis in microglia can cause excessive accumulation of lactic acid in local tissues, which lowers the pH value and activates the pentose phosphate pathway to offset the excessive reactive oxygen species produced by glycolysis and maintain the redox balance of cells. Here, we summarize the potential mechanisms underlying microglial metabolic abnormalities in the pathogenesis of IS (Table 2).

TREM2 plays a crucial role similar to that of various phagocytic receptors, moving among the cytoplasm and membrane to regulate microglial phagocytic function and provide immune protection through anti-inflammatory effects [93, 94]. Additionally, TREM2 influences the metabolic well-being of microglia [79], particularly in maintaining homeostasis of glucose, insulin, cholesterol, HDL, and LDL [95, 96]. Research indicates that TREM2 dysfunction results in reduced cerebral blood flow and decreased glucose metabolism in the brain [80]. In ischemic brain tissue, an increased microglial subpopulation shows elevated levels of insulin-like growth factor 1 (IGF1) and TREM2, along with increased OXPHOS activity [79]. IGF1, a 70-amino acid polypeptide hormone [97], is known for its roles in development, proliferation, survival, antiaging, and neuroprotective effects across various diseases [98, 99]. IGF1 acts as a crucial downstream component of TREM2. The TREM2-IGF1 signaling pathway serves as the foundation for microglial metabolism under cerebral ischemia–reperfusion conditions, where it promotes microglial proliferation and exerts neuroprotective effects by increasing the level of OXPHOS in microglia. Understanding the importance of activating the TREM2-IGF1 signaling pathway in microglia is crucial for the treatment of IS.

Studies indicate a significant increase in microglial serglycin (SRGN) levels in the brain after an ischemic event [81]. The SRGN protein influences microglial activity by interacting with the CD44 receptor. SRGN worsens neuroinflammation following stroke and impedes recovery after IS by enhancing the proinflammatory response mediated by microglia. Furthermore, activation of the NF-kB p65 signaling pathway and enhancement of glycolysis in microglia are induced by SRGN. Previous research has identified CD44 as a known receptor for SRGN, which demonstrates its involvement in the immune response [82, 83] and glucose/lipid metabolism [84, 85]. Targeting SRGN could offer new approaches to reduce brain injury after stroke.

Research has indicated that CKLF1 is significantly upregulated following cerebral ischemia [86]. The investigation revealed that CKLF1 triggers immediate microglial inflammation and shifts in energy processing from OXPHOS to glycolysis, which is dependent on the AMP-AMPK–mTOR–HIF-1α pathway [87]. Subsequently, activated microglia transition into a prolonged tolerant phase due to widespread irregularities in energy metabolism, which lead to decreased immune responses such as cytokine secretion and phagocytosis. The weakening of the immune response from microglia worsens the effects of stroke, but this effect could be improved by blocking or neutralizing CKLF1, which suggests a new direction for the treatment of IS.

Microglia need considerable energy to perform various tasks, such as engulfment and monitoring [100, 101]. Homeostatic microglia mainly use OXPHOS to generate ATP, whereas inflamed microglia rely on glycolysis [102, 103]. Resolvin D1 (RvD1) is a lipid mediator known for its strong anti-inflammatory effects. Derived from docosahexaenoic acid (an omega-3 polyunsaturated fatty acid) within the body [104], RvD1 enhances macrophage phagocytosis and reduces inflammation in experimental autoimmune neuritis [88]. Moreover, RvD1 enhances microglial glutamine uptake and stimulates glutaminolysis to support OXPHOS, increasing ATP production contingent on AMP-AMPK activation, while also shifting energy metabolism from glycolysis to OXPHOS, which ensures an abundant energy supply for microglial phagocytosis [89].

Research has shown that reversing high levels of histone H3 lysine 27 acetylation (H3K27ac) inhibits the activity of overactive brain inflammation-related genes [105]. The enzyme AMPK plays a crucial role in producing ATP through aerobic glycolysis [106, 107]. Increasing phosphoglycerate kinase 1 (PGK1) through the p300/H3K27ac pathway encourages microglia to become proinflammatory and inflamed during oxygen–glucose deprivation by controlling glycolysis [108].

Itaconic acid plays a crucial role as a key metabolite in immune metabolic reprogramming in glucose metabolism, which leads to significant impacts on immunity and host defense [76, 77]. Itaconate and its variants suppress tyrosine kinase protein 1, increase macrophage nuclear factor erythroid 2-related factor 2 protein activity, reduce the expression of proinflammatory proteins, and influence the proinflammatory differentiation of microglia and macrophages poststroke. In IS, itaconate can inhibit the activation of adenosine-activated protein kinases in microglia and macrophages, reduce intracellular lipid synthesis and accumulation, and alleviate inflammatory response-induced injury [78, 109].

3.2 Lipid Metabolism in Microglia

The lipid metabolic pathway is an important part of microglial phenotypic regulation. Lipid synthesis metabolism generates the raw materials for inflammatory mediator precursors such as IL-1β, leukotriene, and prostaglandin. The increased breakdown of lipids can lead to the conversion of microglia into an anti-inflammatory state and increase the efficiency of lipid metabolism in microglia [110]. Following a stroke, CD11c+ microglia demonstrate an increased ability to engulf particles, elevated levels of genes related to lipid metabolism (Abca1, Abcg1, Apoe, Apoc1, and Lpl), and genes that support myelin, thereby accelerating the repair of white matter after an IS [110, 111].

Microglia depend on their lipid metabolism to fulfill their energy requirements during activation and when they execute effector tasks [112]. Under hypoxic conditions, cells react to stress signals by activating the transcription factor HIF-1α. Treatment of the cells with cobalt chloride induces microglial activation through HIF-1α. Sterol regulatory element binding protein 1 (SREBP1) plays a crucial role in controlling lipid production, whereas PPARα oversees the breakdown of fats and the absorption of fatty acids. Research has shown that when activated, microglia need continuous lipid production and fatty acid absorption, as shown by increased levels of SREBP1 and PPARα. By depleting antioxidant defense mechanisms, SREBP1 promotes lipid synthesis and enables alternative activation of cells [90]. The oxidative stress observed during microglial activation, characterized by elevated mtROS and iNOS gene expression, may result from weakened antioxidant defense mechanisms in the cells, which lead to the promotion of the phenotypic switch. Additionally, SREBP1-mediated lipid synthesis also plays a role in regulating their phagocytic activity [90].

Microglia lacking TREM2 show a reduced ability to engulf particles and an increase in the storage of cholesteryl ester, which results in the formation of lipid droplets and an increase in the expression of perilipin-2 (PLIN2) following hypoxia (Table 2). Silencing of TREM2 leads to an increase in lipid production (PLIN2, SOAT1) and a decrease in cholesterol elimination and lipid breakdown (LIPA, ApoE, ABCA1, NECH1, and NPC2), which further pushes microglia toward a proinflammatory phenotype and decreased TGF-β1 levels in an IS model [52]. The overexpression of TREM2 in microglia may help alleviate postischemic neuroinflammation and neuronal cell death, at least partially, through the TGF-β1/Smad2/3 signaling pathway and cholesterol production (Figure 2).

3.3 Amino Acid Metabolism in Microglia

While little is known about how the reprogramming of amino acid metabolism affects the ability of microglia to respond to inflammation and mount an immune response, evidence shows that alterations in amino acid metabolism occur when microglia are activated (Table 2). For example, activated, proinflammatory microglia frequently exhibit increased glutamate synthesis, which, through the tricarboxylic acid cycle, can produce a significant amount of succinate. Elevated succinate has been shown to be a feature of proinflammatory microglia [113]. In contrast, Arg-1 is frequently upregulated by anti-inflammatory microglia; the precise function of this upregulation is not yet known, although it is generally linked to greater microglial phagocytosis, neuroprotective benefits, and improved tissue repair [114].

Altering the metabolism of amino acids can lead to modifications in the inflammatory phenotype and immunological response of microglia. On the one hand, the inflammatory response of microglia may be influenced by changes in important enzyme activities related to amino acid metabolism. One of the essential enzymes in controlling the conversion of arginine to succinate is aspartate aminotransferase (AAT), and blocking AAT activity has been shown to lower the generation of proinflammatory molecules, including NO and IL-6, in microglia [91]. L-arginine is converted to L-citrulline by NOS, and NOS enzyme activity inhibition can reduce the level of hazardous NO produced by microglia [92]. On the other hand, glutamine can function as an alternative energy source for microglia and can regulate the immune system. Increasing glutamine intake by microglia can increase the metabolism of glutamine breakdown, increase ATP synthesis in microglia, and promote microglial phagocytosis of neutrophils, which reduces postischemic stroke neuroinflammation [89].