Microglia-Astrocyte Crosstalk in Post-Stroke Neuroinflammation: Mechanisms and Therapeutic Strategies

Abstract

Stroke is a leading cause of severe disability and mortality worldwide. Glial cells in the central nervous system (CNS) not only provide nutritional support but also play crucial roles in the inflammatory response. Microglia and astrocytes, integral components of the innate immune system, are involved in all stages of stroke and are active participants in inducing post-stroke neuroinflammation. Recent studies have increasingly focused on the potential crosstalk between microglia and astrocytes, identifying it as a promising area for understanding the pathogenesis and therapeutic mechanisms of CNS inflammatory diseases. These cells not only undergo dynamic phenotypic changes but also establish an intimate two-way dialogue by releasing various signaling molecules. This review paper elucidates the spatiotemporal dynamics of microglia and astrocytes in post-stroke neuroinflammation and highlights interaction pathways and potential therapeutic strategies for stroke.

1. INTRODUCTION

Stroke is an acute cerebrovascular disease that significantly threatens human health and quality of life worldwide. It ranks as the second leading cause of death globally, following ischemic heart disease, and is the third leading cause of death and disability [1]. In 2019, the incidence of stroke in China reached 276.7 cases per 100,000 population [2]. Stroke is categorized into hemorrhagic stroke (HS, including intracerebral hemorrhage and subarachnoid hemorrhage) and ischemic stroke (IS). IS is the most common type of stroke, accounting for 87% of all stroke cases and 62.4% of global new cases in 2019 [2].

Primary brain injury occurs immediately after stroke, essentially referring to mechanical brain damage, but it triggers a series of complex pathophysiological events, including cellular toxicity, neurotoxicity, oxidative and nitrosative stress, apoptosis, and inflammatory responses, leading to secondary brain injury (SBI) and further exacerbation [3]. Neuroinflammation is a crucial process during stroke, involving a cascade immune response mediated by cellular and molecular components. This includes the activation of microglia, astrocytes, and endothelial cells, as well as infiltration of peripheral immune cells and the release of inflammatory factors [4, 5]. Damage-associated molecular patterns (DAMPs) are molecules released by stressed or damaged cells, recognized by pattern recognition receptors (PRRs) as danger signals. This recognition triggers innate immune responses and initiates inflammatory cascades [6].

In HS, DAMPs originate from blood components in the subarachnoid space and/or brain parenchyma and by-products of plasma proteins (thrombin, fibrinolytic enzymes, etc.). In IS, DAMPs arise from dead and degenerated neurons due to the microvascular system and the blood-brain barrier (BBB) disruption [7, 8]. DAMPs activate innate and non-immune cells, release cytokines and chemokines, and recruit more inflammatory cells. These cells, in turn, upregulate and actively secrete more DAMPs, forming a positive feedback loop that amplifies the inflammatory response. Ultimately, this cascade leads to brain edema, secondary brain ischemia, ischemic-to-hemorrhagic transformation, neuronal apoptosis, and various forms of irreversible brain damage, resulting in serious adverse outcomes (Fig. 1) [9-11].

Fig. (1).

Sources of post-stroke neuroinflammation and inflammatory cascade. Both HS and IS produce DAMPs during the injury process, promoting immune-inflammatory responses in the brain and activating microglia and astrocytes. Activated microglia and reactive astrocytes release IL-1β, IL-6, IL-17, IL-18, tumor necrosis factor α (TNF-α), reactive oxygen species (ROS), reactive nitrogen species (RNS), and matrix metalloproteinases (MMPs). These induce wider activation of brain-resident cells and recruitment of peripheral cells, including neutrophils, monocytes (which mature into macrophages in the CNS), and lymphocytes. These inflammatory cells continuously produce cytokines, ROS, and RNS, changing the permeability of the BBB, forming a positive feedback loop of inflammation, and exacerbating brain damage. (Created by Figdraw).

Microglia and astrocytes play extensive and critical roles in the development of the CNS. Under physiological conditions, they maintain highly bidirectional communication, crucially regulating aspects such as cell numbers, synaptic transmission, and vascular development [12, 13]. Microglia can regulate the proliferation of astrocytes, influence astrocytic control over neuronal dependence, and affect neurotransmission [14, 15]. Astrocytes, in turn, recruit microglia and promote physiological functions of microglia associated with synapses and neural circuits [16]. After stroke, microglia and astrocytes, as two major groups of reactive glial cells, become activated and polarized, exhibiting close interaction in neuroinflammation. Although previous studies have shown interactions and cascading amplification between microglia and astrocytes in neuroinflammation [17, 18], the mechanisms of crosstalk between them in post-stroke neuroinflammation require further elucidation. In this review, we discuss the pathophysiological changes of microglia and astrocytes after stroke, emphasizing the complex communication between these two cells in neuroinflammation and potential therapeutic strategies related to their crosstalk, providing valuable insights and targets for the pathological research and clinical treatment of stroke.

2. MICROGLIA AND POST-STROKE NEUROINFLAMMATION

2.1. Physiological Function of Microglia

Microglia originate from primitive myeloid progenitor cells within the yolk sac and migrate to the CNS during development, constituting 10-15% of neuroglial cells [19]. They maintain homeostasis of the CNS, phagocytize dead cells or cellular debris, participate in synaptic remodeling and pruning, regulate neuronal connections, and repair damage [20].

As regulatory cells in neuroimmune responses, microglia participate in inflammatory reactions and respond rapidly, acting as scavengers. In the resting state, microglia are inactive, with stationary cell bodies and highly branched processes that establish direct communication with other nerve cells and blood vessels for immune surveillance. However, when confronted with pathological stimuli, microglia become activated and respond swiftly to perform inflammatory functions. They transition from a highly branched morphology to amoeboid cells to cope with various challenges [21].

2.2. The Pathophysiology of Microglia in Post-Stroke Neuroinflammation

The response of microglia to different stages of stroke can be characterized by changes in their morphological function and polarization. Polarization is a phenotypic change in microglia induced by pathological stimulation, mainly including the “classically activated” M1 phenotype or the “selectively activated” M2 phenotype, which plays a dual role in the pro-inflammatory/anti-inflammatory and injury/repair processes [22]. Activation of M1 microglia promotes inflammatory response and leads to neurotoxicity, usually induced by lipopolysaccharide (LPS), Toll-like receptors (TLRs), and interferon-γ [23, 24]. Conversely, M2 microglia exhibit anti-inflammatory effects, participating in debris clearance and tissue repair after injury, and are usually induced by IL-4, IL-13, and TGF-β [25]. Apart from stroke, they are also well-represented in a wide range of neurodegenerative and neurotrauma-induced pathologies. Recent research has found that in addition to the classical pro- and anti-inflammatory phenotypes, there are more complex activation states, including dynamically balanced microglia expressing Tmem 119 and Hexb, as well as other subtypes related to neural repair and stroke-associated microglia (SAM) with antioxidant properties [26, 27]. Although this binary classification is too simplistic due to the overlapping functional states and complex, evolved phenotypes of microglia, we continue to use the M1/M2 phenotype in this article for ease of understanding.

After stroke, microglia undergo temporal and spatial changes and can be activated and change their cellular morphology within minutes [28, 29]. During acute phases (<1 day), activation of microglia in the ischemic region first appeared around the lesion and correlated closely with the extent of neuronal damage. Thirty minutes after the onset of permanent middle cerebral artery occlusion (pMCAO), activated microglia were observed at the periphery of the ischemic lesion, characterized by enlarged cell bodies and thickened processes. Over time, microglia spread more widely and gradually extended into the cortex, peaking at 48 h [30]. Activation of microglia could be observed 3.5 h after MCAO reperfusion, whereas microglia in the ischemic core appear 12 h later [31].

In subacute and chronic phases, microglia undergo dynamic phenotypic changes. Perego et al. [32] observed in pMCAO that microglia showed different morphologies and functions at different time intervals. 24 h after ischemic injury, microglia were activated, showing a branched morphology in the peri-infarct region and an amoeboid morphology in the ischemic core. The rounded microglia were widespread in the ischemic area, likely corresponding to recruited peripheral immune cells. CD68 microglia were initially present at the border area and infiltrated the core area by 7 days, displaying characteristics of phagocytic cells that phagocyte neurons. Markers of M2 microglia (Ym1 and CD206) appeared abundantly in the ischemic core as early as 24 h after injury, with their protective effects persisting for at least 7 days before declining. In MCAO mice, researchers observed consistent expression levels of M1 microglial markers CD32 and pro-inflammatory cytokines (such as IL-1β and TNF-α), significantly increased by day 3, peaked at day 14, followed by a decrease and maintaining stability in the ischemic border area; whereas CD206 and Ym1/2 peaked at day 7 and declined rapidly thereafter [33]. Similarly, Hu et al. [34] found that in transient middle cerebral artery occlusion (tMCAO) mice, the gene expression level of M2 microglia was detected at 1-3 days after ischemia, peaked at 3-5 days, decreased at 7 days, and returned to the pre-injury level by day 14. In contrast, the gene expression of M1 microglia was initially low, starting to rise by day 3 and persisting until 14 days after ischemia. Shu et al. [35] also showed that microglia tended to be polarized into M2 phenotype 3 days after tMCAO.

However, in HS, microglia appear to exhibit an opposite polarization trend as in IS. During acute phases, the activation of microglia first occurred at 1 h after collagenase injection [36] and 4 h after autologous blood injection [37]. Activated microglia participate in hematoma phagocytosis. In vitro experiments indicate that a single microglial cell can phagocytize multiple red blood cells in a very short time, and with the increase of microglial density, multinucleated giant microglia/macrophages may appear around the hematoma and the surrounding area [38, 39]. In a collagenase-induced ICH model, Yang et al. [40] found that amoeboid microglia at the hemorrhagic center exhibited an M1 phenotype and selectively secreted IL-1β. Wan et al. [41] discovered that in ICH constructed by autologous blood injection, M1 microglia were activated early, appeared at 4 h, persistently highly expressed for 1-3 days, and declined after 7 days. In a collagenase-induced ICH model, Lan et al. [42] observed a significant increase in M1 (CD16/32⁺) microglia from 24 to 72 h, albeit with a reduced proportion due to a substantial increase in M2 (Ym-1⁺) microglia. In subarachnoid hemorrhage (SAH), Zheng et al. [43] observed a dynamic polarization process of microglia from M1 to M2 phenotypes, with morphological changes from ramified (1-3 days, M1) to spindle-shaped (bipolar microglia expressing both M1 and M2 markers) and finally to amoeboid shape (5-10 days, M2). Gris et al. [44] found in their study that IL-6 levels rapidly increased in both SAH patients and experimental SAH models.

In summary, the ratio of M1/M2 changes dynamically. Microglia transition from the dominant state of M2 to M1 in IS and M1 to M2 in HS (Fig. 2). This also reflects the difference between HS and IS in neuroinflammation, that is, in the early stage inflammatory response is heavier in HS, while ischemia and hypoxia is heavier in IS. Although there is an inflammatory response in IS, the main role may be to promote repair and neuroprotection. With the increase of time and the accumulation of inflammatory factors, neuroinflammation will further aggravate. However, to date, the exact timing and triggers of the phenotypic changes remain unknown. Different models of stroke, the degree of bleeding/infarction, and the size of the lesion are all factors affecting M1 and M2 polarization. In addition, aging gender has also been shown to influence phenotypic changes [45, 46]. Therefore, it is important to assess the time course of M1 and M2 polarization after stroke to further investigate the timing of the most efficient transition to phenotypes, in order to select the optimal temporal switch to reduce inflammation and improve neurological outcomes.

Fig. (2).

Dynamic changes in microglial polarization levels after IS and HS. (a) After IS, M1 microglia increase during the first 14 days and decrease rapidly. M2 microglia increase from day 1, peak on day 3-5, and return to pre-injury levels on day 14. (b) After HS, M1 microglia appear as early as 4 h after hemorrhage, which significantly increase within 3 days and subsequently decrease. M2 microglia begin to increase on day 1 after HS and persist for 1-2 weeks before decline. Evidence supports a switch of the M1 to M2 phenotype during the first 7 days.

3. ASTROCYTES AND POST-STROKE NEUROINFLAMMATION

3.1. Physiological Function of Astrocytes

Astrocytes originate from glial cells of neural precursors and are the most abundant in the CNS, accounting for approximately 20-40% of glial cells [47]. In the past, astrocytes were considered as a “glue-” like function, providing structural and nutritional support for neurons. In recent years, more and more functions have been elucidated. Astrocytes are the main cells in the neurovascular unit and are essential for the formation and growth of cerebrovascular beds and the maintenance of neurovascular function [48]. Astrocytes regulate the release of active molecules such as glutamate (Glu) and adenosine and modulate the excitability and synaptic transmission of neurons. They also control the strength and plasticity of synapses through gap junctions and provide cellular connections between neuronal circuits and blood vessels, which are helpful in regulating local blood flow, maintaining the dynamic balance of extracellular fluid, responding to neuronal activities, and coordinating glucose metabolism [49].

3.2. The Pathophysiology of Astrocytes in Post-Stroke Neuroinflammation

After stroke, under the stimulation of microglia and inflammatory factors, astrocytes are activated and then rapidly proliferate and become hypertrophic, leading to reactive astrocytosis. This is a graded reaction characterized by the increased expression of intermediate filament proteins, including glial fibrillary acidic protein (GFAP), vimentin, and nestin [50]. GFAP is a structural protein, and its expression changes are associated with the proliferation intensity of reactive astrocytes. Within the time window of 2-6 h after stroke, GFAP levels can distinguish between IS and early ICH [51]. In the early stages of IS, aquaporins (AQPs) regulate the rapid membrane expansion and transport of water, causing astrocytes to swell, which leads to the increase of intracranial pressure and the decrease of cerebral blood flow [52]. After HS, blood extravasation activates the coagulation cascade to produce thrombin, which not only leads to the formation of perihematoma edema but also triggers the sustained activation of protease-activated receptor PAR1 in astrocytes. This activation induces rapid remodeling of astrocyte processes adjacent to glutamatergic synapses, including contraction, flattening, proliferation, and foot processes away from excitatory glutamatergic synapses, potentially affecting long-term neuroplasticity and exacerbating brain injury [53].

Reactive astrocytes play a dual role at different stages of stroke. On one hand, they release a large number of inflammatory factors, MMPs, and chemokines to aggravate inflammation and induce neuronal death; on the other hand, they release trophic factors, lipoxins A4 and B4, excitatory amino acids to inhibit neuroinflammation, and regulate the production of microglial inflammatory mediators to promote neuroprotection, exert anti-inflammatory effects and promote synaptic formation and neuronal survival [54, 55]. After stroke, reactive astrocytes usually present two subtypes: the pro-inflammatory A1 phenotype and the anti-inflammatory A2 phenotype. With the deepening of research, the phenotypes of astrocytes are gradually enriched, and according to their differences in gene expression, they can present a variety of activation states from pro-inflammatory to anti-inflammatory rather than simple A1 and A2 polarized phenotypes [56]. Therefore, we used a pro-inflammatory/anti-inflammatory description in place of the A1/A2 phenotype.

After stroke, astrocytes preferentially differentiate into a “pro-inflammatory” phenotype rather than an “anti-inflammatory” phenotype. In the tMCAO mice, within 14 days, the number of perifocal regions C3d⁺/GFAP⁺ cells in the marginal area of ischemic focus increased gradually, and the expression of inflammatory factors such as TNF and IL-6 increased. On day 3 after the stroke, a large number of pro-inflammatory astrocytes were wrapped around the blood vessel wall and destroyed the integrity of the BBB [57]. In MCAO, the number of pro-inflammatory astrocytes in the penumbra of cerebral ischemia increased continuously to the peak on day 14, and the appearance of anti-inflammatory astrocytes was delayed, peaked on day 7, and decreased on day 14 [58]. In the tMCAO model, researchers found that after 72 h, the anti-inflammatory-related transcription predominated over the pro-inflammatory-specific transcripts, suggesting neuroprotective effects [59]. After ICH, the pro-inflammatory astrocyte marker C3 was active and peaked on day 3 [60]. After experimental SAH, the continuous increase of C3 level indicated that astrocytes preferentially differentiated into a pro-inflammatory phenotype [61, 62]. Obviously, the trend of morphology and phenotype of astrocytes after HS and IS is consistent in time and space, but the transformation and peak time of pro-inflammatory/anti-inflammatory phenotype may be affected by different models and disease severity.

Within days after injury, around the lesion, activated astrocytes further form glial scar by secreting extracellular matrix molecules to participate in the regulation of neuroinflammation. Glial scar is a network formed by glial cell processes and thin filament proteins in plasma, whose formation is highly dependent on hypertrophy, proliferation, and cellular overlap of microglia and astrocytes [63]. Finally, abundant proliferating astrocytes form glial scars between damaged areas and healthy tissue. In the early stages, it reduces excitotoxicity by siphoning K⁺ and Glu uptake. By forming physical and functional walls around the lesion, the glial scar closes the injured site, regulates the plasticity of nerves, and controls the local immune response in space and time to inhibit the spread of inflammation [64]. However, in the late stages, the uncontrolled proliferation of glial scars releases pro-inflammatory factors and expresses a series of molecules that inhibit neuronal migration and axonal regeneration, such as chondroitin sulfate proteoglycans (CSPGs), which is not conducive to nerve regeneration [65]. Therefore, the heterogeneity of phenotype and function of reactive astrocytes after stroke affects the progress of inflammation.

4. MICROGLIA-ASTROCYTE CROSSTALK PROMOTES POST-STROKE NEUROINFLAMMATION

4.1. Microglia Activate Pro-Inflammatory Astrocytes

Microglia are more sensitive to pathogens/damage, whereas astrocyte responses are usually delayed and cannot be fully activated in the absence of microglia [66]. After a stroke, microglia preferentially respond to pathological stimuli and secrete IL-1α, TNF-α, and C1q after activation, triggering reactive signals of astrocytes through soluble and membrane-bound signaling molecules, inducing their transformation into pro-inflammatory astrocytes. This leads to neuronal and oligodendrocyte death and forms a neuroinflammatory cascade [54, 67, 68]. Whether through acute CNS injury or systemic LPS injection, microglial activation induces pure pro-inflammatory astrocytes both in vitro and in vivo [67]. C1q is one of the initiating molecules of the classical complement activation pathway, which produces chemotactic C3a and C5a through the complement cascade. On one hand, this attracts neutrophils and monocytes to migrate to the lesion and activates cells to release more inflammatory mediators. On the other hand, it activates astrocytes toward a pro-inflammatory phenotype and contributes to synaptic toxicity [69, 70]. Astrocytes express the class F scavenger receptor Megf10, a receptor for C1q, and can clear apoptotic neurons through the Megf10/C1q pathway [71]. However, under pathological conditions, C1q levels rise sharply, temporarily increasing ROS and NO levels, promoting pro-inflammatory cytokines, and leading to neuronal death [72]. Additionally, astrocytes release C3a, which binds to the C3a receptors (C3aR) on microglia, thereby triggering microglial secretion of more C1q and pro-inflammatory factors [73]. Deleting C1q in microglia not only prevents the deposition of C3a on astrocytes but also reduces the upregulation of TLR4 in microglia [74]. Activated microglia also release interleukin, monocyte chemotaxis protein-1 (MCP-1), and macrophage colony-stimulating factor (M-CSF), which is crucial in the initial triggering and regulation of astrocytes during acute phases [75].

TNF-α and IL-1 not only act as inflammatory factors to trigger reactive astrogliosis but also act as a pro-inflammatory signal amplifier in microglia-astrocytes crosstalk, amplified by the unique physiological structure of astrocytes. In experimental stroke models, TNF-α and IL-1 were overexpressed as early as 2 h after ICH and 24 h after IS [76, 77]. Astrocytes express IL-1β receptor (IL-1R) [78], and IL-1 stimulates the expression of GFAP and hypertrophy of astrocytes, indicating its important role in mediating astrocyte activation [79]. TNF-α receptors exist in astrocytes, and their activation can inhibit the excitatory amino acid transporter (EAAT) on astrocytes, causing increased extracellular Glu concentrations and neurotoxicity, promoting calcium influx and cellular overload. Simultaneously, they activate microglial-mGluR2, promote further release of TNF-α, and exacerbate inflammatory response [80, 81].

SDF-1α, also known as CXC chemokine ligand 12 (CXCL12), and its receptor CXCR4 have been reported to be upregulated in cerebral ischemic penumbra tissue, which attracts inflammatory cells to release pro-inflammatory factors and aggravate brain injury [82, 83]. SDF-1α is a known stimulator of astrocyte proliferation [84]. Studies have confirmed that astrocytes are involved in SDF-1α/CXCR4 autocrine/paracrine signaling [85]. After a stroke, TNF-α amplifies the Glu release cascade through SDF-1α/CXCR4, increases neuronal excitotoxicity, promotes the continuous release of pro-inflammatory mediators (TNF-α, IL-1β, and IL-6) from microglia and astrocytes, and positively feedback regulates the neuroinflammatory microenvironment [86, 87]. The release of TNF-α stimulates microglia to express high levels of HMGB1 and TLR4, increases inflammatory cytokines and NF-κB activity, and promotes the response of astrocytes [88].

In addition to pro-inflammatory cytokines, mitochondria may become a new mechanism of glial cell interactions in neuroinflammation. Mitochondrial fission and fusion, as the basis of mitochondrial quality control, regulate the activity and function of glial cells, ensuring the dynamic equilibrium state of mitochondria in response to changes in stress conditions [89]. After the stroke, excessive mitochondrial fission triggers apoptosis, which is mediated by dynamin-associated protein 1 (Drp1). The key process is the phosphorylation of Drp1 and its subsequent relocalization to the outer mitochondrial membrane and binding to the Fis1 receptor, which subsequently promotes microglia-induced neuroinflammation [90, 91]. Mitochondrial morphology becomes discontinuous after accelerated fission, leading to mitochondrial fragmentation, and these microglial extracellular mitochondria activated by DAMPs are a driving force for the progression of secondary neuronal damage, participating in communication with astrocytes [92]. Recent research showed that under pathological conditions, DAMPs and neurotoxic proteins further activated microglia, induced excessive mitochondrial fission mediated by Drp1/fis1, and mitochondrial dysfunction. This released fragmented extracellular mitochondria and generated diffusion signals to astrocytes into a neurotoxic phenotype, exacerbating pathogenic inflammation [93]. Liu et al. [94] found that in tMCAO, M1 microglia released damaged and unhealthy mitochondrial debris, transferred to neurons, and fused with neuronal mitochondria, deteriorating neurological prognosis. To date, the potential interaction of intercellular mitochondrial transmission in glia-glia and glia-neuron after stroke remains unclear and warrants further investigation.

4.2. Activated Astrocytes Induce M1 Microglia

Astrocytes activated by microglia lose many physiological functions and release inflammatory cytokines, chemokines, ATP, and specific proteins that act on microglia, thus forming a paracrine feedback loop that aggravates neuroinflammation.

4.2.1. IL-17A/IL-17RA

IL-17A and its receptor IL-17RA are involved in post-stroke inflammation and disruption of the BBB. IL-17A belongs to the IL-17 family (IL-17A~IL-17F), which is a major inflammatory mediator and can induce the expression of inflammatory genes [95]. Astrocytes are the main source of IL-17A production. During acute phases of stroke, IL-17A is involved in microglial activation and neuroinflammation, promoting the expression of TNF-α, IL-1β, and other inflammatory factors and downstream signaling molecule NF-κB p65 [96, 97]. IL-17A also induces chemokines CXCL1 and CXCL2 to participate in the expansion and recruitment of neutrophils, which aggravates neuroinflammation [98, 99]. IL-17RA exists on the surface of microglia and is the main target of the IL-17 signaling pathway [100, 101]. Studies have shown that the knockdown of IL-17A significantly reduces the activation of microglia and promotes their transformation to the M2 phenotype [102]. Therefore, the IL-17A/IL-17RA pathway induces the formation of M1 microglia after stroke.

4.2.2. IL-15/IL-15R

IL-15 is a cytokine that regulates the proliferation, chemotaxis, and survival of immune cells. In the inflammatory CNS, IL-15 is mainly derived from astrocytes [103, 104]. IL-15 is a factor specifically upregulated after stroke and acts as an immune enhancement regulator. Studies have shown that astrocyte-derived IL-15 in the ischemic brain promotes the migration of T cells to inflammatory tissues, affects the activation and effector function of CD8⁺ T cells and NK cells, aggravates brain damage, and knockdown of IL-15 improves the immune response of the damaged brain [103, 105, 106]. In addition, IL-15 can enhance the Th1 response after acute cerebral ischemia, which is manifested by an increase in the number of CD4⁺ T cells [105]. The IL-15 receptor (IL-15R) is a heterotrimeric receptor composed of three chains: IL-15Rα, IL-2/IL15Rβ, and γc [107]. Intracellular IL-15 transmits signals through a “trans-presentation” mode, binding to high-affinity IL-15Rα to form a complex, triggering signal transduction through IL-15Rβ and γc on neighboring cells [108]. Microglia express IL-15R and thus could be receptive to IL-15 from astrocytes to form crosstalk [109]. Recent studies have shown that IL-15 plays a mediator in microglia-astrocyte crosstalk after ICH, increasing the expression of CD86, IL-1β, and TNF-α from microglia and skewing microglia toward a pro-inflammatory phenotype. Astrocyte-derived IL-15 mainly affects the response of microglia. After the elimination of microglia, the aggravation of brain injury caused by IL-15 is attenuated [110].

4.2.3. CCL2/CCR2

Chemokine ligand 2 (CCL2), also known as MCP-1, is the main endogenous agonist of CCR2 and is involved in the inflammatory response after CNS injury. In neuroinflammation, CCL2 is mainly derived from astrocytes and can bind to CCR2 to induce microglial activation [111, 112]. After stroke, CCL2/CCR2 induces microglial recruitment, increases leukocyte infiltration and the expression of inflammatory mediators, further aggravates damage to the BBB, and leads to brain edema and neuronal death [113, 114]. Microglia express CCR2 [115]. In in vitro cell culture experiments, TNF-α stimulation causes astrocytes to release a large amount of CCL2, which enhances the ability of microglia to migrate to the site of injury through the CCL2/CCR2 pathway and induces their polarization to the M1 phenotype. The above phenomenon is inhibited after CCL2 siRNA or the use of CCR2 inhibitors [116].

4.2.4. GM-CSF/GM-CSFR

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is mainly produced by astrocytes, which is a potent activator of microglia and participates in the pro-inflammatory process [111, 117]. GM-CSF exerts its biological function by binding to the GM-CSF receptor complex. GM-CSFR is highly expressed in microglia in the CNS [118]. GM-CSF can cross the BBB and may mediate the pathogenic effects of neuroinflammation [119]. Although GM-CSF itself can not induce the secretion of classic inflammatory cytokines like IL-1β or TNF-α, it can be achieved by acting directly or indirectly on microglia. GM-CSF promotes microglial migration through the upregulation of cathepsin, MMP-9, -11, and -12, thereby triggering excessive inflammatory responses [120]. Studies have shown that GM-CSF increases the expression of TLR4 and CD14 in microglia by activating ERK1/2 and p38 and promotes neuroinflammation by increasing the production of LPS-induced inflammatory mediators (IL-1β, IL-6, TNF-α, NO) [118]. In a microglia-astrocyte co-culture system, GM-CSF stimulates the proliferation of microglia in vitro and induces polarization of M1 microglia [121].

4.2.5. S100B Protein

S100B, a member of the S100 protein family, is predominantly expressed in astrocytes in the brain. It is an alarm protein released during brain injury and acts as an intracellular regulator and extracellular signal and participates in various cellular processes such as cell proliferation, differentiation, apoptosis, inflammation and metabolism [122, 123]. Under physiological conditions, S100B has trophic effects on neurons and promotes Glu uptake. However, it increases after stroke, inducing migration and morphological changes of microglia, upregulating the expression of pro-inflammatory transcription factors, and stimulating the release of MMP-9 and NO, leading to cytotoxic effects and SBI [124]. Extracellular S100B may upregulate chemokine expression through RAGE and stimulate microglial migration, thus participating in the dissemination of inflammation in the brain [125]. Zhou et al. [126] showed that in MCAO, S100B induced excessive production of INOS and high-level release of NO, activated NF-κB, promoted the polarization and migration of M1 microglia, and aggravated inflammatory damage after cerebral ischemia. The study found that the administration of Arundic Acid (AA) to inhibit S100B could prevent the increase in microglial activation after injury [127].

4.2.6. Connectin 43

Connexin 43 (Cx43), composed of gap junction and hemichannel functional units, is one of the main gap junction proteins in astrocytes, involved in maintaining cellular and tissue homeostasis [128]. In the early stages of stroke, Cx43 is involved in indirect interactions between astrocytes and microglia, mediated through a vicious cycle involving Cx43 hemichannel activation. On one hand, activated microglia release pro-inflammatory factors (such as TNF-α and IL-1β), increasing the permeability of astrocytic Cx43 hemichannels, leading to the release of ATP, Ca²⁺, and Glu, and regulating downstream signaling intermediates (including STAT3, p38 MAPK, NF-κB), thereby enhancing the proliferation of reactive astrocytes and activation of M1 microglia, intensifying pro-inflammatory activity [129, 130]. On the other hand, ATP can activate purinergic receptors, known as P2 receptors, to enable the continuous activity of the inflammasome in various brain cell types, including microglia, promote the spread of ischemic brain injury, and aggravate neuroinflammatory injury [131]. After a stroke, P2X7 receptors on microglia are activated by ATP, triggering the release of IL-1β, IL-18, and TNF-α, promoting apoptosis and contributing to deep ion imbalance during neuronal death [132, 133]. Recently, researchers have developed genetically encoded fluorescent sensors for monitoring the spatiotemporal dynamics of ATP in vivo. Results indicated functional specialization and coordination between astrocytes and microglia, with astrocytes sensing damage and encoding injury information and microglia subsequently decoding this information to adapt to various stimuli and drive changes in their activation states [134].

4.2.7. PRDX6-iPLA2

Peroxiredoxin 6 (PRDX6) is the sole 1-Cys member of the peroxiredoxin family, functioning as a dual enzyme with glutathione peroxidase and Ca²⁺-independent phospholipase A2 (iPLA2) activities. Increasing evidence suggests that PRDX6 is predominantly expressed in astrocytes [135, 136]. In primary cultured astrocytes, the iPLA2 activity of PRDX6 induces its own proliferation and increases pro-inflammatory factors [137]. Shanshan et al. [138] observed in OGD/R co-cultures of microglia/neurons and in the MCAO model that PRDX6-iPLA2 is associated with the secretion of neurotoxic inflammatory mediators in microglia, potentially regulating neuroinflammation via TLR2/4. Downregulation of PRDX6-iPLA2 significantly mitigates neuronal damage in co-cultures. Recently, Peng et al. [139] found that after cerebral ischemia-reperfusion injury, PRDX6-iPLA2 activity is significantly upregulated in astrocytes, inducing ROS production and further promoting M1 microglial polarization, revealing the mechanisms of astrocyte-microglia crosstalk and oxidative stress-neuroinflammation crosstalk. Future research should explore how PRDX6 serves as a multifaceted communicator between astrocytes, neurons, microglia, and other brain cells.

5. MICROGLIA-ASTROCYTE CROSSTALK REPAIRS POST-STROKE NEUROINFLAMMATION

The inhibition of inflammation between microglia and astrocytes can be achieved through cytokines, transcription factors, specific proteins, extracellular vesicles, and mitochondria.

5.1. Microglia Regulate Astrocytes

5.1.1. IL-10/IL-10R

IL-10 is a key anti-inflammatory cytokine, produced not only by Th2 cells or Treg cells but also acts as a feedback modulator of various immune responses [140]. In IS, IL-10 is produced by regulatory T cells, macrophages, and microglia [141]. In ICH, microglia-derived IL-10 can trigger a series of intracellular signal transduction events, activating signal transducer and activator of transcription 3 (STAT3)-dependent pathway to accelerate hematoma clearance and inhibit the production of pro-inflammatory cytokines [142, 143]. Clinical trials have shown that IL-10 can serve as an independent predictor for stroke-related infections and may predict the prognosis in patients with acute IS [144, 145]. In IS, IL-10 not only controls the IL-17A-driven inflammatory response in peripheral immunity but also directly suppresses the production of IL-17A in γδ T cells in the ischemic brain [141]. The IL-10 receptor (mainly IL-10 Ra) is widely expressed in various cells. Studies have shown that IL-10R on the surface of astrocytes can bind to IL-10 produced by M2 microglia, stimulate astrocytes to secrete TGF-β, further increase the expression of microglia CXC3R1 and IL-4α, and attenuate microglia activation in the feedback loop, thus exerting anti-inflammatory effects [146]. Moreover, another study has found that IL-10 can alleviate lipid ROS accumulation and iron death in oligodendrocyte precursor cells (OPCs) after ICH, suggesting a novel mechanism of cell crosstalk under inflammatory conditions [147].

5.1.2. IGF-1/IGF-1R

Insulin-like growth factor-1 (IGF-1) in the CNS is primarily derived from microglia and has been shown to promote neurotrophic effects and vascular regeneration after experimental IS and reduce brain injury. Astrocytes and neurons, typically acting as target cells, overexpress the IGF-1 receptor after brain injury, participating in intercellular interactions [148, 149]. IGF-1 receives signals from IGF-1R and exerts its growth and metabolic effects through the downstream PI3K/Akt pathway [150]. Recently, using spatial transcriptomics and transcriptome sequencing, researchers identified potential IGF-1/IGF-1R ligand-receptor pairs between microglia and astrocytes in a collagenase-induced ICH model. This interaction was particularly strong at day 7 after brain injury, concentrated in the core lesion area where the cells were located. Further studies revealed that early after ICH, IGF-1 derived from microglia induced neuroprotective scar formation in astrocytes through mTOR signaling activation [151]. Thus, IGF-1/IGF-1R/mTOR mediates microglia-astrocyte crosstalk and attenuates neuroinflammation.

5.1.3. ZEB1

Zinc finger E-box binding homeobox-1 (ZEB1) is a member of the transcription factor family involved in epithelial-mesenchymal transition (EMT), driving cellular plasticity in tissues and regulating cell differentiation and specific cellular functions [152, 153]. ZEB1 plays a dual role in inducing and eliminating inflammation in microglia. After cerebral ischemia, ZEB1 is highly expressed in microglia and highly associated with its larger branched morphology, which favors the pro-inflammatory phenotype [154]. Conversely, ZEB1 induction promotes neuroprotection and cell survival in the neocortex after cerebral ischemia [155]. Studies have shown that ZEB1 limits inflammation by regulating macrophage metabolism, inhibiting mitochondrial translation and ROS production, and promoting macrophage transition to an immunosuppressive state. Recent research indicated that ZEB1 also acts as a medium in the interaction between microglia and astrocytes to attenuate neuroinflammation. In the tMCAO model, microglia-derived ZEB1 was upregulated in the ischemic penumbra, inhibiting astrocytic CXCL1 production through a TGF-β1-dependent pathway, thereby reducing neutrophil infiltration [156].

5.1.4. Microglial-Derived Extracellular Vesicles

Extracellular vesicles (EVs) refer to double-layered membrane vesicles detached from the cell membrane or secreted by cells. According to their size and biogenesis, they are divided into exosomes, microvesicles, and apoptotic bodies [157]. EVs are a mechanism of intercellular communication, a carrier where cells exchange proteins, lipids, and genetic information [158]. In the CNS, the specific release of EVs is thought to signal brain injury. In tMCAO mice, miR-124 in M2 microglial small EVs reduced glial scar formation and astrocyte proliferation and activation through miR-124/STAT3 signaling. Simultaneously, it decreased Notch1 expression, increased Sox2 expression, participated in the transformation of astrocytes into neural progenitor cells, alleviated neuroinflammation after stroke, and promoted neurological function recovery [159]. Xin et al. [160] experimentally demonstrated that microglial-derived EVs after hypoxia attenuated focal ipsilateral reactive astrogliosis and aggregation of AQP4 on the plasma membrane of cortical astrocytes, and alleviated neuroinflammation in the peri-infarct cortex.

5.2. Astrocytes Induce M2 Microglia

5.2.1. IL-33/ST2

IL-33 belongs to the IL-1 family. Once released upon cellular damage, it binds with the heterodimeric receptor ST2, participating in the immunomodulatory mechanism [161, 162]. CNS injuries can trigger damaged brain cells to release IL-33, primarily produced by oligodendrocytes and astrocytes [163, 164], while ST2 mainly colocalizes with microglia [165]. Under physiological conditions, IL-33 produced by astrocytes is involved in regulating the maturation and reconstruction of neural circuits by microglial cells to clear synapses and maintain synaptic homeostasis [166]. IL-33 is an important endogenous regulator in stroke, which can downregulate the level of pro-inflammatory cytokines, inhibit apoptosis and autophagy, and exert anti-inflammatory effects on brain edema caused by ICH [167]. In IS, activation of the IL-33/ST2 signaling pathway can cause an anti-inflammatory response in microglia, induce M2 microglial polarization, inhibit the expression of pro-inflammatory cytokines, and stimulate microglia to produce IL-10, thereby protecting ischemic neurons and reducing inflammatory responses. Furthermore, IL-4 released from neurons after stroke may synergistically regulate microglial responses with IL-33, providing neuroprotection [168, 169].

5.2.2. ORM2

Orosomucoid (ORM) is located in the endoplasmic reticulum and is an acute phase response protein. It has various biological activities, such as involvement in protein quality control and co-regulation of lipid status, modulation of immunity, and maintenance of capillary barrier integrity [170, 171]. ORM reduces the production of pro-inflammatory cytokines in the ischemic penumbra, decreases malondialdehyde levels, significantly alleviates inflammation, improves BBB permeability, and shifts the balance from oxidative stress to antioxidant defense, thus preventing stroke [172]. ORM2 is the major subtype involved in IS and is believed to be protective. Jo et al. [173] found that in the LPS-induced neuroinflammation model, astrocytes are the major cellular source of ORM2 in the inflamed brain. ORM2 inhibits microglial activation and migration by blocking the interaction between CCL4 and CCR5, which is critical for its anti-inflammatory function.

5.2.3. Astrocyte-Derived Extracellular Vesicles

After stroke, astrocyte-released extracellular vesicles (EVs) serve as vehicles for information transmission to establish communication with microglia. Liu et al. [174] found that in the oxygen-glucose deprivation (OGD) cell model, miR-29a in astrocyte-derived EVs downregulated TP53INP1 and inhibited NF-κB/NLRP3 pathway in microglia-related inflammatory response, reduced ischemia-reperfusion injury, and improved neurological function in rats after ischemia. Moreover, exosomes from astrocytes enriched with miR-873a-5p [175] and miR-148a-3p [176] could significantly inhibit LPS-induced microglial inflammation and promote M2 microglial polarization by reducing the phosphorylation of ERK and NF-κB p65, thus ameliorating neurological deficits.

5.2.4. Mitochondrial Transfer

The mechanism of mitochondrial exchange for glial crosstalk after stroke is gradually being elucidated. Early studies by Hayakawa et al. [177] found that astrocytes released extracellular mitochondrial particles after cerebral ischemia to adjacent neurons, promoted survival and plasticity after injury, which was mediated by a calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling. Recently, Jung et al. [178, 179] discovered that after ICH, astrocytes released intact mitochondria and small bioactive peptide humanin (HN) into neurons, which restored antioxidant defense mediated by the neuronal mitochondrial enzyme manganese superoxide dismutase (Mn-SOD) and enhance neuronal plasticity. Mitochondria and HN released by astrocytes could also enter into microglia, promoting a “reparative” microglial phenotype, enhancing microglial phagocytosis, and reducing neuroinflammation. Thus, mitochondrial transfer has emerged as a novel mechanism in the interaction between microglia and astrocytes (Fig. 3).

Fig. (3).

Pro-inflammatory and anti-inflammatory pathways related to stroke in microglia-astrocyte crosstalk. (Created by Figdraw).

6. COMMUNICATION BETWEEN PERIPHERAL INFLAMMATORY CELLS AND MICROGLIA/ ASTROCYTES IN POST-STROKE NEUROINFLAMMATION

In addition to CNS glial cells, the infiltration of inflammatory cells plays a crucial role in the onset and progression of post-stroke neuroinflammation. Neutrophils are the first peripheral cells to infiltrate the lesion after stroke onset. They enhance neurotoxicity by releasing various mediators such as MMPs, ROS, IL-1β, collagenase, etc., and participate in microglia-astrocyte crosstalk [180]. Pro-inflammatory cytokines stimulate astrocytes to secrete more chemokines, leading to neutrophil migration, which depends on transforming growth factor beta-associated kinase 1 (TAK1) signaling [181]. Upregulation of transient receptor potential canonical 1 (TRPC1) in microglia stimulates the production of CCL5/2, resulting in increased neutrophil infiltration in the brain, thus aggravating neuroinflammation [182]. However, neutrophils are not merely harmful but may exhibit “functional plasticity” properties similar to macrophages, with a dual functional phenotype of pro-inflammatory (N1) and anti-inflammatory (N2) [183]. Recent evidence suggests that after stroke, microglia clear infiltrating neutrophils through phagocytosis, thereby alleviating neutrophil-mediated neurovascular destruction after brain injury [184, 185]. Inhibition of microglia by blocking colony-stimulating factor 1 receptor (CSF1R) significantly increased neutrophil infiltration in the ischemic core, exacerbating inflammatory responses [186]. Zhao et al. [187] found that microglia-derived IL-27 promoted the polarization of polymorphonuclear neutrophils to a less neurotoxic phenotype to limit injury.

After the stroke, astrocytes can promote the migration and infiltration of monocytes from circulation, spleen, and bone marrow into the brain parenchyma, mainly through the CCL2/CCR2 axis. This leads monocytes to differentiate into macrophages at the lesion while also stimulating the production of more M1 microglia, thus exacerbating inflammation [188]. However, monocytes/macrophages (MMs) can transform into an anti-inflammatory phenotype during the post-stroke recovery period, which depends on anti-inflammatory macrophages, particularly under the influence of IL-13, to alleviate neuroinflammation after IS [189, 190]. MMs secrete osteopontin (OPN) in ischemic tissues, inducing astrocytic processes to extend and cover the lesion core, reducing the continuous leakage of the BBB, and aiding in the resolution of edema [191].

Peripheral lymphocytes were stimulated, with an increase in CD4⁺/CD8⁺ T cells and B cells observed within 4 days after stroke [192]. After cerebral ischemia, microglia could stimulate activated CD4⁺ T cells to differentiate into Th1 or Th2 cells, which in turn produced pro-inflammatory or anti-inflammatory cytokines to modulate inflammation [193]. Microglia-derived chemokines CCL2/CCL8 promote cytotoxic CD8⁺ T cell infiltration and aggravate brain injury [194]. Treg cells express high levels of amphiregulin (Areg) and OPN, which are thought to inhibit IL-6 and STAT3 pathways in microglia and astrocytes after stroke and reduce inflammation [195]. However, microglia can, in turn, induce the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in Treg cells through cell-to-cell contact, increasing Sirt2 expression in Treg cells and inhibiting their anti-inflammatory function [196]. Additionally, γδ T cells co-stimulated astrocytes to secrete CXCL1 through IL-17A and TNF-α, resulting in increased recruitment of neutrophils and exacerbation of inflammation [197].

7. THERAPEUTIC STRATEGIES

7.1. Targeting M2 Microglial Polarization

In the early stages of stroke, changes in signaling pathways after brain injury directly impact the type of microglia. Common signaling pathways involved in microglial polarization include AMPK, NF-κB, JAK/STAT, Notch, TLRs, and PPAR-γ. These pathways have been extensively studied and confirmed as therapeutic targets for stroke [198]. Baicalein and α-lipoic acid inhibit microglial NF-κB signaling by regulating the phosphorylation and nuclear translocation of p65, suppressing the expression of M1 markers, promoting M2 polarization, and reducing the release of pro-inflammatory cytokines such as IL-6, IL-18, and TNF-α [199, 200]. Moreover, MMPs and NLRP3 inflammasomes are key factors in regulating microglial polarization. Sinomenine can inhibit microglial caspase-3 activity, attenuate MMP3/9 expression, promote M2 polarization, and regulate neuroinflammation [201]. Mitoquinone promotes the transformation of microglia into the M2 phenotype after ICH by inhibiting the mitochondrial ROS/NLRP3 inflammasome pathway and increasing the high expression of CD36 on cells associated with hematoma absorption, thus improving neurological function [202].

7.2. Targeting Neuroprotective Astrocytes

After stroke, depending on time and environment, astrocytes may exacerbate injury or promote repair. Studies have shown that reduced astrocyte response is often associated with smaller infarct areas. Therefore, therapeutic strategies targeting astrocytes are mainly focused on inhibiting their activation, enhancing their anti-excitotoxicity, and promoting the activation of an anti-inflammatory phenotype. For example, inhibition of cycle-dependent kinases reduces astrocyte proliferation and neuronal death after MCAO [203]. TGN-020, an AQP4 inhibitor, reduces early brain edema, peri-infarct astrocyte proliferation, and infarct volume [204]. The free radical scavenger Edaravone reduces the number of astrocytes and microglia after propofo-induced brain injury through the BDNF/TrkB pathway and reverses the apoptosis and inflammatory response of nerve cells [205]. Ginsenoside Rb1 inhibits astrocyte activation after ischemia, promotes mitochondrial transfer, and supports neuronal survival [206]. Baicalin protects astrocytic Glu synthase protein from oxidative stress during cerebral ischemia-reperfusion injury, promotes Glu uptake, and resists excitotoxicity [207]. Supplementation of exogenous N-3 polyunsaturated fatty acids reduces pro-inflammatory astrocytes and alleviates neuroinflammation in cerebral ischemia/reperfusion stroke [208]. Kruppel-like transcription factor-4 (KLF-4), an evolutionarily conserved zinc finger transcription factor, is upregulated after cerebral ischemic injury and plays an anti-inflammatory role by regulating NF-κB to promote neuroprotective astrocyte polarization [58].

7.3. Acting on Microglia-Astrocyte Crosstalk

After stroke, microglia are the first to respond to inflammation and can induce amplification of inflammation. Then, could the depletion of microglia phenotype/function address the damage caused by intercellular crosstalk? Microglial survival depends on CSF1R signaling. After cerebral ischemia, depleting microglia with the inhibitor PLX3397 significantly increases inflammatory mediators produced by astrocytes, along with increased leukocyte infiltration and cell death, worsening brain infarction, and neurological deficits. PLX3397 itself does not alter the inflammatory state, indicating that the neuroprotective effect of microglia may stem from their role in suppressing astrocyte responses [209]. Similarly, the depletion of microglia increased neuroinflammation after administration of the inhibitors PLX5622 and AFS98 in the acute phase of cerebral ischemia in elderly mice. Although aged microglia are in a dysregulated state, they appear to have beneficial effects in the early stages of IS [210]. Li et al. [211] found that selective depletion of Arg1-positive microglia exacerbated ischemic injury by promoting inflammatory responses. However, Zeyen et al. [212] found that microglia-specific TAK1 depletion could attenuate post-ischemic neuroinflammation and apoptosis in the acute phase. After ICH, CSF1R inhibition effectively depleted microglia, seemingly affecting only the inflammatory state, improving neurological defects and cerebral edema [213]. Recent studies have shown that in the early stages of ICH, sustained microglial depletion leads to glial scar disorder and enhanced neutrophil infiltration. In the chronic stages, glial scars become destructive, at which time PLX3397 treatment provides significant benefits [151]. This suggests to us that, depending on the type of stroke and the complex temporal dynamics and overall net effects of microglia and astrocytes, it is crucial to deplete microglia at precise time points to regulate neuroinflammation effectively.

Current experiments have demonstrated significant therapeutic effects by acting on one type of cell and then indirectly on another. 10% hypertonic saline (HS) can inhibit the activation of microglial NLRP3 inflammasome and act on astrocytes through IL-1β/IL1R1/pNF-κB signaling pathway, down-regulating the expression of VEGF, reducing the permeability of the BBB, and alleviating inflammation and nerve damage after IS [214]. After ICH, crosstalk between astrocytic C3 and microglial C3aR regulates the C3-C3aR signaling pathway and inhibits microglial phagocytosis of myelin fragments. This process can be reversed by cerium nanoparticle (CeNP) treatment [215]. 2-carbonate-cyclophosphatidic acid (2ccPA), a more stable derivative of CPA, mediates communication between microglia and astrocytes and has neuroprotective effects. Astrocytes treated with 2ccPA reduce the secretion of C3 and regulate microglial differentiation to the M2 phenotype [216]. Glutathione S-transferase 1 (GSTM1) promotes a positive feedback pro-inflammatory loop between microglia and astrocytes during brain inflammation, and specific GST inhibitors may be used as therapeutic agents [217]. Comprehensive research on microglia-astrocyte crosstalk may contribute to innovative therapeutic approaches for stroke treatment and will likely become a focus for future drug development.

CONCLUSION AND PERSPECTIVES

Post-stroke neuroinflammation is dynamic, with moderate inflammation exerting neuroprotective effects, helping to maintain the integrity of neuronal structure and function, promoting synaptic formation, and maintaining homeostasis within the CNS. Conversely, excessive inflammation is neurotoxic, leading to cell death and neural dysfunction, becoming a significant trigger or even a driving force for SBI.

The impact of microglia and astrocytes on neuroinflammation depends on distinct phenotypes and functions. Recent research on microglia-astrocyte crosstalk have provided unique insights into the role of the CNS in health and diseases. These cells engage in a molecular dialogue through secretion. Cytokines released by microglia can determine the function and fate of astrocytes. Astrocytes can influence the morphology, activation state, and function of microglia.

Therefore, we aim to further investigate and reveal the molecular mechanisms and pathways regulating neuroinflammation from a new perspective of microglia-astrocyte crosstalk, providing valuable insights into new therapeutic targets for stroke treatment. Additionally, the spatiotemporal dynamics of microglia and astrocytes may be crucial for understanding this crosstalk. Hence, therapeutic strategies should focus on the timing of microglial depletion and emphasize the transformation of microglia and astrocytes to a “protective” functional phenotype. With advancements in technology, the combined application of single-cell and spatial transcriptomics can more accurately and effectively identify different glial cell subtypes involved in interaction dialogues, infer potential mechanisms and target molecules of interactions between different cell types, and construct dynamic cellular maps for disease treatment, which have been successfully applied in research on various CNS diseases [218-220]. Apart from the interactions between microglia and astrocytes, do similar multidimensional crosstalk also exist among them and oligodendrocytes, neurons, and peripheral immune cells? This remains to be considered and studied, with the goal of fully utilizing the beneficial functions of glial cells, reducing inflammatory responses, and preventing secondary injury.

LIST OF ABBREVIATIONS

2ccPA

2-carbonate-cyclophosphatidic Acid

AA

Arundic Acid

AQP4

Aquaporin Protein-4

Areg

Amphiregulin

BBB

Blood-brain Barrier

C3

Complement C3

CCR2

Chemokine Ligand 2 Receptor

CeNP

Ceria Nanoparticle

CNS

Central Nervous System

CSF1R

Colony-stimulating Factor 1 Receptor

CSPGs

Chondroitin Sulfate Proteoglycans

Cx43

Connexin 43

DAMPs

Damage-associated Molecular Patterns

Drp1

Dynamin-associated Protein 1

EAAT

Excitatory Amino Acid Transporter

EMT

Epithelial-mesenchymal Transition

EVs

Extracellular Vesicles

GFAP

Glial Fibrillary Acidic Protein

Glu

Glutamate

GM-CSF

Granulocyte-macrophage Colony-stimulating Factor

HIF-1α

Hypoxia-inducible Factor 1-alpha

HMGB1

High Mobility Group Box-1 Protein

HN

Humanin

HS

Hemorrhagic Stroke

ICH

Intracerebral Hemorrhage

IGF-1

Insulin-like Growth Factor-1

IL

Interleukin

iPLA2

Independent Phospholipase A2

IS

Ischemic Stroke

LPS

Lipopolysaccharide

M-CSF

Macrophage Colony Stimulating Factor

MCP-1

Monocyte Chemotaxis Protein-1

MMPs

Matrix Metalloproteinases

MMs

Monocytes/Macrophages

Mn-SOD

Manganese Superoxide Dismutase

NK

Natural Killer

NLRs

NOD-like Receptors

OGD

Oxygen-glucose Deprivation

OPCs

Oligodendrocyte Precursor Cells

OPN

Osteopontin

ORM

Orosomucoid

PAR1

Protease-activated Receptor 1

pMCAO

Permanent Middle Cerebral Artery Occlusion

PRDX6

Peroxiredoxin 6

PRRs

Pattern Recognition Receptors

RNS

Reactive Nitrogen Species

ROS

Reactive Oxygen Species

SAH

Subarachnoid Hemorrhage

SAM

Stroke-associated Microglia

SBI

Secondary Brain Injury

STAT3

Signal Transducer and Activator of Transcription 3

TAK1

Transforming Growth Factor Beta-associated Kinase 1

TGF-β

Transforming Growth Factor-β

TLRs

Toll-like Receptors

tMCAO

Transient Middle Cerebral Artery Occlusion

TNF-α

Tumor Necrosis Factor-alpha

TRPC1

Transient Receptor Potential Canonical 1

ZEB1

Zinc Finger E-box Binding Homeobox-1

AUTHORS’ CONTRIBUTIONS

The authors confirm their contribution to the paper as follows: T.S. wrote the first draft of the manuscript. B.K. contributed to conceptualization and manuscript editing. Y.S. contributed to visualization. W.Z. supervised the work. It is hereby acknowledged that all authors have accepted responsibility for the manuscript’s content and consented to its submission. They have meticulously reviewed all results and unanimously approved the final version of the manuscript.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

This work was supported by the grants from the National Natural Science Foundation of China (No. 82074540).

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.