Mechanism of action and therapeutic targeting of microglia in autism spectrum disorder Advanced Neurology

Autism spectrum disorder (ASD) is a complex mental illness with a high incidence and considerable impact. More than half of the affected individuals have self-harm behaviors, resulting in high mortality and morbidity. The impact of ASD on education and employment opportunities, the need for family care and support, as well as the burden on families and society is enormous. The underlying pathogenesis of ASD is still unclear, and effective interventions are lacking. Microglia are key immune cells in the central nervous system (CNS), and they function far beyond classical innate immunity, as they can affect normal neuronal activity by secreting cytokines and pruning synapses through phagocytosis. On the one hand, the abnormal activity of microglia may contribute to the development of ASD; on the other hand, it provides a potential target for intervention and treatment. In this review, we comprehensively analyze the mechanism of action of microglia in ASD development and summarize the current methods for targeting microglia in treating ASD.


Introduction
Autism spectrum disorder (ASD), also known as an autistic disorder, is a developmental brain disorder. The main symptoms are social handicaps, communication disorders, narrow interests, and stereotypical repetitive behaviors [1] . ASD begins before the age of three and manifests significantly thereafter. The vast majority of children require longterm rehabilitation training and special education support. One-third of individuals with ASD may remain mute all their lives, one-half have no sense of safety or need to be restrained in their actions, nearly two-thirds of children with ASD experience bullying, and approximately one-third engage in self-harm behaviors, such as head banging, arm biting, and skin scratching [2] . The average cost for individuals with ASD is approximately $60,000/year throughout their childhood [3] and increases with the onset of intellectual disability. According to the latest screening data on ASD from the Centers for Disease Control and Prevention, one in 54 children is diagnosed with ASD, with a 24% increase in prevalence from 2016 to 2018 [4] .
A century ago, microglia were first recognized as a distinct cell population in the central nervous system (CNS) [5] and were primarily thought to be phagocytes responsible https://doi.org/10.36922/an.v1i3.167

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Microglia in autism spectrum disorder for removing debris during CNS development and in pathological conditions. Microglia-mediated immune inflammation was believed to affect the stability of the CNS [6] . With the advances in genetic technologies and single-cell analysis, the connection between microglia and neurons has become more apparent. Besides being central to the inherent immune response, microglia are involved in a variety of neuropsychiatric diseases; functional abnormalities of microglia have been confirmed in anxiety, depression, and other mental disorders. Therefore, the mechanism of action of microglia in mental illness is one of the trending topics in current research. The modulation of neuronal activity by microglia is evident in many brain diseases [7] .
The dysfunction of microglia may be one of the underlying mechanisms in ASD [8] . Aberrant activity of microglia, abnormal levels of associated inflammatory factors, and certain cellular pathways have been observed in autistic patients and animal models. Both genetic and environmental factors that affect microglia may be involved in the etiology of ASD [9] . The deletion or amplification of certain genes may cause ASD, and changes in the peripheral environment may affect microglia, further aggravating autism-like behaviors.
In this review, we propose that microglia are related to the development and progression of ASD. Several widely accepted theories on the etiology of ASD and ASD models are discussed. We focus on the mechanistic role of microglia in ASD from the perspective of phagocytosis and synaptic pruning, which affect neuronal activity, through the release of inflammatory factors and their influence on inflammatory pathways. We also summarize several intervention approaches for ASD by modulating microglia and analyze the current challenges in treating ASD by regulating microglia in hope that this review will make modest contributions to the early diagnosis and treatment of ASD.

Etiology of ASD and its pathogenesis
The etiology of ASD is still unknown. Epidemiological studies, screening for many possible risk factors, could not identify a direct contributor to the development of ASD. Although the etiology of the disease is not fully understood, genetic factors, manifested as deletion or abnormal insertion of specific genes, and geneenvironment interactions might lead to autistic-like behaviors. For example, genes such as CNTNAP2, NLGN3, and SHANK3 have been shown to be associated with ASD. Cntnap2 knockout mice, Nlgn3 knock-in mice, and partial knockout of Shank3 gene are the common genotypes used to construct ASD animal models. In addition, maternal immune activation (MIA) is believed to be a cause of ASD [10] . Studies have demonstrated that infection during pregnancy, especially in the first trimester, is a risk factor for the development of ASD in offspring [11] . MIA models were induced using polyriboinosinic polyribocytidylic acid (poly[I: C]), lipopolysaccharides (LPS), valproic acid treatment, simulated viral and bacterial infections, as well as other environmental factors to activate the maternal immune system [12] and test possible autistic-like behavioral manifestations in offspring ( Figure 1).
Stimulation of certain physicochemical factors during pregnancy also leads to a significantly increased risk of ASD in offspring. It has been reported that exposure to valproic acid during pregnancy increases the probability of ASD in offspring by 50% [13] . Studies of the early pregnancy diets have shown that propionic acid salt might accumulate in the gastrointestinal tract of pregnant women who consume processed foods that are rich in propionate. Propionate can pass through the placental barrier, bind with G-protein receptors 41 (GPR41) on glial progenitor cells, and cause impaired neural differentiation. Furthermore, the expression of downstream phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is inhibited, and protein kinase B (PKB) pathway is activated to promote the proliferation and differentiation of glia. Subsequently, glial cells continue to produce inflammatory cytokines and release glial fibrillary acidic protein (GFAP) after maturation, thus affecting the development of the fetal nervous system.

Role of microglia in ASD
Microglia, the innate immune cells of the CNS, have high plasticity and can react rapidly during pathological processes. Microglia have been implicated to play a crucial role in the development of ASD associated with CNS inflammation [14] . Recent advances have shown that microglia function far beyond the classical innate immune response and are involved in various processes, such as neuronal excitation, synaptic pruning, and remodeling [15,16] . A growing number of studies have demonstrated that the biological role of microglia has a close association with ASD [17] . As early as 2005, the significant activation of microglia and astrocytes in the brains of autistic patients has been reported by Diana et al. [18] . Since microglia activation is the main cellular response to CNS dysfunction, it is conceivable that abnormal glial cell is inextricably linked to ASD.

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Microglia in autism spectrum disorder importance of the active engulfment of synaptic structures by microglia during neuronal development to maintain normal neuronal functions [20] . In their physiological state, microglia have a role in removing pathogens and maintaining CNS stability, directing the remodeling of brain cells after infection, removing debris in pathogenic infection, as well as restoring affected intracellular and extracellular connections. In addition, 50% of neurons undergo programmed cell death during normal brain development, and microglia dispose of these neuronal corpses through phagocytosis. However, under specific circumstances, the impairment of microglial functions, which include phagocytosis and pruning, may lead to abnormalities in neuronal function [21] , thus resulting in certain autistic manifestations.
At the postsynaptic level, previous studies have identified significant neurological changes in the brain of ASD patients, such as neuronal overgrowth and microglia activation in the frontal cortex [18] . Animal experiments have revealed that autistic mice have significantly higher dendritic spine densities [22] , which could be the result of pruning abnormalities and decreased microglial ability to phagocytose synapses. In addition, the impaired synaptic connections between neurons following infection, which are caused by microglia, may also contribute to ASD [23] .
At the presynaptic level, several studies have shown that the balance between excitatory (E) and inhibitory (I) synapses is disrupted in ASD patients. Appropriate pruning of synapses by microglia is vital to ensure the function of excitatory and inhibitory neurons. Therefore, the aberrant pruning effect of microglia on the E/I balance may be one of the pathological reasons for ASD. It has been confirmed in animal experiments that abnormal microglial phagocytosis will break the balance of E-I synapses and eventually lead to the development of neurodevelopmental disorders and advanced brain dysfunction, such as Alzheimer's disease and ASD [24] . However, the role of E/I signaling in ASD is still controversial. Some studies have found that the function of excitatory synapses is inhibited in Shank2-and Shank3-deficient mice [25] . In contrast, other studies have shown that the synaptic E/I balance in the hippocampus inclines toward excitability [26] . Recent studies have observed the preferential elimination of excitatory synapses by microglia in pathological and physiological brains of mature rats. The impairments of microglia in pruning excitatory synapses would result in a predominance of excitatory signaling compared to inhibitory signaling in the ASD brain. The intervention of the abnormal pruning process will be a practical approach for ASD treatment in the future [16] .

Microglia contribute to the progression of ASD by mediating inflammatory pathways and cytokines
Microglia are key immune cells of the CNS, and the neuroinflammation that is mediated by microglia is

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Microglia in autism spectrum disorder centrally involved in CNS injury and disease processing. Microglia can be categorized into two phenotypes, the pro-inflammatory M1 type and the anti-inflammatory M2 type [27] . M1 microglia, which induce neurotoxicity, can secrete pro-inflammatory cytokines and chemokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. The neuroprotective M2 phenotype contributes to regulating inflammation with specific cytokines, such as IL-10 and transforming growth factor (TGF)-β, as well as neurotrophic growth factors, such as brain derived neurotrophic factor (BDNF) and glial cellderived neurotrophic factor (GDNF) [28] . The balance of M1/M2 paradigm underlies different neuropathological conditions in ASD and is essential for CNS hemostasis. It is widely believed that an abnormal inflammatory response induced by M1 microglia may be an etiology of ASD. Microglia regulate neuroinflammation through specific inflammatory pathways, such as nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), IL-1β, IL-6, IL-8, IL-17, and TNF. The dysregulation of these pathways will affect neuronal activity and result in ASD-like symptoms.

Inflammatory pathways
The inflammatory pathway of NF-κB that is regulated by microglia plays an important role in mediating immune response [29] . The initiation of the NF-κB signaling pathway and the transcription of cytokines involved in directing the immune response are required following an infection to clear the invading pathogenic bacteria. Therefore, the NF-κB signaling pathway is closely associated with inflammatory diseases [30] and regulates the expression of genes encoding immune response proteins [31] . Inflammatory cytokines, infection markers, or stress-activated protein kinases can induce the activation of the NF-κB pathway [32] . Within the CNS, the abnormal activation of the NF-κB signaling cascade leads to an abnormal expression of pro-inflammatory cytokines, which may disrupt normal neuronal activity [33] . A large number of literature works have reported that the activation of microglial NF-κB pathway may be involved in the regulation of ASD [34] .
The MAPK signaling pathway also plays a key role in inflammation and can be activated by cytokines, stress mediators, and inflammatory markers. Microglia activation can also induce the MAPK signaling cascade, which is primarily influenced by the activation of toll-like receptors during inflammation, resulting in the phosphorylation of transcription factors [35] . Once phosphorylated, the transcription factors translocate to the nucleus and transcribe genes encoding pro-inflammatory cytokines, such as IL-6 and TNF-α, thus promoting a neuroinflammatory environment [17] and leading to an increased risk of ASD. Besides, IL-6 cytokines form a positive feedback loop by activating MAPK signaling, thus exacerbating inflammation.

Inflammatory cytokines
The increased release of inflammatory factors is closely associated with the development of ASD. The expression of inflammatory molecules, such as IL-1β, IL-6, IL-8, IL-17, and TNF, has been found to be elevated in ASD, and it has been suggested that the severity of the ASD phenotype might be positively correlated with the proinflammatory cytokine concentration [36] . Among them, pro-inflammatory cytokines IL-1β and IL-6, released by activated microglia, might have a role in neuronal damage and significantly affect ASD [37] . In addition, maternal infection or inflammatory immune activation during pregnancy may drive microglia activation [18] and promote pro-inflammatory cytokine release, which would, in turn, exacerbate the inflammatory response.
IL-1β is a key pro-inflammatory cytokine that is involved in various autoimmune inflammatory responses and cellular activities, such as cell multiplication, differentiation, and apoptosis. The synthesis and release of IL-1β are tightly regulated, and its expression is upregulated [38] in acute inflammatory, chronic inflammatory, and autoinflammatory diseases [39] . Microglia can produce large amounts of IL-1β within the CNS [40] . Abnormal IL-1β levels may be responsible for neurological deficits of the brain in ASD. In a study of serum extracellular vesicles in children with ASD, Tsilioni et al. [41] found that the IL-1β levels in these children were remarkably higher than the controls following microglia activation. Another study [42] found elevated IL-1β levels in children with degenerative ASD and that IL-1β regulates the proliferation of neural precursor cells, suggesting that elevated IL-1β levels in the brain of ASD patients may alter neuronal development [43] and affect postnatal neural development [36] .
IL-8 is a pro-inflammatory cytokine, secreted by various cells, including macrophages and microglia. IL-8 functions primarily as a neutrophil chemotactic factor in the blood. This pro-inflammatory cytokine that is released from microglia increases in response to pro-inflammatory stimuli. Elevated peripheral IL-8 levels have been observed in psychiatric disorders, such as major depression, bipolar affective disorder, schizophrenia, sleep disorders, ASD, anxiety disorders, and dementia [44] . In a clinical study conducted by Bryn et al. [45] , serum IL-8 concentrations were significantly elevated in children with ASD compared to healthy controls; this finding validates the role of peripheral IL-8 associated with microglia in ASD. Another study found that ASD patients have higher IL-8 levels in the https://doi.org/10.36922/an.v1i3.167

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Microglia in autism spectrum disorder frontal cerebral cortex [46] and that the local inflammation caused by IL-8 can affect frontal cortical processing in these patients. The frontal cortex is critical for cognitive, emotional, and social behavior [47] . Its abnormal function is closely tied to the pathological mechanisms of ASD [48] and may lead to abnormal cognitive-emotional manifestations.
IL-6 is one of the most important neuroimmune factors that are produced over a short period of time by activated microglia in response to infection and tissue injury. It promotes host defense by stimulating acute phase, hematopoietic, and immune responses [49] . IL-6 has long been shown to be associated with physiological brain development and neurological disorders, such as schizophrenia, major depression, and Alzheimer's disease [50] . Studies have shown that ASD patients have elevated IL-6 levels in their blood, which is strongly associated with neuronal cell adhesion damage, migration, and synapse formation [51] . In a study conducted by Smith et al. [52] , ASD-like behavioral traits were found in the offspring of pregnant female rats following IL-6 injection. ASD behavior was prevented by simultaneous injection of anti-IL-6 antibodies in another experimental group, suggesting that IL-6 and abnormal microglia could influence ASD.
Using the adenoviral gene delivery approach, Wei et al. [53] developed a mouse model that overexpressed IL-6 centrally and confirmed that the overexpression of IL-6 is an important mediator of autistic-like behaviors. When IL-6 levels were elevated in the brains of mice, autistic-like behaviors were observed. Elevated IL-6 levels have also been reported to cause abnormal changes in the shape, length, and distribution pattern of dendritic spines, suggesting that elevated IL-6 levels may mediate ASD-like behaviors through an imbalance of neural circuits and impairment of synaptic plasticity. In the CNS, astrocytes, microglia, neurons, and endothelial cells of the cerebral microvascular system are the cellular sources of IL-6, and its levels are significantly affected by abnormal microglia activation.

Microbial-gut-brain axis and microglia
In the past few decades, the "microbial-gut-brain axis" has garnered widespread attention from researchers [54] , leading to the discovery of the two-way communication between the gut and brain through pathways involving neural, endocrine, and immune systems. Intestinal microbiota is extensively involved in each pathway of the brain-gut axis, influencing neural development, cognition, and behavior by regulating the two-way communication between the gut and the CNS. Studies have shown that gut microbes play an important role in brain function [55] . Many studies have explored the effect of intestinal microbes on microgliaassociated neuroinflammation and found immune abnormalities in the gastrointestinal tract of autistic patients, such as increased intestinal immunoglobulin (Ig) G, intestinal CD8 lymphocyte, and T-cell infiltration in the mucosal lamina propria. The deposition of IgG1 and IgG4 has been observed in the basement membrane of intestinal epithelium, along with an increased production of TNF-α in intestinal lymphocytes. Peripheral circulating immune cells may penetrate the blood-brain barrier (BBB) and subsequently affect neurons and glial cells, thereby perpetuating the immune response. Microglia respond to local signals within the brain and receive inputs from the periphery, including the gastrointestinal tract. There is evidence showing altered microbial community composition in nervous system diseases. Recent clinical research has revealed that gut microbes play a critical role in regulating microglial cell maturation and function [56] . However, compared with the physiological state, when mucosal rupture occurs as a result of gastrointestinal inflammation or other factors, many intestinal bacterialrelated antigens, cytokines, and chemokines that damage the BBB are released continuously. The hyperactivity of the peripheral immune system promotes CNS inflammation, on which microglia are affected, thus altering the synaptic pruning of neurons and triggering ASD [57] (Figure 2).

Therapeutic strategies targeting microglia
In the previous section, we discuss the key roles microglia play in the neuroinflammatory process of ASD. As a dynamic cellular mediator in the CNS, microglia can disrupt the balance of the nervous system by affecting the microenvironment and deranging the expression of important neuroinflammatory molecules [27,58] . Preclinical assessment is anticipated to benefit from treatments targeting microglia dysfunction.

Microglia and inflammatory interventions
Microglia are the primary immune cells involved in neurodegenerative diseases of the CNS, such as ASD [59] . Microglia express various immune receptors that secrete numerous cytokines and chemokines. The malfunctioning of these molecules in the pathologic signaling pathways of ASD provides neuroscientists and clinicians with potential treatment targets.

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Microglia in autism spectrum disorder through the secretion of reactive oxygen species (ROS) and BDNF, which engages the Trk receptors that modulate the activities of various synapses. Synaptic pruning by microglia is impaired if CX3CR1-CX3CL1 interactions are imbalanced [61] . By blocking inflammatory microglia functions, humanized antibodies or other reagents that target the aforementioned molecular markers may have beneficial effects. The inverse functions of M1/M2 microglia phenotypes provide us with a promising therapeutic idea that emphasizes on the balance of polarizations. Shifting the polarization from M1 to M2 helps in neural regeneration and regulates neuroinflammation in neurodevelopmental diseases, like ASD. The simultaneous inhibition of M1 microglia and acceleration of M2 microglia transformation might be useful for treating ASD [62] . However, due to the lack of animal experiments using M1 depressants like cyclooxygenase inhibitors, and anti-inflammatory drugs like aspirin, for treating ASD, the eligibility and availability of these drugs still require further testing. Recently, the research focus has been on microglia autophagy. It has been reported that the loss of mammalian target of rapamycin (mTOR)-dependent macro-autophagy can lead to ASDlike spine densities and a decrease in presynaptic markers, like light chain protein 3-II (LC3-II) [63] ; additionally, malfunctioning autophagy can also be induced by infected microglia [64] . Hence, the potential relationship between mTOR-dependent autophagy and infected microglia may provide researchers with interesting targeting methods ( Figure 3).

Targeting microglia in maternal immune activation
Infection during prenatal period induces pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-β, causing neuroinflammation effectors that activate microglia [65] . Coadministration of anti-IL-6 and -IL-1β antibodies has also been demonstrated in certain animal models. Suramin has shown to be effective in improving core ASD behaviors by recovering dysregulated purinergic metabolism in the poly I: C-exposed MIA model of adult ASD mice [66] (Figure 4).

Metabolic deficiency and microglia abnormalities
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential in maternal diet. A deficiency of n-3 PUFAs alters the offspring's microglia lipid composition and oxylipin signature, which are implicated in inflammation, resulting in microglia polarization toward a phagocytic phenotype, possibly by the overexpression of 12-hydroxyeicosatetraenoic (12-HETE) [67,68] . Phagocytic Figure 2. Involvement of microbial-gut-brain axis in autism spectrum disorder (ASD) development. The microbial-gut-brain axis plays a role in ASD development by regulating body immunity. On the left is the physiological state: the secreted metabolites from intestinal flora activate the peripheral immune system, causing the production of inflammatory factors, which reach the central nervous system through the BBB and act on microglia for normal phagocytosis and pruning. On the right is the pathological state: the integrity of intestinal epithelial cells is destroyed, and the metabolites entering the periphery are significantly increased, enhancing the peripheral immune response, and affecting the activity and physiological function of microglia. https://doi.org/10.36922/an.v1i3.167

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Microglia in autism spectrum disorder microglia produce a cascade of neuroinflammation cytokines that impair spine refinement or synaptic pruning. Supplementing with n-3 PUFAs from weaning to adulthood has led to significant improvements in ASD-like social deficits in ASD mouse models [69] . Therefore, n-3 PUFAs supplementation through fish-oil capsules containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with vitamin E may be a pivotal treatment for ASD patients (Figure 3).

Gut-brain axis and microglia
The gut-brain axis plays an intrinsic role in brain homeostasis. Microbiome-microglia crosstalk research has generated novel therapeutic approaches for ASD patients. There have been reports of several microbiota metabolisms that impact the physiological characteristics of microglia. As bacterial metabolites derived from microbial fermentation, the three main short-chain fatty acids (SCFAs) -propionate, butyrate, and acetate -can be beneficial or detrimental for ASD treatments [63] . Using antibiotics against Clostridia and Bacteroidetes, the main producers of propionate can help prevent the increased activation of microglia. The treatment with butyrate or its byproducts generated by Clostridium butyricum promotes anti-inflammatory effects in microglia, contributing to the improvement of neuropsychiatric symptoms associated with ASD [70] . It has been suggested that p-Cresol, which is derived from Blautia hydrogenotrophica and Clostridium spp., induces microglia activation and neuroinflammation [71] . Recolonizing patients with various microbiota or feeding SCFAs can sufficiently modulate microglia activation and alleviate ASD syndrome [72] .

Challenges
ASD is a psychosocial condition that persists throughout life. ASD patients experience social, speech, and behavioral changes that can severely disrupt their daily life and work. At present, there are many challenges in the treatment and prevention of ASD. Since the disorder cannot be cured, it becomes a lifelong problem for patients. Furthermore, the detection of ASD is not feasible at an early stage or through pregnancy screening. The diagnosis of ASD mainly relies on behavioral observations, and its symptoms cannot be detected under the age of three, thus missing the critical period of intervention.
MIA during pregnancy is one of the well-recognized mechanisms that have been successfully used to construct animal models. Altered cytokine expressions could facilitate the identification of ASD phenotypes and provide Figure 3. Treatment of autism spectrum disorder (ASD) through inflammatory and metabolic methods. Loss of omega-3 polyunsaturated fatty acids (n-3 PUFAs) leads to changes in the lipid composition and oxygen-lipid characteristics of progeny microglia, ultimately polarizing microglia toward the phagocytic phenotype and resulting in the secretion of neuroinflammatory cytokines that impair spine density and/or synaptic pruning, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Supplementation of n-3 PUFAs from weaning to adulthood improves ASD-like social deficits in a mouse ASD model. Therefore, fish-oil capsules, containing n-3 fatty acids, with vitamin E supplements may be one of the key treatments for ASD patients. Shifting the polarization from M1 to M2 has potential application using different kinds of drugs. https://doi.org/10.36922/an.v1i3.167

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Microglia in autism spectrum disorder biomarkers of response to treatment [73,74] . A timely analysis of gestational samples is an excellent opportunity for pathological diagnosis and future behavioral interventions in the early ASD childhood. However, the average age of ASD children at which atypical behaviors are first noticed by the parents is 4.6 years [75] . This means that sample analysis during pregnancy may not be a suitable solution due to parental acceptance. Some animal experiments have shown promising results concerning therapeutic targeting T helper 17 (Th17) cells in preventing inflammationinduced ASD-like behaviors in offspring of susceptible mothers [76] . However, the specific inflammatory factors that cause ASD are poorly understood, and most existing immunomodulatory tools are ineffective. There are still many challenges in reversing the pathological changes in gestation to ameliorate ASD-like behaviors in offspring; however, fostering routine sample analysis and clinical trials would help address these challenges.
Microglia are innate immune cells of the CNS. An important pathogenic mechanism of ASD is the decreased ability of microglia to engulf and prune unwanted synapses, resulting in CNS inflammation. A number of studies have demonstrated that microglia release IL-1β, IL-6, IL-8, and other inflammatory factors, and these factors are significantly increased in the peripheral blood and CNS of autistic patients. Insights into possible targets associated with microglia would aid the development of potential medical therapies for various CNS disorders. A recent study identified a novel pharmacological antineuroinflammatory agent and GIBH-130, for Alzheimer's disease through microglia-based phenotypic screening, the method by which novel favorable targets in ASD models may be introduced [77] . Further studies are needed to confirm the specificity of inflammatory factor release and the intricate interactions between microglia and inflammation. Besides, glial cell modulation cannot be precisely controlled, and microglia modulation may have other adverse effects on the CNS, thus posing a challenge for ASD treatment.

Conclusion
Our work offers a summary of the main pathological mechanisms of microglia in ASD and several targeted therapeutic methods. There are limitations in the existing mechanisms, such as the ambiguous pruning preference for which type of synapses and the lack of explicit causeeffect relationship between studies, like MIA and microglia polarization. Focusing on new insights such as glial cell interactions and autophagy may bring promising results. As for effective interventions, most therapeutic methods are still In the early stage of pregnancy, the exposure of the mother to external stimuli, such as rubella virus, bacteria, and Toxoplasma, leads to the activation of microglia and the maternal immune system, as well as an increased secretion of cytokines. Treatments that are commonly used include the prevention and removal of pathogens to reduce maternal infection by stimulating the immune system and inhibiting maternal immune activation signaling; a concomitant administration of anti-interleukin (IL)-6 and -IL-1β antibodies in poly(I: C) models could take effect in animal models. https://doi.org/10.36922/an.v1i3.167

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Microglia in autism spectrum disorder in the theoretical stage and have not been translated from ongoing animal experiments to clinical trials. The precise regulation of microglia should be the focus of future research, but elucidating the explicit circuits still remains a priority.
The emphasis of future research on ASD should include a range of relevant mechanisms: (1) the involvement of microglia in the development of ASD, and the molecular mechanisms of microglia activation, which are crucial for precise intervention; (2) the modulation of cytokines secreted by microglia, including IL-1β, IL-6, and IL-8 in both peripheral blood and CNS of ASD patients, and the inflammatory pathways they participate in, since there might be association between the role of inflammatory factors and the abnormal phagocytic pruning by microglia; (3) the characterization of microglial activity and marker molecules in the early stages of ASD, which is crucial for the early identification and intervention of ASD.

Acknowledgment
Language editing was performed by editors of scientificmanuscriptediting.com.
The schematic diagrams were prepared using FigDraw resources. The relevant authorization code is as follows: TUPUSd4c58, TARSR9c992, APSSRe7bbe, and ITART05260.