A novel in vitro model for investigating oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions: Impact of microglial depletion and repopulation.

Experimental models of multiple sclerosis (MS) have significantly contributed to our understanding of pathophysiology and the development of therapeutic interventions. Various in vivo animal models have successfully replicated key features of MS and associated pathophysiological processes, shedding light on the sequence of events leading to disease initiation, progression, and resolution. Nevertheless, these models often entail substantial costs and prolonged treatment periods. In contrast, in vitro models offer distinct advantages, including cost-effectiveness and precise control over experimental conditions, thereby facilitating more reproducible results. We have developed a novel in vitro model tailored to the study of oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions, which encompasses all the cell types present in the central nervous system (CNS). Of note, our model enables the evaluation of microglial cell commitment through a protocol involving their depletion and subsequent repopulation. Given that the development and survival of microglia are critically reliant on colony-stimulating factor-1 receptor (CSF-1R) signaling, we have employed CSF-1R inhibition to effectively deplete microglia. This versatile model holds promise for the assessment of potential therapies aimed at promoting oligodendroglial differentiation to safeguard and repair myelin, hence mitigate neurodegenerative processes.

Microglia modulate hippocampal synaptic transmission and sleep duration along the light/dark cycle.

Microglia, the brain's resident macrophages, actively contribute to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor-1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1GFP/GFP mice recapitulate what found in PLX5622-treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase-dependent manner.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion.

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Partial microglial depletion is associated with impaired hippocampal synaptic and cognitive function in young and aged rats.

The role of microglia in mediating age-related changes in cognition and hippocampal synaptic function was examined by microglial depletion and replenishment using PLX3397. We observed age-related differences in microglial number and morphology, as well as increased Iba-1 expression, indicating microglial activation. PLX3397 treatment decreased microglial number, with aged rats exhibiting the lowest density. Young rats exhibited increased expression of pro-inflammatory cytokines during depletion and repopulation and maintenance of Iba-1 levels despite reduced microglial number. For aged rats, several cytokines increased with depletion and recovered during repopulation; however, aged rats did not fully recover microglial cell number or Iba-1 expression during repopulation, with a recovery comparable to young control levels rather than aged controls. Hippocampal CA3-CA1 synaptic transmission was impaired with age, and microglial depletion was associated with decreased total synaptic transmission in young and aged rats. A robust decline in N-methyl-d-aspartate-receptor-mediated synaptic transmission arose in young depleted rats specifically. Microglial replenishment normalized depletion-induced synaptic function to control levels; however, recovery of aged animals did not mirror young. Microglial depletion was associated with decreased context-object discrimination memory in both age groups, which recovered with microglial repopulation. Aged rats displayed impaired contextual and cued fear memory, and microglial replenishment did not recover their memory to the level of young. The current study indicates that cognitive function and synaptic transmission benefit from the support of aged microglia and are hindered by removal of these cells. Replenishment of microglia in aging did not ameliorate age-related cognitive impairments or senescent synaptic function.

Microglial activation contributes to cognitive impairments in rotenone-induced mouse Parkinson's disease model.

BACKGROUND: Cognitive decline occurs frequently in Parkinson's disease (PD), which greatly decreases the quality of life of patients. However, the mechanisms remain to be investigated. Neuroinflammation mediated by overactivated microglia is a common pathological feature in multiple neurological disorders, including PD. This study is designed to explore the role of microglia in cognitive deficits by using a rotenone-induced mouse PD model. METHODS: To evaluate the role of microglia in rotenone-induced cognitive deficits, PLX3397, an inhibitor of colony-stimulating factor 1 receptor, and minocycline, a widely used antibiotic, were used to deplete or inactivate microglia, respectively. Cognitive performance of mice among groups was detected by Morris water maze, objective recognition, and passive avoidance tests. Neurodegeneration, synaptic loss, α-synuclein phosphorylation, glial activation, and apoptosis were determined by immunohistochemistry and Western blot or immunofluorescence staining. The gene expression of inflammatory factors and lipid peroxidation were further explored by using RT-PCR and ELISA kits, respectively. RESULTS: Rotenone dose-dependently induced cognitive deficits in mice by showing decreased performance of rotenone-treated mice in the novel objective recognition, passive avoidance, and Morris water maze compared with that of vehicle controls. Rotenone-induced cognitive decline was associated with neurodegeneration, synaptic loss, and Ser129-phosphorylation of α-synuclein and microglial activation in the hippocampal and cortical regions of mice. A time course experiment revealed that rotenone-induced microglial activation preceded neurodegeneration. Interestingly, microglial depletion by PLX3397 or inactivation by minocycline significantly reduced neuronal damage and α-synuclein pathology as well as improved cognitive performance in rotenone-injected mice. Mechanistically, PLX3397 and minocycline attenuated rotenone-induced astroglial activation and production of cytotoxic factors in mice. Reduced lipid peroxidation was also observed in mice treated with combined PLX3397 or minocycline and rotenonee compared with rotenone alone group. Finally, microglial depletion or inactivation was found to mitigate rotenone-induced neuronal apoptosis. CONCLUSIONS: Taken together, our findings suggested that microglial activation contributes to cognitive impairments in a rotenone-induced mouse PD model via neuroinflammation, oxidative stress, and apoptosis, providing novel insight into the immunopathogensis of cognitive deficits in PD.

Microglial deletion and inhibition alleviate behavior of post-traumatic stress disorder in mice.

BACKGROUND: Alteration of immune status in the central nervous system (CNS) has been implicated in the development of post-traumatic stress disorder (PTSD). However, the nature of overall changes in brain immunocyte landscape in PTSD condition remains unclear. METHODS: We constructed a mouse PTSD model by electric foot-shocks followed by contextual reminders and verified the PTSD-related symptoms by behavior test (including contextual freezing test, open-field test, and elevated plus maze test). We examined the immunocyte panorama in the brains of the naïve or PTSD mice by using single-cell mass cytometry. Microglia number and morphological changes in the hippocampus, prefrontal cortex, and amygdala were analyzed by histopathological methods. The gene expression changes of those microglia were detected by quantitative real-time PCR. Genetic/pharmacological depletion of microglia or minocycline treatment before foot-shocks exposure was performed to study the role of microglia in PTSD development and progress. RESULTS: We found microglia are the major brain immune cells that respond to PTSD. The number of microglia and ratio of microglia to immunocytes was significantly increased on the fifth day of foot-shock exposure. Furthermore, morphological analysis and gene expression profiling revealed temporal patterns of microglial activation in the hippocampus of the PTSD brains. Importantly, we found that genetic/pharmacological depletion of microglia or minocycline treatment before foot-shock exposure alleviated PTSD-associated anxiety and contextual fear. CONCLUSION: Our results demonstrated a critical role for microglial activation in PTSD development and a potential therapeutic strategy for the clinical treatment of PTSD in the form of microglial inhibition.

Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling.

BACKGROUND: Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or "primed" by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo. METHODS: OHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated. RESULTS: Microglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1ß in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1ß that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol. CONCLUSIONS: Microglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.

Bulk and single-cell RNA-seq analyses reveals canonical RNA editing associated with microglial homeostasis and its role in sepsis-associated encephalopathy.

Previous studies have demonstrated the roles of both microglia homeostasis and RNA editing in sepsis-associated encephalopathy (SAE), yet their relationship remains to be elucidated. In the current study, we analyzed bulk and single-cell RNA-seq (scRNA) datasets containing 107 brain tissues and microglia samples of mice with microglial depletion and repopulation to explore canonical RNA editing associated with microglia homeostasis and evaluated its role in SAE. Analysis of brain RNA-Seq of mice revealed hallmarks of microglial repopulation, including peak expressions of Apobec1 and Apobec3 at Day 5 and dramatically changed B2m RNA editing. Significant time-dependent changes in brain RNA editing during microglial depletion and microglial repopulation was primarily observed in synaptic genes, such as Tbc1d24 and Slc1a2. ScRNA-Seq revealed heterogeneous RNA editing among microglia subpopulations and their distinct changes associated with microglia homeostasis. Moreover, repopulated microglia from LPS-induced septic mice exhibited intensified up-regulation of Apobec1 and Apobec3, with distinct RNA editing responses to LPS, mainly involved in immune-related pathways. The hippocampus from septic mice induced by peritoneal contamination and infection showed upregulated Apobec1 and Apobec3 expression, and altered RNA editing in immune-related genes, such as B2m and Mier1, and nervous-related lncRNA Meg3 and Snhg11, both of which were repressed by microglial depletion. Moreover, expression of complement-related genes, such as C4b and Cd47, were substantially correlated with RNA editing activity in microglia homeostasis and SAE. Our study demonstrates canonical RNA editing associated with microglia homeostasis, and provides new insight into its potential role in SAE.

Established and emerging techniques for the study of microglia: visualization, depletion, and fate mapping.

The central nervous system (CNS) is an essential hub for neuronal communication. As a major component of the CNS, glial cells are vital in the maintenance and regulation of neuronal network dynamics. Research on microglia, the resident innate immune cells of the CNS, has advanced considerably in recent years, and our understanding of their diverse functions continues to grow. Microglia play critical roles in the formation and regulation of neuronal synapses, myelination, responses to injury, neurogenesis, inflammation, and many other physiological processes. In parallel with advances in microglial biology, cutting-edge techniques for the characterization of microglial properties have emerged with increasing depth and precision. Labeling tools and reporter models are important for the study of microglial morphology, ultrastructure, and dynamics, but also for microglial isolation, which is required to glean key phenotypic information through single-cell transcriptomics and other emerging approaches. Strategies for selective microglial depletion and modulation can provide novel insights into microglia-targeted treatment strategies in models of neuropsychiatric and neurodegenerative conditions, cancer, and autoimmunity. Finally, fate mapping has emerged as an important tool to answer fundamental questions about microglial biology, including their origin, migration, and proliferation throughout the lifetime of an organism. This review aims to provide a comprehensive discussion of these established and emerging techniques, with applications to the study of microglia in development, homeostasis, and CNS pathologies.

Microglial Regulation of Sleep and Wakefulness.

Sleep serves a multitude of roles in brain maturation and function. Although the neural networks involved in sleep regulation have been extensively characterized, it is increasingly recognized that neurons are not the sole conductor orchestrating the rhythmic cycle of sleep and wakefulness. In the central nervous system, microglia have emerged as an important player in sleep regulation. Within the last two decades, microglia have gained substantial attention for carrying out numerous nonimmune tasks that are crucial for brain development and function by co-opting similar mechanisms used in their conventional immune functions. Here, we highlight the importance of microglia in sleep regulation with recent findings reporting an arrhythmic sleep/wake cycle in the absence of microglia. Although the underlying mechanisms for such regulation are still being uncovered, it is likely that microglial contributions to the rhythmic control of the sleep/wake cycle come from their influence on synaptic strength and neuronal activity. We review the current literature to provide speculative signaling pathways and suggest key questions for future research. Advancing our knowledge of the mechanistic contribution of microglia to sleep regulation will not only further our insight into this critical biological process but also be instrumental in providing novel therapeutic strategies for sleep disorders.

Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism.

Microglia, the resident immune cells of the central nervous system, are intimately involved in the brain's most basic processes, from pruning neural synapses during development to preventing excessive neuronal activity throughout life. Studies have reported both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, less is known about microglia-endothelial cell interactions in the healthy brain. To investigate the role of microglia at a healthy BBB, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed the BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for the maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism. This effect was independent from microglial depletion, suggesting that PLX5622 has off-target effects on brain vasculature.

Partial Depletion of Microglia Attenuates Long-Term Potentiation Deficits following Repeated Blast Traumatic Brain Injury in Organotypic Hippocampal Slice Cultures.

Blast-induced traumatic brain injury (bTBI) has been a health concern in both military and civilian populations due to recent military and geopolitical conflicts. Military service members are frequently exposed to repeated bTBI throughout their training and deployment. Our group has previously reported compounding functional deficits as a result of increased number of blast exposures. In this study, we further characterized the decrease in long-term potentiation (LTP) by varying the blast injury severity and the inter-blast interval between two blast exposures. LTP deficits were attenuated with increasing inter-blast intervals. We also investigated changes in microglial activation; expression of CD68 was increased and expression of CD206 was decreased after multiple blast exposures. Expression of macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1ß, monocyte chemoattractant protein (MCP)-1, interferon gamma-inducible protein (IP)-10, and regulated on activation, normal T cell expressed and secreted (RANTES) increased, while expression of IL-10 decreased in the acute period after both single and repeated bTBI. By partially depleting microglia prior to injury, LTP deficits after injury were significantly reduced. Treatment with the novel drug, MW-189, prevented LTP deficits when administered immediately following a repeated bTBI and even when administered only for an acute period (24 h) between two blast injuries. These findings could inform the development of therapeutic strategies to treat the neurological deficits of repeated bTBI suggesting that microglia play a major role in functional neuronal deficits and may be a viable therapeutic target to lessen the neurophysiological deficits after bTBI.

Contextual Fear Learning and Extinction in the Primary Visual Cortex of Mice.

Fear memory contextualization is critical for selecting adaptive behavior to survive. Contextual fear conditioning (CFC) is a classical model for elucidating related underlying neuronal circuits. The primary visual cortex (V1) is the primary cortical region for contextual visual inputs, but its role in CFC is poorly understood. Here, our experiments demonstrated that bilateral inactivation of V1 in mice impaired CFC retrieval, and both CFC learning and extinction increased the turnover rate of axonal boutons in V1. The frequency of neuronal Ca2+ activity decreased after CFC learning, while CFC extinction reversed the decrease and raised it to the naïve level. Contrary to control mice, the frequency of neuronal Ca2+ activity increased after CFC learning in microglia-depleted mice and was maintained after CFC extinction, indicating that microglial depletion alters CFC learning and the frequency response pattern of extinction-induced Ca2+ activity. These findings reveal a critical role of microglia in neocortical information processing in V1, and suggest potential approaches for cellular-based manipulation of acquired fear memory.

Partial depletion and repopulation of microglia have different effects in the acute MPTP mouse model of Parkinson's disease.

OBJECTIVES: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive and selective degeneration of dopaminergic neurons. Microglial activation and neuroinflammation are associated with the pathogenesis of PD. However, the relationship between microglial activation and PD pathology remains to be explored. MATERIALS AND METHODS: An acute regimen of MPTP was administered to adult C57BL/6J mice with normal, much reduced or repopulated microglial population. Damages of the dopaminergic system were comprehensively assessed. Inflammation-related factors were assessed by quantitative PCR and Multiplex immunoassay. Behavioural tests were carried out to evaluate the motor deficits in MPTP-challenged mice. RESULTS: The receptor for colony-stimulating factor 1 inhibitor PLX3397 could effectively deplete microglia in the nigrostriatal pathway of mice via feeding a PLX3397-formulated diet for 21 days. Microglial depletion downregulated both pro-inflammatory and anti-inflammatory molecule expression at baseline and after MPTP administration. At 1d post-MPTP injection, dopaminergic neurons showed a significant reduction in PLX3397-fed mice, but not in control diet (CD)-fed mice. However, partial microglial depletion in mice exerted little effect on MPTP-induced dopaminergic injuries compared with CD mice at later time points. Interestingly, microglial repopulation brought about apparent resistance to MPTP intoxication. CONCLUSIONS: Microglia can inhibit PD development at a very early stage; partial microglial depletion has little effect in terms of the whole process of the disease; and microglial replenishment elicits neuroprotection in PD mice.

Evaluation of the Neuroprotective Effect of Microglial Depletion by CSF-1R Inhibition in a Parkinson's Animal Model.

PURPOSE: Neuroinflammation in Parkinson's disease (PD) is known to play a pivotal role in progression to neuronal degeneration. It has been reported that colony-stimulation factor 1 receptor (CSF-1R) inhibition can effectively deplete microglia. However, its therapeutic efficacy in PD is unclear still now. PROCEDURES: To elucidate this issue, we examined the contribution of microglial depletion to PD by behavioral testing, positron emission tomography (PET) imaging, and immunoassays in sham, PD, and microglial depletion PD model (PLX3397 was administered to PD groups, with n = 6 in each group). RESULTS: The microglial depletion in PD model showed improved sensory motor function and depressive-like behavior. NeuroPET revealed that PLX3397 treatment resulted in partial recovery of striatal neuro-inflammatory functions (binding values of [18F]DPA-174 for PD, 1.47 ± 0.12, p < 0.01 vs. for PLX3397 in PD: 1.33 ± 0.26) and the dopaminergic (binding values of 18F-FP-CIT for PD, 1.32 ± 0.07 vs. for PLX3397 in PD: 1.54 ± 0.10, p < 0.01) and glutamatergic systems (binding values of [18F]FPEB for PD: 9.22 ± 0.54 vs. for PLX3397 Tx in PD: 9.83 ± 0.96, p > 0.05). Western blotting for microglia showed similar changes. CONCLUSION: Microglial depletion has inflammation-related therapeutic effects, which have beneficial effects on motor and nonmotor symptoms of PD.

Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity.

Investigators are increasingly interested in using microglial depletion to study the role of microglia under pathologic conditions. Liposome-encapsulated clodronate is commonly used to eliminate macrophage populations because it causes functionally irreversible inhibition and apoptosis once phagocytized by macrophages. Recent studies have shown that microglia can be depleted in disease models by injecting clodronate liposomes into the brain parenchyma. However, it is unclear whether intracerebral administration of clodronate liposomes is a practical method of eliminating microglia under physiologic conditions or whether microglial depletion induces damage to other brain cells. In this study, injecting 1 µL of clodronate liposomes (7 µg/µL) into the striatum of mice caused ablation of microglia at 1 day that persisted for 3 days. Microglia reappeared in the boundary regions of microglia elimination after 5 days. Importantly, we observed an increase in proinflammatory cytokine levels and an increase in neural/glial antigen 2 and glial fibrillary acidic protein expression in the perilesional region. In contrast, expression levels of myelin basic protein, microtubule-associated protein 2, and postsynaptic protein-95 decreased in the periphery of regions where microglia were depleted. Moreover, clodronate liposome administration decreased the density and integrity of blood vessels in the perilesional regions. In cultured primary neurons, clodronate liposome exposure also attenuated ATP synthesis. Together, these findings suggest that intracerebral administration of clodronate liposomes into brain parenchyma can deplete microglia, but can also damage other brain cells and blood vessel integrity.

Microglia Are Essential to Protective Antiviral Immunity: Lessons From Mouse Models of Viral Encephalitis.

Viral encephalitis is a rare but clinically serious consequence of viral invasion of the brain and insight into its pathogenesis is urgently needed. Important research questions concern the involvement of the host innate immune response in pathogenesis, key to which is the role played by microglia, resident macrophages of the brain parenchyma. Do microglia have a protective function, by coordinating the innate immune response to viral infection, or do they drive pathogenic neuroinflammation? Here we synthesize recent data from mouse models of acute viral encephalitis, which reveal an unambiguously protective role for microglia. Depletion of microglia, via blockade of colony-stimulating factor 1 receptor (CSF1R) signaling, led to increased viral replication accompanied by more severe neurological disease and heightened mortality. Whilst the underlying mechanism(s) remain to be defined, microglial interactions with T cells and phagocytosis of infected neurones appear to play a role. Paradoxically, the production of inflammatory cytokines was increased in several instances following viral infection in microglia-depleted brains, suggesting that: (i) cells other than microglia mediate inflammatory responses and/or (ii) microglia may exert a regulatory function. Under certain circumstances the microglial antiviral response might contribute negatively to longer-term neurological sequelae, although fewer studies have focused on this aspect in encephalitis models. Understanding regulation of the microglial response, and how it contributes to disease is therefore a priority for future studies. Collectively, these findings demonstrate the central role of microglia in pathogenesis, suggesting the exciting possibility that defects of microglial function might contribute to encephalitis susceptibility and/or outcome in humans.