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 cx3cr1^GFP/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.     

Sialoadhesin promotes neuroinflammation‐related disease progression in two mouse models of CLN disease

CLN diseases are mostly fatal lysosomal storage diseases that lead to neurodegeneration in the CNS. We have previously shown that CD8+ T‐lymphocytes contribute to axonal perturbation and neuron loss in the CNS of Ppt1^?/? mice, a model of CLN1 disease. We now investigated the role of the inflammation‐related cell adhesion molecule sialoadhesin (Sn) in Ppt1^?/? and Cln3^?/? mice, a model of the most frequent form, CLN3 disease. Microglia/macrophages in the CNS of both models showed an upregulation of Sn and markers for proinflammatory M1 polarization and antigen presentation. Sn+ microglia/macrophages associated with SMI32+ axonal spheroids and CD8+ T‐lymphocytes. To analyze their pathogenic impact, we crossbred both models with Sn‐deficient mice and scored axonal degeneration and neuronal integrity using immunohistochemistry, electron microscopy and optical coherence tomography. Degenerative alterations in the retinotectal pathway of Ppt1^?/?Sn^?/? and Cln3^?/?Sn^?/? mice were significantly reduced. Ppt1^?/?Sn^?/? mice also showed a substantially improved clinical phenotype and extended lifespan, attenuated numbers of M1‐polarized microglia/macrophages and reduced expression levels of proinflammatory cytokines. This was accompanied by an increased frequency of CD8+CD122+ T‐lymphocytes in the CNS of Ppt1^?/?Sn^?/? mice, the regulatory phenotype of which was demonstrated by impaired survival of CD8+CD122? effector T‐lymphocytes in co‐culture experiments. We show for the first time that increased Sn expression on microglia/macrophages contributes to neural perturbation in two distinct models of CLN disease. Our data also indicate that a rarely described CD8+CD122+ T‐cell population can regulate the corresponding diseases. These studies provide insights into CLN pathogenesis and may guide in designing immuno‐regulatory treatment strategies. GLIA 2016;64:792–809     

Microglia control glutamatergic synapses in the adult mouse hippocampus

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1^?/? mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.     

Cx3cr1‐deficiency exacerbates alpha‐synuclein‐A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha‐synuclein (α‐SYN). Mutations of the gene that codes for α‐SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild‐type α‐SYN (α‐SYN^WT) or mutated α‐SYN (α‐SYN^A53T) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α‐SYN^WT or α‐SYN^A53T in wild type (Cx3cr1^+/+) or deficient (Cx3cr1^–/–) mice for CX3CR1 using unilateral intracerebral injection of adeno‐associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT‐PCR in this model. Interestingly, the expression α‐SYN^WT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α‐SYN^A53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1^–/– mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro‐inflammatory markers, which were exacerbated in Cx3cr1^–/– mice overexpressing α‐SYN^A53T. Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α‐SYN^WT or α‐SYN^A53T. Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α‐SYN^A53T.     

Microglial phagocytosis and activation underlying photoreceptor degeneration is regulated by CX3CL1‐CX3CR1 signaling in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation‐bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well‐defined. In this study, we investigate the role of CX3CR1, a microglia‐specific receptor, in regulating microglia‐mediated degeneration using the well‐established rd10 mouse model of RP. We found that in CX3CR1‐deficient (CX3CR1^GFP/GFP) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1‐sufficient (CX3CR1^GFP/+) rd10 littermates. CX3CR1‐deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at‐risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1‐CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1‐CX3CR1 signaling is a molecular mechanism capable of modulating microglial‐mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479–1491     

CX3CL1 is neuroprotective in permanent focal cerebral ischemia in rodents

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A?R). We show that, in the presence of the A?R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A?R?/? mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1?/? and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1?/? ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1?/? microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.     

Abnormal microglial activation in the Cstb−/− mouse,a model for progressive myoclonus epilepsy,EPM1

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1) is an autosomal‐recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss‐of‐function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb^?/? mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb^?/? mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb^?/? mouse brain, expression of the inflammatory marker p‐p38 MAPK and the proportion of both pro‐inflammatory M1 and anti‐inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb^?/? mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro‐inflammatory inducible nitric oxide synthase (iNOS) and anti‐inflammatory arginase 1 (ARG1) in Cstb^?/? mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb^?/? microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1. GLIA 2015;63:400–411     

Reduced microglial CX3CR1 expression delays neurofibromatosis‐1 glioma formation

Although traditional models of carcinogenesis have largely focused on neoplastic cells, converging data have revealed the importance of non‐neoplastic stromal cells in influencing tumor growth and progression. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1)‐associated optic glioma, we now demonstrate that stromal microglia express the CX3CR1 chemokine receptor, such that reduced CX3CR1 expression decreases optic nerve microglia. Moreover, genetic reduction of Cx3cr1 expression in Nf1 optic glioma mice delays optic glioma formation. Coupled with previous findings demonstrating that microglia maintain optic glioma growth, these new findings provide a strong preclinical rationale for the development of future stroma‐directed glioma therapies in children. ANN NEUROL 2013;73:303–308     

CX3 chemokine receptor 1 deficiency leads to reduced dendritic complexity and delayed maturation of newborn neurons in the adult mouse hippocampus

Previous studies have shown that microglia impact the proliferation and differentiation of neurons during hippocampal neurogenesis via the fractalkine/CX3 chemokine receptor 1 (CX3CR1) signaling pathway. However, whether microglia can influence the maturation and dendritic growth of newborn neurons during hippocampal neurogenesis remains unclear. In the present study, we found that the number of doublecortin-positive cells in the hippocampus was decreased, and the dendritic length and number of intersections in newborn neurons in the hippocampus were reduced in transgenic adult mice with CX3CR1 deficiency (CX3CR1^GFP/GFP). Furthermore, after experimental seizures were induced with kainic acid in these CX3CR1-deficient mice, the expression of c-fos, a marker of neuronal activity, was reduced compared with wild-type mice. Collectively, the experimental findings indicate that the functional maturation of newborn neurons during hippocampal neurogenesis in adult mice is delayed by CX3CR1 deficiency.     

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression

Microglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1^+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth.To fill this gap, we generated APP^swe/PSEN1^dE9:CX3CR1^+/eGFP mice and analyzed these mice for Alzheimer’s like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-β (Aβ) deposition just starts, at intermediate stage during Aβ accumulation phase and at more advanced stages when Aβ plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APP^swe/PSEN1^dE9 model and demonstrated that the APP^swe/PSEN1^dE9:CX3CR1^+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aβ plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different.     

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1^+/+, CX3CR1^+/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1^+/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.     

CX3CL1 and CX3CR1 in the GL261 murine model of glioma: CX3CR1 deficiency does not impact tumor growth or infiltration of microglia and lymphocytes

Human glioblastoma multiforme (GBM) is the most malignant form of human brain tumors. A characteristic of GBM is the marked presence of tumor infiltrated microglia/macrophages and lymphocytes. The goal of this study was directed toward understanding the role of the chemokine system CX3CL1 and its receptor CX3CR1 in the GL261 murine model of malignant glioma. In situ hybridization analysis identified CX3CL1 and CX3CR1 expression in GL261 tumors. The impact of CX3CR1 deletion on the growth of intracranial GL261 gliomas and associated immune cell infiltration was evaluated in CX3CR1 gene-disrupted C57BL/6 mice. A slight increase in the tumor growth rate in CX3CR1-/- mice was evident with similar numbers of microglia and CD4+, CD8+, FoxP3+, or Ly49G2+ lymphocytes within tumors established in CX3CR1 +/- and -/- mice. These data indicate that CX3CR1 has little or no effects on either gliomagenesis or the migration of microglia and lymphocytes into GL261 tumors.     

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.     

Identification of a chronic non‐neurodegenerative microglia activation state in a mouse model of peroxisomal β‐oxidation deficiency

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β‐oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2^?/? microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2^?/? microglia were compared with those from SOD1^G93A mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1^G93A spinal cord, neurons were intact in Mfp2^?/? brain and Mfp2^?/? microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2^?/? microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2^?/? microglia were immunologically activated but not polarized to a pro‐ or anti‐inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2^?/? brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity. GLIA 2015;63:1606–1620     

Microglia replenished OHSC: A culture system to study in vivo like adult microglia

Recent data suggest that ramified microglia fulfil various tasks in the brain. However, to investigate this unique cell type cultured primary microglia are only a poor model. We here describe a method to deplete and repopulate organotypic hippocampal slice cultures (OHSC) with ramified microglia isolated from adult mouse brain creating microglia‐replenished OHSC (Mrep‐OHSC). Replenished microglia integrate into the tissue and ramify to a degree indistinguishable from their counterparts in the mouse brain. Moreover, wild‐type slices replenished with microglia from TNFα‐deficient animals provide similar results as OHSC prepared from microglia‐specific TNFα‐knockout mice (CX3CR1^cre/TNFα^fl/fl). Furthermore, this study demonstrates that replenished microglia in OHSC maintain original functions and properties acquired in vivo. Microglia from ERCC1^Δ/ko mice, a mouse model of accelerated aging, maintain enhanced Mac2 expression and their activated phenotype after replenishment to wild‐type OHSC tissue. Thus, the present study demonstrates that Mrep‐OHSC are a unique tool to construct chimeric brain slices allowing studying the function of different phenotypes of in vivo like microglia in a tissue culture setting. GLIA 2016 GLIA 2016;64:1285–1297     

Microglial depletion aggravates the severity of acute and chronic seizures in mice

Microglia are the resident immune cells of the center nervous system and participate in various neurological diseases. Here we determined the function of microglia in epileptogenesis using microglial ablation approaches. Three different microglia-specific genetic tools were used, CX3CR1^CreER/+:R26^iDTA/+, CX3CR1^CreER/+:R26^iDTR/+, and CX3CR1^CreER/+:Csf1r^Flox/Flox mice. We found that microglial depletion led to worse kainic acid (KA)-induced status epilepticus, higher mortality rate, and increased neuronal degeneration in the hippocampus. In KA-induced chronic spontaneous recurrent seizures, microglial depletion increased seizure frequency, interictal spiking, and seizure duration. Therefore, microglial depletion aggravates the severity of KA-induced acute and chronic seizures. Interestingly, microglial repopulation reversed the effects of depletion upon KA-induced status epilepticus. Our results demonstrate a beneficial role of microglia in suppressing both acute and chronic seizures, suggesting that microglia are a potential therapeutic target for the management of epilepsy.     

Neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth

Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.     

Resident microglia,rather than blood‐derived macrophages,contribute to the earlier and more pronounced inflammatory reaction in the immature compared with the adult hippocampus after hypoxia‐ischemia

The mechanisms of neuronal injury after hypoxia–ischemia (HI) are different in the immature and the adult brain, but microglia activation has not been compared. The purpose of this study was to phenotype resident microglia and blood‐derived macrophages in the hippocampus after HI in neonatal (postnatal day 9, P9) or adult (3 months of age, 3mo) mice. Unilateral brain injury after HI was induced in Cx3cr1^GFP/+Ccr2^RFP/+ male mice on P9 (n = 34) or at 3mo (n = 53) using the Vannucci model. Resident microglia (Cx3cr1‐GFP+) proliferated and were activated earlier after HI in the P9 (1–3 days) than that in the 3mo hippocampus, but remained longer in the adult brain (3–7 days). Blood‐derived macrophages (Ccr2‐RFP+) peaked 3 days after HI in both immature (P9) and adult (3mo) hippocampi but were twice as frequent in adult brains, 41% vs. 21% of all microglia/macrophages. CCL2 expression was three times higher in the P9 hippocampi, indicating that the proinflammatory response was more pronounced in the immature brain after HI. This corresponded well with the higher numbers of galectin‐3‐positive resident microglia in the P9 hippocampi, but did not correlate with CD16/32‐ or CD206‐positive resident microglia or blood‐derived macrophages. In conclusion, resident microglia, rather than infiltrating blood‐derived macrophages, proliferate and are activated earlier in the immature than in the adult brain, but remain increased longer in the adult brain. The inflammatory response is more pronounced in the immature brain, and this correlate well with galectin‐3 expression in resident microglia. GLIA 2015;63:2220–2230