Microglial Deramification Research Articles

Regulation of Microglial Activation by Wnt/β-Catenin Signaling After Global Cerebral Ischemia in Mice.

Microglia are immunocompetent cells in the central nervous system. Following cerebral ischemia, microglia will be rapidly activated and undergo proliferation, morphological transformation, and changes in gene expression and function. At present, the regulatory mechanisms of microglial activation following ischemia remain largely unclear. In this study, we took advantage of CX3CR1GFP/+ fluorescent mice and a global cerebral ischemia-reperfusion model to investigate the mechanisms of microglial activation following different degrees of global ischemia. Our results showed that the proliferation of microglia was gated by the degree of ischemia. Marked microglial de-ramification and proliferation were observed after 60 min of ischemia but not in transient ischemia (20 min). Immunohistology, qRT-PCR, and Western blotting analysis showed that microglial activation was accompanied with a reduction in Wnt/β-catenin signaling after cerebral ischemia. Downregulation of Wnt/β-catenin signaling using Wnt antagonist XAV939 during 20 min ischemia promoted microglial de-ramification and proliferation. In contrast, enhancing Wnt/β-catenin signaling using Wnt agonist LiCl during 60 min ischemia-reduced microglial de-ramification and proliferation. Importantly, we found that Wnt agonist inhibited inflammation in the ischemic brain and was conducive to animal behavioral recovery. Collectively, these data demonstrated that Wnt/β-catenin signaling played a key role in microglial activation following cerebral ischemia, and regulating microglial activation may be a potential therapeutic strategy for the treatment of ischemic stroke.

Sustained microglial activation and accelerated elimination of dendritic spines during acute sleep deprivation and restoration

<p>Sleep is a fundamental physiological condition strongly regulated by a variety of neuroanatomical and neurochemical systems. Recent studies have indicated that catch-up sleep falls short of effectiveness to counteract the negative consequences of sleep debt however, the underlying mechanisms remain poorly understood. In this study, by using various transgenic fluorescent mouse models as well as techniques including intravital two-photon imaging and immunofluorescence staining of brain sections, we have documented morphological and functional changes of microglia during acute sleep deprivation (ASD) and subsequent short-term recovery sleep (RS). In these cerebral resident immune cells, we observed sustained microglial de-ramification, reduction of process motility and enhancement of microglial phagocytosis across brain regions. Given the intimate connections between microglia activity and neuronal plasticity, we also investigated synaptic plasticity and demonstrated an accelerated elimination of dendritic spines during both ASD and subsequent RS. Furthermore, untargeted metabolomic analyses revealed extensive whole-brain metabolic changes during ASD, and that a substantial number of metabolites and pathways failed to recover within a short period of RS. It is tempting to speculate that the disturbed cerebral metabolic homeostasis contributes to the sustained microglial activation and accelerated elimination of dendritic spines during this process. This study reveals the adverse effects of sleep loss on neuroimmunomodulation and neuronal plasticity, and implicates potential mechanisms underlying how irregular sleep schedules lead to neurological disorders.</p>

Functionality of a Peripheral Blood Derived Microglia-like Cell System for Drug Discovery to Treat Sickle Cell Related Chronic Pain

Background: Abnormal microglial activation is emerging as a clinically promising drug target to treat chronic pain in sickle cell disease (SCD). But research involving microglia has been stymied by difficulty in obtaining human CNS microglia and differences between human and animal neurobiology. To address such limitations, we have developed an in vitro culture system to induce human microglia-like cells (MLC) from peripheral blood mononuclear cells. In this study, we further validated the functionality of the cultured peripheral blood derived microglia-like cell (PB-MLC) and evaluate the pathological characteristics captured in the PB-MLC model system, which is an important requirement for disease model systems used in drug discovery. Methods: Human peripheral blood mononuclear cells were cultured with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL) and cholesterol (1.5 µg/mL) for 7 days to induce Human peripheral blood derived microglia-like cells (HPB-MLC). HPB-MLC were morphologically analyzed by phase contrast microscopy, phenotyped by immunofluorescence staining with anti-TMEM119, P2RY12, and Iba1 antibodies. To validate that the HPB-MLC culture system recapitulates important pathological features, we compared the microglial morphology, activation states and phagocytic activity of HPB-MLC from individuals with SCD with or without chronic pain, individuals with headache and normal donors without pain: Cells were stimulated with 100 ng/ml LPS for 24 hours, and secretion of TNF-alpha, IL-1beta, and IL-6 was measured by ELISA. The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with fluorescent microscopy and imaging flow cytometry. The model system was also validated by comparing cultured PB-MLC to CNS derived microglia cells using Sprague-Dawley rats: Rat brain derived microglia (BDM) and rat peripheral blood monocytes were isolated and then cultured with murine IL-34 (100 ng/ml), murine GM-CSF (10 ng/ml). TGF-β2 (2 ng/mL), and cholesterol (1.5 µg/mL) for 7 days. The cultured cells were morphologically analyzed as above; phenotyped with anti-TMEM119, P2RY12, and Iba1 antibodies. Rat BDM, rat PB-MLC and HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL) for 24 hours; TNF-alpha secretion was measured by ELISA. Results: Rat PB-MLC resemble BDM morphologically and express microglia specific markers. HPB-MLC express microglia specific markers (TMEM119, P2RY12, Iba1), exhibit microglial phagocytic activity. HPB-MLC retain the patient pain phenotype: Cultures from donors without chronic pain show more ramified morphology and lower phagocytic activity than cultures from individuals with chronic pain (Figure 1); HPB-MLC can be activated by LPS, causing microglial deramification and increased the expression of microglial activation marker CD68. HPB-MLC from patients with pain are more easily activated and secrete more inflammatory cytokines in response to LPS; SCD pain and headache samples exhibit different pro-inflammatory cytokine profile. To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the microglial inhibitor, minocycline. The compound significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner (Figure 2). Conclusions: We have developed and validated a microglial-like cell culture system from the peripheral blood of patients with sickle cell disease that retains the patient pain phenotype. HPB-MLC are physiologically functional and exhibit mature microglial features, including LPS-induced activation, microglial specific morphological transition, microglial phagocytic activity, and drug response to a well-studied microglia inhibitor, minocycline. As a cost-effective, efficient, and patient-specific microglia model, HPB-MLC culture system can be applied to identify target compounds that normalize microglia activation, and potentially treat chronic pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Resistance to noise-induced gap detection impairment in FVB mice is correlated with reduced neuroinflammatory response and parvalbumin-positive neuron loss

Exposure to loud noises results in neuroinflammatory responses in the central auditory pathway. Noise-induced neuroinflammation is implicated in auditory processing deficits such as impairment in gap detection. In this study, we examined whether strain differences between the FVB and C57BL/6 mice in noise-induced impairment in gap detection are correlated with strain differences in neuroinflammatory responses. We found that noise induced more robust TNF-α expression in C57BL/6 than in FVB mice. Noise-induced microglial deramification was observed in C57BL/6 mice, but not in FVB mice. Furthermore, noise exposure resulted in a reduction in parvalbumin-positive (PV+) neuron density in the C57BL/6 mice, but not in FVB mice. These results suggest that neuroinflammatory responses and loss of PV+ neurons may contribute to strain differences in noise-induced impairment in gap detection.

Pharmacological Targeting of CSF1R Inhibits Microglial Proliferation and Aggravates the Progression of Cerebral Ischemic Pathology.

Ischemic stroke can induce rapid activation of the microglia. It has been reported that the microglia’s survival is dependent on colony-stimulating factor 1 receptor (CSF1R) signaling and that pharmacological inhibition of CSF1R leads to morphological changes in the microglia in the healthy brain. However, the impact of CSF1R inhibition on neuronal structures and motor ability after ischemia–reperfusion remains unclear. In this study, we investigated microglial de-ramification, proliferation, and activation after inhibition of CSF1R by a tyrosine kinase inhibitor (ki20227) in a mouse model of global cerebral ischemia induced by bilateral common carotid artery ligation (BCAL). In addition to microglial morphology, we evaluated the mRNA expression of cytokines, chemokines, and inflammatory receptors. Our results show that pharmacological inhibition of CSF1R in ischemic mice resulted in the blockade of microglial proliferation and a shift in microglial morphology reflected by excessive de-ramification and a more activated phenotype accompanied by an enhanced innate immune response. Furthermore, we show that pharmacological inhibition of CSF1R in ischemic mice resulted in the aggravation of neuronal degeneration and behavioral impairment. Intravital two-photon imaging revealed that although pharmacological inhibition of CSF1R did not affect the recovery of dendritic structures, it caused a significant increase in spine elimination during reperfusion in ischemic mice. These findings suggest that pharmacological inhibition of CSF1R induces a blockade of microglial proliferation and causes acute activation of the microglia accompanied by a severe inflammatory response. It aggravates neuronal degeneration, loss of dendritic spines, and behavioral deficits after transient global cerebral ischemia.

Diffusible Tumor Necrosis Factor-Alpha (TNF-α) Promotes Noise-Induced Parvalbumin-Positive (PV+) Neuron Loss and Auditory Processing Impairments.

Neuroinflammation has been implicated in noise-induced auditory processing disorder and tinnitus. Certain non-auditory neurological disorders can also increase the levels of proinflammatory cytokines in the brain. To investigate the impact of increased brain proinflammatory cytokine levels on the central auditory pathway, we infused recombinant TNF-α into the right lateral cerebral ventricle, and examined auditory processing and cytoarchitecture of the auditory cortex. Microglial deramification was observed in the auditory cortex of mice that had received both TNF-α infusion and exposure to an 86-dB noise, but not in mice that had received either TNF-α infusion or noise exposure alone. In addition, we observed reduced cortical PV+ neuron density and impaired performances in gap detection and prepulse inhibition (PPI) only in mice that received both TNF-α infusion and the noise exposure. These results suggest that disease-related increase in brain proinflammatory cytokine release could be a risk factor for noise-induced auditory processing disorder and tinnitus.

DPP-4 inhibitor reduces striatal microglial deramification after sensorimotor cortex injury induced by external force impact.

Dipeptidyl peptidase-4 inhibitors (or gliptins), a class of antidiabetic drugs, have recently been shown to have protective actions in the central nervous system. Their cellular and molecular mechanisms responsible for these effects are largely unknown. In the present study, two structurally different gliptins, sitagliptin and vildagliptin, were examined for their therapeutic actions in a controlled cortical impact (CCI) model of moderate traumatic brain injury (TBI) in mice. Early post-CCI treatment with sitagliptin, but not vildagliptin, significantly reduced body asymmetry, locomotor hyperactivity, and brain lesion volume. Sitagliptin attenuated post-CCI microglial deramification in the ipsilateral dorsolateral (DL) striatum, while vildagliptin had no effect. Sitagliptin also reduced striatal expression of galectin-3 and monocyte chemoattractant protein 1(MCP-1), and increased the cortical and striatal levels of the anti-inflammatory cytokine IL-10 on the ipsilateral side. These data support a differential protective effect of sitagliptin against TBI, possibly mediated by an anti-inflammatory effect in striatum to preserve connective network. Both sitagliptin and vildagliptin produced similar increases of active glucagon-like peptide-1 (GLP-1) in blood and brain. Increasing active GLP-1 may not be the sole molecular mechanisms for the neurotherapeutic effect of sitagliptin in TBI.

Inhibition of Microglial Activation in the Amygdala Reverses Stress-Induced Abdominal Pain in the Male Rat.

Background & AimsPsychological stress is a trigger for the development of irritable bowel syndrome and associated symptoms including abdominal pain. Although irritable bowel syndrome patients show increased activation in the limbic brain, including the amygdala, the underlying molecular and cellular mechanisms regulating visceral nociception in the central nervous system are incompletely understood. In a rodent model of chronic stress, we explored the role of microglia in the central nucleus of the amygdala (CeA) in controlling visceral sensitivity. Microglia are activated by environmental challenges such as stress, and are able to modify neuronal activity via synaptic remodeling and inflammatory cytokine release. Inflammatory gene expression and microglial activity are regulated negatively by nuclear glucocorticoid receptors (GR), which are suppressed by the stress-activated pain mediator p38 mitogen-activated protein kinases (MAPK).MethodsFisher-344 male rats were exposed to water avoidance stress (WAS) for 1 hour per day for 7 days. Microglia morphology and the expression of phospho-p38 MAPK and GR were analyzed via immunofluorescence. Microglia-mediated synaptic remodeling was investigated by quantifying the number of postsynaptic density protein 95–positive puncta. Cytokine expression levels in the CeA were assessed via quantitative polymerase chain reaction and a Luminex assay (Bio-Rad, Hercules, CA). Stereotaxic infusion into the CeA of minocycline to inhibit, or fractalkine to activate, microglia was followed by colonic sensitivity measurement via a visceromotor behavioral response to isobaric graded pressures of tonic colorectal distension.ResultsWAS induced microglial deramification in the CeA. Moreover, WAS induced a 3-fold increase in the expression of phospho-p38 and decreased the ratio of nuclear GR in the microglia. The number of microglia-engulfed postsynaptic density protein 95–positive puncta in the CeA was increased 3-fold by WAS, while cytokine levels were unchanged. WAS-induced changes in microglial morphology, microglia-mediated synaptic engulfment in the CeA, and visceral hypersensitivity were reversed by minocycline whereas in stress-naïve rats, fractalkine induced microglial deramification and visceral hypersensitivity.ConclusionsOur data show that chronic stress induces visceral hypersensitivity in male rats and is associated with microglial p38 MAPK activation, GR dysfunction, and neuronal remodeling in the CeA.

Microglia-like Cells Derived from Hematopoietic Stem and Progenitor Cells Are a Model System to Investigate Chronic Pain in Sickle Cell Disease

Background: Patients with sickle cell disease (SCD) experience acute pain episodes in early childhood; over 50% of adult SCD patients go on to develop chronic pain. In animal models, microglia, derived from hematopoietic stem cells, play an important role in the transition from acute to chronic pain; in patients with chronic pain microglia are often activated with minimal stimuli, releasing TNF-alpha and stimulating neurons. Research involving microglia has been stymied by difficulty in obtaining and culturing microglia, particularly from humans. We therefore propose to culture microglia-like cells from peripheral blood mononuclear cells (PBMC) from pediatric patients with SCD with and without chronic pain (defined as pain for more than 50% of days for at least 3 months) and from normal blood donors.Objectives: Our goal was to determine if our cultured cells exhibited microglia morphology and surface markers, could be activated with inflammatory stimuli, and if donor characteristics were retained in the culture derived microglia-like cells.Methods: PBMC were obtained from peripheral blood by Ficoll separation from three unique patients with SCD and no history of chronic pain (SCD CP-), peripheral blood from three unique patients with SCD with history of chronic pain (SCD CP+), or buffy coat from three human blood donors. PBMC were cultured with recombinant human GM-CSF (10 ng/ml; R&D Systems, MN), recombinant human IL-34 (100 ng/ml; R&D Systems), as previously described (Ohgidani et al, 2014) to induce ramified microglia. On day 7 of culture, cells were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry by staining with CD11b and CD45 antibodies, indirect immunofluorescence performed with anti-CX3CR1antibodies and imaging by confocal laser scanning microscope. Microglia-like cells from 3 normal blood donors, 3 SCD CP- and 3 SCD CP+, were stimulated by 100 ng/mL LPS for 24 hours, beginning on day 6 of culture. Deramification (activation) was assessed by immunofluorescence using anti-Iba1, and by flow using anti-CD68.Results: PBMCs from patients with SCD ± CP and normal blood donors developed microglial morphology, namely, soma bodies with branched collaterals, after incubation with GM-CSF and IL-34. When subjected to flow cytometry, treated cells were CD11bhigh and CD45low; fluorescence microscopy revealed CX3CR1 positivity in the cultured cells, all consistent with accepted microglia phenotype. PBMCs untreated with GM-CSF and IL-34 maintained their round morphology, and were CD11blow, CD45high by FACS.Microglia-like morphology differed significantly between PBMC sources. Microglia-like cells from SCD CP+ had shorter and fewer branches than normal individuals; branching of microglia-like cells from SCD CP- were intermediate in number and length, greater than to SCD CP+ but less than normal individuals.When treated with LPS, nearly 100% of microglia-like cells derived from patients with SCD CP+ became amoeboid in shape, indicating microglial deramification, or activation, compared to approximately 25% of microglial-like cells derived from normal human blood donors or SCD CP-.We also assessed deramification through flow measurement of CD68, a marker found on activated microglia and found that the microglia-like cells derived from HSPCs from SCD CP+ patients had significantly higher CD68 positivity compared to microglia-like cells derived from HSPCs from normal human blood donors or SCD CP-, and significantly higher Iba1 positivity as measured by immunofluorescence.Conclusions: We established the microglia-like nature of the cultured PBMCs derived from patients with SCD±CP and normal blood donors through morphologic examination and confirmation of characteristic surface markers via flow and fluorescence microscopy, and through demonstration of activation with LPS. Our microglial-like cells derived from patients with SCD CP+ were activated at higher percentages by LPs treatment than microglial-like cells derived from normal human blood donors and SCD CP-, suggesting that donor characteristics are retained by the microglial-like cells developed in culture. We propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat and prevent chronic pain. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic stress induced disturbances in Laminin: A significant contributor to modulating microglial pro-inflammatory tone?

Over the last decade, evidence supporting a link between microglia enhanced neuro-inflammatory signalling and mood disturbance has continued to build. One issue that has not been well addressed yet are the factors that drive microglia to enter into a higher pro-inflammatory state. The current study addressed the potential role of the extracellular matrix protein Laminin. C57BL6 adult mice were either exposed to chronic stress or handled for 6 consecutive weeks. Changes in Laminin, microglial morphology and pro-inflammatory cytokine expression were examined in tissue obtained from mice exposed to a chronic restraint stress procedure. These in vivo investigations were complemented by an extensive set of in vitro experiments utilising both a primary microglia and BV2 cell line to examine how Laminin influenced microglial pro-inflammatory tone. Chronic stress enhanced the expression of Laminin, microglial de-ramification and pro-inflammatory cytokine signalling. We further identified that microglia when cultured in the presence of Laminin produced and released significantly greater levels of pro-inflammatory cytokines; took longer to return to baseline following stimulation and exhibited enhanced phagocytic activity. These results suggest that chronic restraint stress is capable of modulating Laminin within the CNS, an effect that has implications for understanding environmental mediated disturbances of microglial function.

Capillary blood flow around microglial somata determines dynamics of microglial processes in ischemic conditions

Microglia are the resident immune cells in the brain. Under normal conditions, resting ramified microglia constantly extend and retract fine processes while performing immunological surveillance. In ischemia, microglia become activated as demonstrated by morphological changes during deramification leading to transformation from ramified to amoeboid form. In vivo two-photon microscopy of enhanced green fluorescent protein (EGFP)-expressing microglia in mouse neocortex was used to examine microglial dynamics during the early periods of focal and global ischemia. A penumbra-like "area-at-risk" surrounded by a square-shaped area of severely hypoperfused tissue was created by laser-induced photothrombosis. The dynamics of microglial processes in the area-at-risk was strongly correlated with capillary blood flow (BF) measured within 10 μm of microglial somata. Changes in BF around distal microglial processes (>30 μm from somata) had no effect on microglial dynamics. A severe reduction of capillary BF near somata by 84% ± 6% resulted in initiation of microglial deramification, suggesting activation. A moderate decrease in BF near somata by 22% ± 5% or increase by 87% ± 10%, reflecting a redistribution of capillary BF, had no effect on microglial morphology. Complete BF loss during cardiac arrest (CA) or transient bilateral common carotid artery occlusion (BCCAO) entirely stalled all microglial processes without structural changes. Reperfusion after BCCAO induced recovery of microglial dynamics to preocclusion values. These findings suggest that during ischemia, the severe drop in BF around microglial somata coincides with morphological activation. However, this activation requires some residual BF, because complete perfusion loss (as during BCCAO and CA) did not support microglial deramification.

Physiological mechanisms of lysophosphatidylcholine-induced de-ramification of murine microglia.

Activation of microglial cells, the resident macrophages of the brain, occurs rapidly following brain injury. De-ramification, i.e. transformation from ramified into amoeboid morphology is one of the earliest manifestations of microglial activation. In the present study, we identified the physiological mechanisms underlying microglial de-ramification induced by lysophosphatidylcholine (LPC). Patch-clamp experiments revealed activation of non-selective cation currents and Ca(2+)-dependent K(+) currents by extracellular LPC. LPC-activated non-selective cation channels were permeable for monovalent and divalent cations. They were inhibited by Gd(3+), La(3+), Zn(2+) and Grammostola spatulata venom, but were unaffected by diltiazem, LOE908MS, amiloride and DIDS. Ca(2+) influx through non-selective cation channels caused sustained increases in intracellular Ca(2+) concentration. These Ca(2+) increases were sufficient to elicit charybdotoxin-sensitive Ca(2+)-dependent K(+) currents. However, increased [Ca(2+)](i) was not required for LPC-induced morphological changes. In LPC-stimulated microglial cells, non-selective cation currents caused transient membrane depolarization, which was followed by sustained membrane hyperpolarization induced by Ca(2+)-dependent K(+) currents. Furthermore, LPC elicited K(+) efflux by stimulating electroneutral K(+)-Cl(-) cotransporters, which were inhibited by furosemide and DIOA. LPC-induced microglial de-ramification was prevented by simultaneous inhibition of non-selective cation channels and K(+)-Cl(-) cotransporters, suggesting their functional importance for microglial activation.

In vitro model of microglial deramification: ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures.

Changes in the morphology of ramified microglia are a common feature in brain pathology and culminate in the appearance of small, rounded, microglia-derived phagocytes in the presence of neural debris. Here, we explored the effect of adding brain cell membranes on the morphology of alphaMbeta2-integrin (CD11b/CD18, CR3) positive microglia cultured on a confluent astrocyte substrate as an in vitro model of deramification. Addition of brain membranes led to a loss of microglial ramification, with full transformation to small, rounded, macrophages at 20-40 microg/ml. Time course studies showed a rapid response, with first effects at 1-3 hours, and full transformation at 24-48 hours. Removal of cell membranes and exchange of the culture medium led to a similarly rapid process of reramification. Comparison of cell membranes from different tissues at 20 microg/ml showed strong transforming effect for the brain, more moderate for kidney and liver, and very weak for spleen and skeletal muscle. Fluorescent labeling of brain membranes revealed uptake by almost all rounded macrophages, by a subpopulation of glial fibrillary acidic protein (GFAP)-positive astrocytes, but not by ramified microglia. Phagocytosis of inert fluorobeads did not lead to a transformation into macrophages but their phagocytosis was inhibited by brain membranes, pointing to a saturable uptake mechanism. In summary, addition of brain cell membranes and their phagocytosis leads to a rapid and reversible loss of ramification. The differences in transforming activity from different tissues and the absence of effect from phagocytosed fluorobeads suggest, however, the need for a second stimulus following the phagocytosis of cell debris.