An Overview of in vitro Methods to Study Microglia

Timmerman, Raissa; Burm, Saskia M.; Bajramović, Jeffrey J.

doi:10.3389/fncel.2018.00242

Cited by 175 publications

(157 citation statements)

References 137 publications

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“…In this regard, many authors in the last few years have reported that there are several substances of natural origin, as well as of a synthetic nature with the ability of inhibiting, both in vitro and in vivo, the PI3K/Akt inflammatory signaling pathway and, consequently, reducing the microglia activation and the inflammatory responses associated with this pathway. A number of in vitro models, including microglia cell lines, stem cell derived microglia cultures, and primary dissociated cell cultures, were performed in order to study the role of microglia in neurodegenerative disorders and, in particular, relevant to PI3K signaling (for more detail, see [73]). This anti-inflammatory beneficial effect could be linked to the ability of inhibiting PI3K/Akt signaling pathways [74,75].…”

Section: Pi3k Role In Neuroinflammationmentioning

confidence: 99%

Microglia Mediated Neuroinflammation: Focus on PI3K Modulation

et al. 2020

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Immune activation in the central nervous system involves mostly microglia in response to pathogen invasion or tissue damage, which react, promoting a self-limiting inflammatory response aimed to restore homeostasis. However, prolonged, uncontrolled inflammation may result in the production by microglia of neurotoxic factors that lead to the amplification of the disease state and tissue damage. In particular, specific inducers of inflammation associated with neurodegenerative diseases activate inflammatory processes that result in the production of a number of mediators and cytokines that enhance neurodegenerative processes. Phosphoinositide 3-kinases (PI3Ks) constitute a family of enzymes regulating a wide range of activity, including signal transduction. Recent studies have focused attention on the intracellular role of PI3K and its contribution to neurodegenerative processes. This review illustrates and discusses recent findings about the role of this signaling pathway in the modulation of microglia neuroinflammatory responses linked to neurodegeneration. Finally, we discuss the modulation of PI3K as a potential therapeutic approach helpful for developing innovative therapeutic strategies in neurodegenerative diseases.

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Section: Pi3k Role In Neuroinflammationmentioning

confidence: 99%

Microglia Mediated Neuroinflammation: Focus on PI3K Modulation

et al. 2020

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“…Laboratory experimentation may generate different activation profiles from those found in live human brain tissue following a stroke, as in vitro cultures commonly demonstrate amoeboid morphologies and are generally considered to exhibit signs of low-level inflammatory activation [169]. Therefore, it may be more scientifically sound to caution against attempts to classify microglial populations as exhibiting a distinct phenotype and instead view microglial "activation" as specific to individual cells [60].…”

Section: Challenges For Stroke Therapies Targeting Microgliamentioning

confidence: 99%

Post-Ischaemic Immunological Response in the Brain: Targeting Microglia in Ischaemic Stroke Therapy

et al. 2020

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Microglia, the major endogenous immune cells of the central nervous system, mediate critical degenerative and regenerative responses in ischaemic stroke. Microglia become “activated”, proliferating, and undergoing changes in morphology, gene and protein expression over days and weeks post-ischaemia, with deleterious and beneficial effects. Pro-inflammatory microglia (commonly referred to as M1) exacerbate secondary neuronal injury through the release of reactive oxygen species, cytokines and proteases. In contrast, microglia may facilitate neuronal recovery via tissue and vascular remodelling, through the secretion of anti-inflammatory cytokines and growth factors (a profile often termed M2). This M1/M2 nomenclature does not fully account for the microglial heterogeneity in the ischaemic brain, with some simultaneous expression of both M1 and M2 markers at the single-cell level. Understanding and regulating microglial activation status, reducing detrimental and promoting repair behaviours, present the potential for therapeutic intervention, and open a longer window of opportunity than offered by acute neuroprotective strategies. Pharmacological modulation of microglial activation status to promote anti-inflammatory gene expression can increase neurogenesis and improve functional recovery post-stroke, based on promising preclinical data. Cell-based therapies, using preconditioned microglia, are of interest as a method of therapeutic modulation of the post-ischaemic inflammatory response. Currently, there are no clinically-approved pharmacological options targeting post-ischaemic inflammation. A major developmental challenge for clinical translation will be the selective suppression of the deleterious effects of microglial activity after stroke whilst retaining (or enhancing) the neurovascular repair and remodelling responses of microglia.

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“…The next challenge was to examine APC activity in different cell types, and to determine whether APC is secreted into the medium or attached to the cells. Two cells types were used; N9 cells as a model for microglia, and C6 cells as a model for astrocytes [24,25]. In order to distinguish between medium and cells derived activity, the serum-free medium was transferred into empty wells 24 h after initiating the experiment.…”

Section: Cell Culturesmentioning

confidence: 99%

A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF

Golderman

Gofrit

Maggio

et al. 2020

IJMS

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Background: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Results: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl2. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. Conclusions: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF.

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An Overview of in vitro Methods to Study Microglia

Cited by 175 publications

References 137 publications

Microglia Mediated Neuroinflammation: Focus on PI3K Modulation

Microglia Mediated Neuroinflammation: Focus on PI3K Modulation

Post-Ischaemic Immunological Response in the Brain: Targeting Microglia in Ischaemic Stroke Therapy

A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF

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