Co‐Culture of Neurons and Microglia

Roqué, Pamela J.; Costa, Lucio G.

doi:10.1002/cptx.32

Cited by 37 publications

(25 citation statements)

References 55 publications

(107 reference statements)

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“…While these models have provided significant insights into neuroinflammatory processes [15], these models contain inherent limitations, most notably the inability to observe the effects of membrane-bound or cell proximity-dependent mechanisms and the fact that the concentration of secreted cytokines transferred between cultures may not be physiologically relevant. An alternative model involves seeding microglia over a previously established primary neuron culture to observe the effect of this cell-cell interaction over a short period of time (24-72 h) [16][17][18]. In addition to the limited time-scale of this model, the culture media used to support the microglia prior to their addition to the neuronal culture typically contains a high concentration of serum, likely causing the microglia to be in an already activated state before their addition to the neuronal cell cultures [19].…”

Section: Introductionmentioning

confidence: 99%

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation

Goshi

Morgan

Lein

et al. 2020

J Neuroinflammation

132

101

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Background: Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required. Methods: Primary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co-and tri-cultures were also compared. Results: The tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture. Conclusions: The tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono-and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.

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Section: Introductionmentioning

confidence: 99%

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation

Goshi

Morgan

Lein

et al. 2020

J Neuroinflammation

132

101

View full text Add to dashboard Cite

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“…Therefore, (i) a rather high number of animals must be sacrificed for each experiment. Keeping in mind the necessity for enforced application of high animal welfare standards, an ethical evaluation and consideration of the scientific significance, e.g., a scientific validation for harm-benefit analysis for each and every experiment has to be considered [100,101]; (ii) in order to warrant a sufficient cell number and appropriate purity of neonatal microglia for in vitro approaches, different methods have been described and comprehensively reviewed [99,102,103]. The herein used method is based on the most frequently used differential adherence method.…”

Section: Discussionmentioning

confidence: 99%

Impact of ambient temperature on inflammation-induced encephalopathy in endotoxemic mice—role of phosphoinositide 3-kinase gamma

Lang

Ndongson-Dongmo

Lajqi

et al. 2020

J Neuroinflammation

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Background Sepsis-associated encephalopathy (SAE) is an early and frequent event of infection-induced systemic inflammatory response syndrome. Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and inflammation-related microglial activity. In homeotherms, variations in ambient temperature (Ta) outside the thermoneutral zone lead to thermoregulatory responses, mainly driven by a gradually increasing sympathetic activity, and may affect disease severity. We hypothesized that thermoregulatory response to hypothermia (reduced Ta) aggravates SAE in PI3Kγ-dependent manner. Methods Experiments were performed in wild-type, PI3Kγ knockout, and PI3Kγ kinase-dead mice, which were kept at neutral (30 ± 0.5 °C) or moderately lowered (26 ± 0.5 °C) Ta. Mice were exposed to lipopolysaccharide (LPS, 10 μg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection)—evoked systemic inflammatory response (SIR) and monitored 24 h for thermoregulatory response and blood–brain barrier integrity. Primary microglial cells and brain tissue derived from treated mice were analyzed for inflammatory responses and related cell functions. Comparisons between groups were made with one-way or two-way analysis of variance, as appropriate. Post hoc comparisons were made with the Holm–Sidak test or t tests with Bonferroni’s correction for adjustments of multiple comparisons. Data not following normal distribution was tested with Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Results We show that a moderate reduction of ambient temperature triggers enhanced hypothermia of mice undergoing LPS-induced systemic inflammation by aggravated SAE. PI3Kγ deficiency enhances blood–brain barrier injury and upregulation of matrix metalloproteinases (MMPs) as well as an impaired microglial phagocytic activity. Conclusions Thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range exacerbates LPS-induced blood–brain barrier injury and neuroinflammation. PI3Kγ serves a protective role in suppressing release of MMPs, maintaining microglial motility and reinforcing phagocytosis leading to improved brain tissue integrity. Thus, preclinical research targeting severe brain inflammation responses is seriously biased when basic physiological prerequisites of mammal species such as preferred ambient temperature are ignored.

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“…This reductionist in vitro system allows for preliminary questions to be asked about microglial neurogenic potential in the cortex and can be used for future studies with other microglial cell lines and primary microglial cells. These studies do not negate the role of microglia in other areas of the brain such as the cerebellum studied using similar in vitro methods [ 79 ] .…”

Section: Discussionmentioning

confidence: 99%

Microglia induce neurogenic protein expression in primary cortical cells by stimulating PI3K/AKT intracellular signaling in vitro

et al. 2021

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Emerging evidence suggests that microglia can support neurogenesis. Little is known about the mechanisms by which microglia regulate the cortical environment and stimulate cortical neurogenesis. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following mechanical injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and survival. Analyses of cortical cell proliferation, cell death, neurogenic protein expression, and intracellular signaling were performed on uninjured and injured cortical cells in co-culture with microglial cell lines. Microglia soluble cues enhanced cortical cell viability and proliferation cortical cells. Co-culture of injured cortical cells with microglia significantly reduced cell death of cortical cells. Microglial co-culture significantly increased Nestin + and α-internexin + cortical cells. Multiplex ELISA and RT-PCR showed decreased pro-inflammatory cytokine production by microglia co-cultured with injured cortical cells. Inhibition of AKT phosphorylation in cortical cells blocked microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro. This in vitro model system allows for assessment of the effect of microglial-derived soluble signals on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following mechanical injury. These data suggest that microglia cells can downregulate inflammatory cytokine production following activation by mechanical injury to enhance proliferation of new cells capable of neurogenesis via activation of AKT intracellular signaling. Increasing our understanding of the mechanisms that drive microglial-enhanced cortical neurogenesis during homeostasis and following injury in vitro will provide useful information for future primary cell and in vivo studies.

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Co‐Culture of Neurons and Microglia

Cited by 37 publications

References 55 publications

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation

A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation

Impact of ambient temperature on inflammation-induced encephalopathy in endotoxemic mice—role of phosphoinositide 3-kinase gamma

Microglia induce neurogenic protein expression in primary cortical cells by stimulating PI3K/AKT intracellular signaling in vitro

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