Abstract
BackgroundInteractions 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.MethodsPrimary 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.ResultsThe 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.ConclusionsThe 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.
Highlights
Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system
To address the shortcomings of existing in vitro models of neuroinflammation, we developed an enhanced cell culture model comprised of the three major cell types associated with neuroinflammation—neurons, astrocytes, and microglia
The tri-culture supports neurons, astrocytes, and microglia in vitro Primary cortical cells taken from neonatal rats were cultured in our previously described neuron-astrocyte coculture media [25], or in our tri-culture media consisting of the co-culture media supplemented with 100 ng/mL IL-34, 2 ng/mL Transforming growth factor beta (TGF-β) and 1.5 μg/mL cholesterol
Summary
Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. Current cell culture models of neuroinflammation typically consist of cultures of individual cell types with conditioned media from one cell type transferred to cultures of another cell type [12,13,14] 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. There is a need for new, multicellular culture systems that are capable of modeling the neuroinflammatory impact of crosstalk between different cells in the CNS This need was highlighted in a recent review on the current tools and methods for studying glia [12]
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