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

Abstract

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.

Neuronal–Glial Interaction in a Triple-Transgenic Mouse Model of Alzheimer’s Disease: Gene Ontology and Lithium Pathways

“…Experimental models have also shown that overactive GSK3β is also associated with reactive astrocytosis and microgliosis, an effect that may also be attenuated by lithium (Tang et al, 2016). In a recent study conducted by our group, we showed that that chronic treatment with lithium chloride at subtherapeutic concentrations was able to modify the secretion of pro-and anti-inflammatory interleukins in cocultures of cortical and hippocampal neurons with glial cells, and this effect was associated with the preservation of culture integrity (De-Paula et al, 2016b;Goshi et al, 2020). Gene Ontology (GO) is a bioinformatics approach to ascertain and to unify the representations of gene-and gene product attributes across species, focusing on the related functions of these interactions.…”

Honing the Double-Edged Sword: Improving Human iPSC-Microglia Models

“…A primary rodent tri-culture system of neurons, astrocytes and microglia in serum-free conditions (using TIC-supplemented media) shows promise in modeling different neuroinflammatory scenarios ( 88 ), in line with both in vivo and in vitro studies ( 77 , 89 , 90 ). However, TIC factors could not be omitted in the tri-cultures ( 88 ), indicating that endogenous secretion from the cultured astrocytes was not sufficient to retain a viable and physiologically active microglial phenotype. Interestingly, co-culture with hiPSC-neurons and astrocytes is capable of shifting the transcriptional profile of hiPSC-microglia further towards an ex vivo state than TIC media, although the gap between in vitro and ex vivo microglia is not fully closed ( 29 ).…”

“…Microglia and astrocytes operate in concert, both possessing that "double-edged sword" capacity to either support neuronal recovery (simplistically, "helpful" M2 and A2 phenotypes) or cause neuronal death ("harmful" M1 and A1 reactive phenotypes) [(77) reviewed by [87)]. A primary rodent tri-culture system of neurons, astrocytes and microglia in serum-free conditions (using TIC-supplemented media) shows promise in modeling different neuroinflammatory scenarios (88), in line with both in vivo and in vitro studies (77,89,90). However, TIC factors could not be omitted in the tri-cultures (88), indicating that endogenous secretion from the cultured astrocytes was not sufficient to retain a viable and physiologically active microglial phenotype.…”

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology

“…Their potential has been further expanded by the establishment of co-cultures, where multiple cell types are cultured together with the use of semi-permeable membrane inserts. Neuronal co-cultures allow investigation of the interplay between neurons and other different cell types, such as astrocytes [ 16 , 17 ], microglia [ 18 , 19 ], or both in the so-called tri-culture system [ 20 ], up to more complex heterogeneous cultures such as those including neurons, glia, endothelium and glioma cells [ 21 ], or those addressing multi-cellular tissue units, like neurogenic niches [ 22 , 23 ] and the blood–brain barrier (BBB) [ 24 ].…”