Abstract
Microglia are central nervous system (CNS) resident immune cells that have been implicated in neuroinflammatory pathogenesis of a variety of neurological conditions. Their manifold context-dependent contributions to neuroinflammation are only beginning to be elucidated, which can be attributed in part to the challenges of studying microglia in vivo and the lack of tractable in vitro systems to study microglia function. Organotypic brain slice cultures offer a tissue-relevant context that enables the study of CNS resident cells and the analysis of brain slice microglial phenotypes has provided important insights, in particular into neuroprotective functions. Here we use RNA sequencing, direct digital quantification of gene expression with nCounter® technology and targeted analysis of individual microglial signature genes, to characterize brain slice microglia relative to acutely-isolated counterparts and 2-dimensional (2D) primary microglia cultures, a widely used in vitro surrogate. Analysis using single cell and population-based methods found brain slice microglia exhibited better preservation of canonical microglia markers and overall gene expression with stronger fidelity to acutely-isolated adult microglia, relative to in vitro cells. We characterized the dynamic phenotypic changes of brain slice microglia over time, after plating in culture. Mechanical damage associated with slice preparation prompted an initial period of inflammation, which resolved over time. Based on flow cytometry and gene expression profiling we identified the 2-week timepoint as optimal for investigation of microglia responses to exogenously-applied stimuli as exemplified by treatment-induced neuroinflammatory changes observed in microglia following LPS, TNF and GM-CSF addition to the culture medium. Altogether these findings indicate that brain slice cultures provide an experimental system superior to in vitro culture of microglia as a surrogate to investigate microglia functions, and the impact of soluble factors and cellular context on their physiology.
Highlights
As the central nervous system (CNS)-resident immune cell in the parenchyma, microglia have emerged as a promising cellular target in the context of many diseases including Alzheimer’s disease (AD) and other neuroinflammatory/neurodegenerative conditions such as multiple sclerosis (MS) (Hemmer et al, 2015; Jansen et al, 2019; Liu et al, 2019; Rostalski et al, 2019; Bellenguez et al, 2020)
We demonstrate organotypic brain slice cultures provide a means of studying microglia in a tissue environment closely mimicking the in vivo brain by preserving tissue architecture and cellular composition
While the data we present are confirmatory in nature and support the use of slice cultures to allow for the maintenance of a microglia signature and function more akin to what is seen in vivo, we hope that these results will serve as foundation for future studies focused on microglia biology as a valid experimental system and alternative to in vivo studies to generate novel biological insights
Summary
As the CNS-resident immune cell in the parenchyma, microglia have emerged as a promising cellular target in the context of many diseases including Alzheimer’s disease (AD) and other neuroinflammatory/neurodegenerative conditions such as multiple sclerosis (MS) (Hemmer et al, 2015; Jansen et al, 2019; Liu et al, 2019; Rostalski et al, 2019; Bellenguez et al, 2020). In contrast to other cell types, such as neurons or peripheral immune cells, well-validated in vitro culture methods that would enable insights into microglia function are lacking This represents a significant barrier to development of meaningful therapeutics that modify microglia function in a disease setting. Several gene expression signatures associated with microglia identity, inflammation, proliferation and specific disease models have been determined through meta-analysis of gene expression profiling data (Friedman et al, 2018). Their relevance to human disease remains uncertain (Mathys et al, 2019)
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