Organotypic Brain Slice Culture Microglia Exhibit Molecular Similarity to Acutely-Isolated Adult Microglia and Provide a Platform to Study Neuroinflammation

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.

Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

“…The MerTK mechanistic experiment was performed in organotypic slice cultures, enabling isolating the pharmacological effects of the MerTK inhibitor to the PVN, rather than the whole brain or whole body. Organotypic slice cultures are not a perfect reflection of the in vivo context, as they do involve initial microglial activation and the loss of extrahypothalamic synaptic connections ( Czapiga and Colton, 1999 ; Delbridge et al, 2020 ). However, most inflammatory markers and neuronal death rates stabilize at very low levels by 1 week in culture, the time point at which we begin pharmacological treatments.…”

“…However, most inflammatory markers and neuronal death rates stabilize at very low levels by 1 week in culture, the time point at which we begin pharmacological treatments. Importantly, organotypic slice cultures are far superior to dissociated cultures in approximating the in vivo condition, because of the relative preservation of tissue architecture and cellular composition ( Czapiga and Colton, 1999 ; Delbridge et al, 2020 ; Weinhard et al, 2018 ). Off-target actions of the MerTK inhibitor should be considered: UNC2025 has a ~40-fold greater selectivity for MerTK over Axl and Tyro3.…”

Microglial TNFα controls synaptic GABAARs, sleep slow waves and memory consolidation

“…The latter system closely mimics the in vivo brain by preserving tissue architecture and cellular composition. In these slices microglia retain their 3D ramified morphology and their functional properties (De Simoni et al, 2008; Delbridge et al, 2020; Weinhard et al, 2018). They further conserve their ability to regulate synapses (Cantaut-Belarif et al, 2017; Pascual et al, 2012).…”

The Impact of Obesity on Microglial Function: Immune, Metabolic and Endocrine Perspectives

“…Although in vitro culture conditions can be improved through the addition of TGF-β2, IL-34 and cholesterol [ 429 ], the homeostatic profile of microglia is severely affected by in vitro culture [ 24 , 103 ]. Instead, ex vivo culture of brain slices more successfully preserves the physiological profile of microglia [ 430 ].…”