Abstracts

Rationale: Microglia are highly adaptable innate immune cells in the brain. They respond to different pathogens with tailored actions and progress through a series of activated phenotypes to restore normal brain function and avoid chronic neuronal dysfunction. During viral encephalitis, the virus and collateral cell damage activate microglia to produce cascades of defensive inflammatory cytokines. But these cytokines also adversely increase neuronal excitability, possibly leading to acute seizures and an increased risk of chronic epilepsy. Newly activated pro-inflammatory microglia have been shown to express a different set of genes for damage receptors and purinergic receptors than quiescent microglia. Therefore, our goal was to determine if the damage-responsive microglia phenotype was maintained following a CNS infection that induces seizures and neuronal damage.

Methods: We used two-photon microscopy in acute hippocampal brain slices to examine the functional ability of microglia to detect damage signals from either a nearby laser burn or exogenous application of ATP, and to engage calcium signaling pathways in the days and weeks after viral brain infection in the Theiler’s murine encephalomyelitis virus (TMEV) mouse model of temporal lobe epilepsy (TLE). The GCaMP5G calcium signal in microglia structures, including processes and cell somas, was uniquely detected using the Suite2p point-weighted calcium signal tool pack.

Results: Two days after brain infection, GCaMP5G-expressing microglia have less calcium activity in their cell bodies after a nearby laser burn. By 5 and 15 days after brain infection, microglia have dramatic decreases in both somatic and process calcium signals, and less extension of microglia processes toward the burn area in ex vivo acute brain slices. Yet, microglial ability to detect extracellular ATP was only depressed at 5 days and was restored by 15 days. These activated microglia were less responsive to damage, had decreased intracellular calcium events in different subcellular regions, and reduced extension of microglia processes into acutely damaged brain regions. After viral brain infection, P2ry12 gene expression was decreased at 5 and 15 days in Iba1-positive microglia/macrophages, while the pro-inflammatory cytokine Tnfa was elevated at 2, 5, & 15 days.

Conclusions: The reduced sensitivity of the microglia damage response during viral infection may beneficially limit the scale of the innate immune response in the brain while pathological seizures, neuronal damage, and inflammatory cytokine are prevalent.

Funding: Please list any funding that was received in support of this abstract.: This work was funded by R37 NS065434 (KSW), NSF GRFP (LAB), and the Skaggs Scholars Fellowship (GJW).