Abstracts

Rationale: Inflammation is a key component of many acquired forms of epilepsy; however, we do not understand how triggering events lead to potentially irreversible anatomical changes and epileptic seizures. Tumor necrosis factor-a (TNF) is produced primarily by resident microglia to coordinate the inflammatory immune response against viral and bacterial invaders. Adversely, TNF is elevated during the acute stage of epileptogenesis in the TMEV (Theiler’s murine encephalomyelitis virus) model, and is hypothesized to contribute to epileptogenesis by increasing excitatory receptor expression in neurons. Many attempts have been employed to characterize microglia heterogeneity and structural changes during disease including measurements of soma size, branch length, branch complexity, branch diameter, 2D/3D area, and branch tip movement. This is the first study to selectively express a structural marker (cytosolic TdTomato) and a genetically encoded calcium sensor (GCAMP5G) in CX3CR1 microglia and macrophages to measure phenotype changes in innate immune cells viral-induced seizure development. Methods: Mice were bred to express cytosolic TdTomato and the GCAMP5G calcium sensor (JAX 024477) using tamoxifen inducible Cre expression under the CX3CR1 receptor (JAX 021160) on a C57BL/6J background.  Male and female mice were given 100 mg/kg i.p. tamoxifen (TAM) in peanut oil (10 mg/ml) four times. Four to ten days after the last tamoxifen dose, mice were injected intracortically with 2x10⁵ plaque forming units (pfu) of TMEV or PBS (n=4 per group). Mice were weighed daily and graded twice daily for handling induced seizures on a modified Racine scale between 3-7 days post-injection (dpi). At 2, 5, and 14 dpi, acute brain slices were imaged at least 60 µm below the slice surface with 2-photon microscopy in CA1 hippocampus. Branch diameter and the distance to the soma were measured on 3 branches per cell and for 3 cells per group (n=12) using ImageJ. Results: The branch diameter displayed a significant difference overall between TMEV and PBS samples (p=0.0398), as well as overall between timepoints (p=0.0492) for branches measured proximal to the soma using a two-way ANOVA. The distance to the soma for distal branches displayed a significant difference between TMEV and PBS at 2 dpi (p<0.05 Bonferroni post hoc). Within the TMEV group, distal branches at 2 dpi had greater lengths than branches at 5 and 14 dpi (p<0.05 Bonferroni post hoc). Conclusions: Resident microglia and infiltrating macrophages change their phenotype after CNS infection to fight pathogens, but they also adversely contribute to epileptogenesis. We have initially measured a thickening of processes close to the cell body and gradual reduction in branch length for CX3CR1 expressing cells in TMEV infected animals. Elucidating the role of microglia and macrophages in initiating and in maintaining neuroinflammation could help us design better therapeutic strategies to reduce proconvulsant signals and to prevent epileptogenesis. Funding: Skaggs Research Fellowship