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-α (TNF) is produced primarily by resident microglia to coordinate the inflammatory immune response against viral and bacterial invaders. Adversely, TNF is elevated during a stage of acute seizures in the TMEV (Theiler’s Murine Encephalomyelitis Virus) model, and is hypothesized to contribute to epileptogenesis by increasing excitatory receptor expression in neurons. Previous work in global knock out (KO) mice supports a link between seizure development and the pro-inflammatory TNF receptor 1 (TNFR1). However, it is unclear how glial cells respond to pathologic TNF levels and whether their response contributes to seizure development. Therefore, we knocked out the pro-inflammatory TNFR1 in microglia and monitored for changes in seizure development using a viral infection mouse model. Methods: Mice were bred to KO TNFR1-flox (EM 07099) using tamoxifen inducible Cre expression under the microglial CX3CR1 promoter (JAX 021160) on a C57BL/6J background.  Male and female CX3CR1 creERT2/+ TNFR1 flox/flox mice were given 150 mg/kg i.p. tamoxifen (TAM) in peanut oil three times at 4-6 weeks old. Littermate controls either did not receive TAM (Cre+/peanut oil) or did not express Cre (Cre-/TAM). Four days later, mice were injected intracortically with 2x105 plaque forming units (pfu) of the Daniels strain of TMEV. Mice were graded twice daily between 3-7 days post-injection (dpi) for handling induced seizures on a modified Racine scale by experimenters blinded to the groups. At 14-15 dpi, brains were fixed in formalin and sections stained using antibodies for neurons (NeuN), astrocytes (GFAP), and microglia/macrophages (Iba-1). Results: The incidence, severity, and frequency of acute seizures was compared for WT controls and TNFR1 KO in microglia. The number of mice with seizures in each group was not significantly different at 71% (12/17 mice), 56% (5/9 mice), and 42% (8/19 mice) for the TNFR1 KO, Cre+/TAM control, and Cre-/no TAM control, respectively (Fisher’s Exact Test). The weighted average total seizure burden was distributed similarly for TNFR1 KO mice and the control groups. Also, the average cumulative seizure burden at each timepoint was very similar for TNFR1 KO mice and the control groups. The mice in each group gained weight in a similar manner, and no adverse reactions were noted to the infection. Conclusions: Neither a pro-epileptogenic, nor a protective mechanism was demonstrated by abolishing TNFR1 signaling in microglia during an acute viral infection in mice. Although TNF is produced in large amounts by activated microglia, it seems TNFR1 feedback in microglia does not alter the progression of brain damage that leads to seizures in this viral model. From a therapeutic perspective, this finding is helpful as drugs that decrease signaling via TNFR1 are unlikely to interfere with microglial viral clearance.  Funding: KSW: University of UtahRSF: NIH 5R01NS065714