Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: The ketogenic diet is a treatment for epilepsy; it elevates β-hydroxybutyrate levels. We investigated the effect of (β-HB) ketone body on continuous hippocampal stimulation-induced status epilepticus in c57BL/6 mice and in vitro coronal sections of GCamp7-AAV9 injected hippocampus after high potassium perfusion.

Methods: The 7-8 weeks old C57Bl/6 mice were implanted with insulated stainless-steel electrodes in the left hippocampus and bilateral supra-dural cortical electrodes, a reference electrode at cerebellum. A week after implantation, animals received continuous hippocampal stimulation to induce status epilepticus. The animals in self-sustaining status epilepticus were injected intraperitoneally with β-HB (1g/kg) 15 minutes after stimulation, and the seizure duration and severity were evaluated. Separately, the 4-6 weeks old c57BL/6 mice were injected with Cam-Cre driven GCamp7-AAV (pGP-AAV-syn-FLEX-jGCaMP7s-WPRE: pENN.AAV.CamKII0.4.Cre.SV40) into the hippocampus. 10-14 days after injection, the 300µm coronal sections of the hippocampus were sliced using a vibratome in well-oxygenated ice-cold slicing buffer (95% O₂/ 5% CO₂; 4°C). Following incubation in well-oxygenated artificial cerebrospinal fluid (aCSF), they were visualized under green fluorescence filters to locate the CA1 region. The CA1 neural network was stimulated by applying aCSF with high potassium (5mM) for 15 min. We further exposed the neurons to 2.5mM β-HB in 5mM K⁺ aCSF for 15 min followed by a 15 min high K⁺ containing aCSF washout to affirm the effect of β-HB on neural stimulation.

Results: β-HB treatment significantly reduced SE duration (mean SE duration in saline vs β-HB treated mice: 205.6 and 104.3 min; n=8 each, P = 0.0454; unpaired t-test). Interestingly, seizures though shorter were more severe in the β-HB -treated group (median of saline and β-HB: 3.66 and 2.9;n=12, P< 0.0001; Mann Whitney U test) compared to saline-injected mice. In the in vitro analysis, high K⁺ solutions caused increased fluorescence across CA1 and some neurons showed flickering fluorescence. We observed that the β-HB application significantly reduced flourescent intensity and stopped flickering in the CA1 region of the hippocampal slices as compared to the baseline (5mM K⁺ aCSF) (%ΔF/F Baseline vs. β-HB:15.91 and -7.874%,n=3, P< 0.0001; Brown-Forsythe and Welch ANOVA test). Similarly, the β-HB washout with high K⁺ reversed its effect (%ΔF/F β-HB vs washout: -7.874 and 8.034%;n=3, P< 0.0001; Brown-Forsythe and Welch ANOVA test).