Abstracts

Rationale: Exogenous ketone esters (KE) may serve as an alternative to the ketogenic diet (KD) in the treatment of medically intractable epilepsy (PMID: 23552496, 27546058, 28790891 & 30930744). KE are metabolized in the blood and other tissues to ketone bodies (KB) and thereby elevate blood BHB levels. While earlier studies have suggested that sustained ketosis is required to achieve seizure control, Olson et al. (PMID: 29804833) recently proposed that changes to the gut microbiome are responsible for the anti-seizure effects of the ketogenic diet, not ketosis per se. Further, although KE-induced seizure control is attributed to ketosis, changes to the gut microbiome have not yet been characterized following KE treatment. We, therefore, sought to determine whether KE supplementation induces microbiome changes, in addition to rendering anti-seizure effects, in spontaneously epileptic mice. Methods: Male wild-type (WT) and Kcna1-null (KO) mice (C3HeB/FeJ background) were used in this study (N=24). There were four experimental groups (N=6, each): (1) WT mice treated with water (WT-W), (2) WT mice administered KE (WT-KE), (3) KO mice treated with water (KO-W), and (4) KO mice treated with KE (KO-KE). Mice in each group were administered 5 mL/kg of treatment (W or KE) daily over 12 days. Blood samples for BHB and glucose levels were collected by tail-prick at time t=0, 1, 4, 8, and 12 hours post-gavage on day 1, and then immediately prior to gavage every other day thereafter. Animals were implanted with screw electroencephalography (EEG) electrodes and monitored for 12 days. Seizures were scored using a modified Racine scale. Seizure burden was calculated as the quotient of the summed Racine scores and the length of the observation period. Baseline and end-of-treatment fecal samples were collected for 16s rRNA metagenomic sequencing and analysis. Results: There was a significant increase in blood BHB, and a decrease in blood glucose, levels relative to baseline (t=0) in the KO-KE group 1 hour after gavage. These effects dissipated within 4 hours. Interestingly, KE treatment did not affect blood BHB or glucose chronically. Further, there was no effect of genotype (WT vs. KO) on blood chemistry acutely or chronically. KE supplementation ameliorated seizure frequency and burden in Kcna1-null mice (p<.05). Microbiome analyses revealed a significant increase in Lactobacilli abundance following KE treatment. In addition, we observed a significant effect of genotype (WT vs. KO) on the host gut microbial composition. Conclusions: Collectively, our data further support the growing experimental literature that KE alone can provide anti-seizure effects. Moreover, sustained ketosis may not be necessary for such effects, as transient increases in BHB are shown in the present study to confer anti-seizure efficacy. Finally, while gut microbiome changes following the KD in animals and humans with epilepsy appear somewhat discordant (PMID: 29804833, 27594980, 30701077), the present study suggests that the bacterial species Lactobacilli may play a contributory role in seizure control. Funding: Branch Out Neurological Foundation Undergraduate Research Grant, CANADIAN INSTITUTES OF HEALTH RESEARCH, ALBERTA CHILDREN’S HOSPITAL RESEARCH INSTITUTE