Acute TrkB inhibition rescues phenobarbital-resistant seizures in a mouse model of neonatal ischemia

Abstract

Neonatal seizures are commonly associated with hypoxic-ischemic encephalopathy. Phenobarbital (PB) resistance is common and poses a serious challenge in clinical management. Using a newly characterized neonatal mouse model of ischemic seizures, this study investigated a novel strategy for rescuing PB resistance. A small-molecule TrkB antagonist, ANA12, used to selectively and transiently block post-ischemic BDNF-TrkB signaling in vivo, determined whether rescuing TrkB-mediated post-ischemic degradation of the K(+)-Cl(-) co-transporter (KCC2) rescued PB-resistant seizures. The anti-seizure efficacy of ANA12 + PB was quantified by (i) electrographic seizure burden using acute continuous video-electroencephalograms and (ii) post-treatment expression levels of KCC2 and NKCC1 using Western blot analysis in postnatal day (P)7 and P10 CD1 pups with unilateral carotid ligation. ANA12 significantly rescued PB-resistant seizures at P7 and improved PB efficacy at P10. A single dose of ANA12 + PB prevented the post-ischemic degradation of KCC2 for up to 24 h. As anticipated, ANA12 by itself had no anti-seizure properties and was unable to prevent KCC2 degradation at 24 h without follow-on PB. This indicates that unsubdued seizures can independently lead to KCC2 degradation via non-TrkB-dependent pathways. This study, for the first time as a proof-of-concept, reports the potential therapeutic value of KCC2 modulation for the management of PB-resistant seizures in neonates. Future investigations are required to establish the mechanistic link between ANA12 and the prevention of KCC2 degradation.

Keywords: Bumetanide; KCC2; PB resistance; hypoxic-ischemic encephalopathy; neonatal seizure.

Figure 1. Effect of ANA12+PB on the total seizure burden at P7 and P10 I

Schematics of the experimental design. Experimental paradigm for the acute ligation surgery and synchronous video-EEG recordings with the time-course of drug administrations and the durations for each procedure. Arrowheads indicate time-points of drug administration after carotid-ligation and brain-harvesting for 24h WB data. II. A–D. Total time spent seizing on EEG was quantitated as seizure burden at P7 and P10 over a 3h period, and was represented by bar graphs (black bar=baseline, gray=2^nd h, and white = 3^rd h) A&C) vehicle (5% DMSO) and ANA12-alone treatment groups. The data from A&C were pooled to form a ligate-control group represented in B&D. B&D) ligate control vs. ANA12+PB+BTN. Within-group comparison was done using repeated measures ANOVA (i.e., *=P<0.05; **=P<0.01; ***=P<0.001). Brackets (@) denote significant between-group comparisons at each hour (independent sample t-tests; P<0.05).

Figure 2. ANA12 rescued PB-resistance at P7 and improved PB-efficacy at P10

The effect of ANA12 on PB-efficacy was evaluated as percent PB seizure suppression over baseline: % PB seizure suppression = 100*[Post-PB seizure burden/baseline seizure burden]. A. PB by itself failed to subdue ischemic seizures at P7. ANA12+PB significantly subdued ischemic seizures by 62% (One-way ANOVA; *** = P<0.001). B. PB alone subdued seizures by 68% at P10. ANA12+PB improved this efficacy to 85%. This improved PB-efficacy however was not statistically significant when compared to the efficacy of PB alone. The sample size for saline+PB group was n=13 and n=11 for P7 and P10 respectively; n for ligate control and treated group was same as listed in Fig. 1 (see Suppl. Table 1). Sz: Seizure.

Figure 3. The anti-seizure efficacy of ANA12+PB was not different by sex

The efficacy of ANA12+PB on total seizure burden was evaluated by sex at P7 and P10. A&C. Males at both ages of P7 and P10 responded significantly to ANA12+PB. However, at P10, BTN administration following PB weakened the statistical significance of seizure suppression achieved by PB. B&D. ANA12+PB efficacy was significant in females at both P7 and P10. Additionally, a significant temporal increase in the total seizure burden over time-course of 3h period was detected in P7 females, but was absent in P7 males. At P7, BTN aggravated PB-subdued seizures in females such that PB-efficacy was lost. ANA12+ PB efficacy was not significantly different between males and females. (Repeated measures ANOVA; *= P<0.05; ** =P<0.01; ***=P<0.001).

Figure 4. ANA12+PB rescued KCC2 expression but had no effect on NKCC1 expression at 24h

A–D. Bar graphs represent mean expression of KCC2 and NKCC1 in ipsilateral hemisphere normalized to the contralateral at 24h after ischemia. β-actin was used as an internal control. The protein bands for both co-transporters were: 1) normalized to the level of actin for the same sample, 2) followed by normalization of ipsilateral co-transporter expression levels to contralateral hemisphere of the same pup A&C. The post-ischemic degradation of KCC2 expression (~20%) was prevented in the ANA12+PB+BTN treated group at P7 and P10. B&D. Post-ischemic expression levels of NKCC1 in the treated group were not significantly different from ligate controls (One-way ANOVA; *=P<0.05; ***=P<0.001).

Figure 5. Significant correlation between rescue of KCC2 degradation at 24h and seizure suppression was detected

The percent change of KCC2 expression of an ipsilateral hemisphere [% change = 100*(ipsi - contra/contra KCC2 expression)] was correlated to the percent seizure suppression [% baseline seizure burden = 100*(baseline seizure burden - post-PB seizure burden)/baseline seizure burden)]. The color gradient (blue or red) applied to the background represents the data distribution in the positive (blue) or negative (red) direction of KCC2 expression after ligation/treatment. The dotted gray lines within each scatter plot represent the mean values for percent seizure suppression in each group (x axis). A&C. The ligated control group at both ages of P7 and P10 showed the post-ischemic degradation of KCC2 expression evident by predominant distribution of dots on the red gradient. B&D. The post-ischemic KCC2 expression of the treated group was rescued at both ages of P7 and P10, as seen in the positive-shift of the dots towards the blue gradient (red arrows). The correlation between the percent KCC2 expression and percent seizure suppression (black arrows) was significant at P10 (Spearman’s test; P=0.04) but not at P7. When P7 data were binned by baseline seizure burden (<1200sec vs. ≥1200sec), the correlation became significant for the pups that seized <1200sec (p=0.02; data not shown).