Dose-dependent reversal of KCC2 hypofunction and phenobarbital-resistant neonatal seizures by ANA12

B. M. Carter,B. J. Sullivan,J. R. Landers,S. D. Kadam

doi:10.1038/s41598-018-30486-7

Abstract

Neonatal seizures have an incidence of 3.5 per 1000 newborns; while hypoxic-ischemic encephalopathy (HIE) accounts for 50–60% of cases, half are resistant to 1st-line anti-seizure drugs such as phenobarbital (PB). Tyrosine receptor kinase B (TrkB) activation following ischemic injury is known to increase neuronal excitability by downregulation of K-Cl co-transporter 2 (KCC2); a neuronal chloride (Cl−) co-transporter. In this study, three graded doses of ANA12, a small-molecule selective TrkB antagonist, were tested in CD1 mice at P7 and P10 following induction of neonatal ischemia by a unilateral carotid ligation. The PB loading dose remained the same in all treatment groups at both ages. Evaluation criteria for the anti-seizure efficacy of ANA12 were: (1) quantitative electroencephalographic (EEG) seizure burden and power, (2) rescue of post-ischemic KCC2 and pKCC2-S940 downregulation and (3) reversal of TrkB pathway activation following ischemia. ANA12 significantly rescued PB resistant seizures in a dose-dependent manner at P7 and improved PB efficacy at P10. Additionally, female pups responded better to lower doses of ANA12 compared to males. ANA12 significantly reversed post-ischemic KCC2 downregulation and TrkB pathway activation at P7 when PB alone was inefficacious. Rescuing KCC2 hypofunction may be critical for preventing emergence of refractory seizures.

Highlights

Phenobarbital (PB), a barbiturate that prolongs the opening of GABA receptors, remains the most common first-line anti-seizure drug (ASD)
This study investigated the efficacy of graded doses of ANA12 (Table 1) in modulating the Tyrosine receptor kinase B (TrkB) pathway and associated K-Cl co-transporter 2 (KCC2) hypofunction, in a model of neonatal ischemic seizures
Rescue of PB-resistance was associated with a dose-dependent rescue of KCC2 downregulation; 3

Summary

Introduction

Phenobarbital (PB), a barbiturate that prolongs the opening of GABA receptors, remains the most common first-line anti-seizure drug (ASD). Ischemic neonatal seizures are often refractory to PB and unresponsive to adjunct ASDs1. Potassium-chloride cotransporter 2 (KCC2) is the primary neuronal Cl− extruder and maintains the [Cl−]i gradient that allows strong, hyperpolarizing GABAergic inhibition[14]. Acute KCC2 downregulation is one of the hallmarks of excitotoxic neuronal injuries like neonatal ischemic insults and may underlie the emergence of PB refractoriness[25,26]. The loss of vGLUT reduces glutamatergic neurotransmission, causing severe deficits to cognition, and increases in seizure susceptibility[33,34]. PB alone of vesicular GABA transporter (vGAT) following unilateral ischemia in Sprague-Dawley rats[35]. To investigate vGLUT, and contrast it with vGAT, both were quantified in this neonatal ischemic seizure model

Methods

Results

Conclusion