Detected in 1 to 3.5 per 1000 newborns, neonatal convulsions refer to the seizures occurring within the first 28 days of life. Neonatal seizures, if poorly managed, can result in severe neurodevelopmental outcomes that threaten cognition, motor function, and even life. The associated pathologies include, but are not limited to hypoxic-ischemic encephalopathy (HIE), stroke, intracranial hemorrhage, brain malformation, infarction, prenatal and neonatal infections. However, HIE has been the most prevalent cause of neonatal seizures, and the HIE-associated seizures pose a great challenge to its current therapy because those have higher propensity of showing a stubborn refractoriness to conventional antiepileptic drugs (AEDs) provided as a first-line therapy in clinics.
The refractoriness in neonatal seizures can be mainly attributed to a neuronal chloride gradient that does not generate hyperpolarization as much as the one established in the adult nervous system. When the ion channels located at neuronal membrane open, the net ion influx or efflux is determined by both electrical and chemical gradient: an electrical gradient of a certain threshold voltage that a neuron wants to maintain and a chemical gradient that is determined by the net concentration of ions at extracellular and intracellular environment. The mature nervous system maintains a relatively low intracellular chloride concentration such that an opening of chloride channels results in an influx of negatively charged chloride ions which ultimately renders a post-synaptic inhibition in central nervous system (CNS). In contrast, the immature nervous system has a relatively higher intracellular chloride concentration that results in less hyperpolarization or even depolarization in some cases. Hence, the HIE-associated neonatal seizures are not efficaciously modulated by conventional anti-convulsants, phenobarbital and phenytoin, that target GABAA receptors to open the chloride channels to induce neuronal hyperpolarization and halting seizures eventually. The difference in the chloride gradient also contributes to the neonatal hyperexcitability that leads to a higher seizure susceptibility observed in seizing neonates, especially in the first 2 days in the neonatal period.
The depolarizing chloride gradient has been shown to play a critical role in neurodevelopment such as neuronal migration, proliferation, and maturation. The critical switch of neuronal chloride gradient from depolarizing to hyperpolarizing occurs within neonatal period, and cation chloride co-transporters (CCCs) are one of the pivotal players that drive the neuronal chloride gradient toward its adult level. In early developmental stage, NKCC1 pumps in chloride ions into a neuron to build up a high intracellular chloride concentration which results in a depolarizing gradient necessary for the associated neurodevelopment. In the later development, KCC2 pumps out chloride ions to lower the intracellular chloride concentration which renders a hyperpolarizing gradient when the chloride channels are opened. KCC2 expression is neuron-specific15 whereas NKCC1 expression is relatively ubiquitous, therefore the ratio of these two CCCs are critical for the efficacy of anti-convulsants that depend on chloride ion influx. The well-studied developmental profile of CCCs in human and rodents suggests: 1) the high expression level of NKCC1 during early development decreases throughout neonatal period, and stabilizes at the lowest level after neonatal period, 2) the lower expression of KCC2 during early development gradually increases throughout neonatal period, and stabilizes at the highest level by the end of adolescent stage. Thus, in seizing adults with fully matured KCC2 expression in mature CNS, the delivery of conventional GABAA-modulating AEDs drives an ideal hyperpolarization driven by the chloride influx upon channel opening. However, in seizing neonates with lower KCC2 expression in a developing CNS, refractoriness to traditional AEDs is often observed. Importantly, KCC has a caudal-rostral expression pattern that the establishment of hyperpolarizing chloride gradient starts at the spinal cord, and reaches the brain at last. This relates to a neonate-specific phenomenon of electroclinical dissociation where a neonate undergoes electrographic seizures without behavioral manifestation.
Designing and investigating an efficient therapy for treating refractory neonatal seizures is challenging because there are intrinsic difficulties in dissociating the harmful effects of hypoxic-ischemia and seizures. Many animal models have been proposed to examine the refractory neonatal seizures that mimic HIE condition such as in vitro chemo-convulsive, in vivo hypoxic, and ischemic model. Ischemic models, with the highest clinical relevance among many HIE models, have provided a crucial insight on the role of CCCs in designing an optimal pharmacotherapy for refractory seizures in neonates. Recent animal studies have focused on targeting NKCC1 using Bumetanide, a potent NKCC1 blocker, to control refractory seizures. Under ischemia, an acute upregulation of NKCC1 expression occurs such that an increased chloride influx causes hyperexcitability and more refractoriness to conventional AEDs. Prevention of chloride influx by blocking NKCC1 with Bumetanide may establish a neuronal environment that enables GABAA modulating AEDs to act efficaciously upon an ischemic insult. However, more studies are needed to ensure: 1) the safety of Bumetanide in neonates as an adjunct therapy, and 2) the true additional efficacy of Bumetanide on refractory seizures. Indeed, chronic delivery of bumetanide may induce an overwhelming diuresis in neonates with HIE suffering other pathophysiological complications such as energy failure and altered homeostasis. Accordingly, new methods for treating neonatal seizures are needed.