Abstracts

Rationale: Status epilepticus (SE) is life-threatening neurological emergency characterized by a prolonged seizure, and thereafter becomes resistant to benzodiazepine treatment. The loss of inhibitory activity of GABAergic receptors has recently spotlighted as a pathophysiological mechanism of drug refractory SE. Persisting seizure activity induces the attenuation of GABAergic inhibition by internalization of GABAA receptors, resulting in the change of intracellular chloride concentration ([Cl-]i). The activations of Na–K–Cl cotransporter 1 (NKCC1) and K–Cl cotransporter 2 (KCC2) regulate [Cl-]i to maintain the balance of neuronal Cl- homeostasis; however, it remains unclear whether prolonged SE causes the expressional changes of cotransporters in the hippocampal neurons. Thus, we investigated the dynamic alterations of NKCC1 and KCC2 in the experimental models of prolonged SE. Methods: We used in vitro primary neuronal culture in Mg2+-free media and in vivo adult male mice pilocarpine model. Temporal alteration of cotransporters during prolonged SE was investigated by western blotting and immunostaining for total and phosphorylated NKCC1 and KCC2. Results: The activation of KCC2 was significantly decreased as SE proceeds, whereas no significant change in NKCC1 activation was detected in Mg2+-free-induced SE model of cultured hippocampal neurons. Moreover, the expression of membrane KCC2 was clearly reduced, while cytoplasmic KCC2 was gradually increased as SE proceeds, suggesting that the internalization of KCC2 might be induced by prolonged SE. In contrast, immunoreactivities of membrane NKCC1 was enhanced over time after SE. Similar to the expressional changes of cotransporters during prolonged SE in vitro, the levels of KCC2 were reduced until 4 hour after SE, while NKCC1 levels were upregulated at the same time period in the hippocampus treated by pilocarpine in vivo. Although no significant changes in the phosphorylation of NKCC1 was found, the levels of phospho-KCC2 was significantly decreased until 2 hours after SE, and then its levels was significantly increased during SE compared to its control levels. Conclusions: Our observation showed that persisting seizure activity triggered the dynamic alterations of NKCC1 and KCC2, such as the change of cellular distribution and activation in the hippocampal neurons. These findings suggest that differential alteration of NKCC1 and KCC2 might be involved in the change of GABAergic inhibition as SE continues, and the control in alteration of NKCC2 and KCC2 could be a potential therapeutic target for benzodiazepine-refractory SE. Funding: This research was supported by grants of the National Research Foundation of Korea (NRF-2015R1D1A1A01059901) funded by the Korea Government.