Abstracts

Rationale: Aquaporin-4 (AQP4) is a transmembrane protein that regulates cellular water transport and potentially ion homeostasis in the brain. The functions of AQP4 make it a likely candidate to regulate seizure onset and development. Previous studies have demonstrated prolonged seizure duration in AQP4-/- mice but the relevance of this finding to epileptogenesis has been unclear. Therefore, we aimed to examine the role of AQP4 in epileptogenesis using the intrahippocampal kainic acid model of epilepsy. Methods: Kainic acid (100 nl, 20 mM) or saline (100 nl, 0.9%) was injected into the dorsal hippocampus of AQP4+/+ and AQP4-/- adult male mice to induce status epilepticus (SE). These mice were then monitored with continuous (24 hrs/day for up to 14 days) digital video and electroencephalography (EEG) recordings for the development of spontaneous seizures. Immunohistochemistry was performed at 1, 4, 7, 14 and 30 days after the initial SE to determine changes in hippocampal AQP4, GFAP, NG2 and glutamine synthase (GS) immunoreactivity. Results: In this model, spontaneous seizures developed between post-SE day 3 and 5; however, AQP4-/- mice exhibited seizures more frequently than AQP4+/+ mice (post-SE day 3 seizure frequencies for AQP4-/-, 5.8 +/- 1.4 and AQP4+/+, 1.8 +/- 2.2) in the early epileptogenic period. These consisted of spontaneous focal seizures (grades 1-3 in the Racine classification). Saline-injected controls never demonstrated spontaneous seizure activity. Based upon densitometric analyses, AQP4 immunoreactivity was significantly downregulated on post-SE day 1. This initial downregulation was followed by a slow return to near-normal levels over the next 30 days. In the AQP4+/+ mice, downregulation of GFAP occurred at post-SE day 1 and slowly returned toward baseline over the next 7 days. In AQP4-/- mice, there was no early downregulation of GFAP; however, transient upregulation of GFAP by post-SE day 7 and 14 was observed. NG2 immunoreactivity was significantly downregulated in both AQP4+/+ and AQP4-/- mice after SE and throughout the study period (to day 30). No significant change from baseline in GS immunoreactivity was observed in either AQP4+/+ or AQP4-/- mice. Conclusions: During the early epileptogenic period, AQP4-/- mice had more frequent seizures than AQP4+/+ mice. In addition, coordinate regulation of AQP4 and other glial antigens following SE was observed. Significant downregulation of AQP4 following SE suggests that the initial SE insult may lead to the development of a transient “functional knockout” of AQP4 and a dysregulation of water homeostasis. In addition, marked downregulation of NG2 may suggest alterations in “NG2 glia” during epileptogenesis. These alterations in important glial functional molecules may contribute to increased excitability during the early epileptogenic phase. Further study of these and related glial molecules may lead to novel therapeutic targets with fewer potential side effects.