Abstracts

RATIONALE: Numerous [italic]in vivo[/italic] and [italic]in vitro[/italic] studies have demonstrated the neuroprotective properties of topiramate (TPM). An increase of intracellular calcium has been implicated in the process of cell death. Electrophysiological experiments have demonstrated that TPM affects the function of several ion channels including kainate receptors and voltage - gated calcium channels. Therefore, we hypothesized that the neuroprotective effect of TPM may be related to an attenuation of the rise of intracellular calcium concentration ([Ca²⁺][sub]i[/sub]) after excitatory amino acid application.
METHODS: Populations of cells (mixed neuronal and glial) from cortical and hippocampal regions were isolated from E18 rat pups. Cells were cultured at a density of 30k/well in 96-well plates and maintained for 8 and 9 days before the day of experiments. Ca²⁺ imaging experiments were performed using a fluorescence-imaging plate reader (FLIPR) assay kit and protocol. Cells were pre-incubated for 10 minutes with TPM (1 - 100 [mu]M) before the addition of glutamate (1 [ndash] 100 [mu]M), AMPA (3 - 300 [mu]M), kainate (10 - 1000 [mu]M), NMDA (3 - 300 [mu]M), or KCl (10 and 50 mM).
RESULTS: TPM failed to reduce the increases of [Ca²⁺][sub]i[/sub] evoked by glutamate, AMPA, and NMDA. However, TPM did attenuate kainate and KCl induced increases of [Ca²⁺][sub]i[/sub]. Increasing the concentration of KCl and kainate decreased this effect of TPM. For example, in cortical cells maintained 8 days in culture, 100 [mu]M TPM reduced peak increases of [Ca²⁺][sub]i[/sub] evoked by 10 and 50 mM KCl to 68 [plusminus] 4 and 85 [plusminus] 6 % of control, respectively. Also, TPM effects on increases of [Ca²⁺][sub]i[/sub] differed slightly between cortical and hippocampal cultures and 8 and 9 day old cultures. For example, in hippocampal cells maintained 8 days in culture, 100 [mu]M TPM reduced peak increases of [Ca²⁺][sub]i[/sub] evoked by 30, 100, 300, and 1000 [mu]M kainate to 41 [plusminus] 8.7, 65.9 [plusminus] 16, 80 [plusminus] 4, and 69 [plusminus] 14 % of control, respectively, while in cortical cells maintained 8 days in culture, 100 [mu]M TPM reduced peak increases of [Ca²⁺][sub]i[/sub] evoked by kainate to 47.7 [plusminus] 7.8, 89.3 [plusminus] 6.4, 89.2 [plusminus] 4.3, and 96 [plusminus] 4.3 % of control, respectively.
CONCLUSIONS: These results suggest that TPM may reduce excitability and excitotoxicity by attenuating increases of [Ca²⁺][sub]i[/sub]. The precise mechanism through which TPM exerts these effects has not been established; however, it is hypothesized that the demonstrated inhibitory activity of TPM at non-NMDA receptors and voltage - gated calcium channels contributes to its efficacy in this study.
[Supported by: Johnson & Johnson Pharmaceutical Research and Development, LLC.]; (Disclosure: Salary - Johnson & Johnson Pharmaceutical Research and Development, LLC, Grant - Johnson & Johnson Pharmaceutical Research and Development, LLC)