Backgrounds
There are no reports referring to the cellular functional evaluation of the cortical neurones protected with both edaravone and hypothermia against hypoxic insults (HI). In this study, we compared the neuroprotective effects of the combined administration of edaravone and hypothermia with each individual administration on rat primary cultured cortical neurones against prolonged HI.

Materials and Methods
Neurones prepared from E17 Wistar rats were used after 10-11 days in culture. Shortly before 24h HI, 50µM edaravone was administrated to the neurones. During HI, neurones were exposed to mild hypothermia (32°C). As a cellular function, the degrees of glutamate (Glu) -induced intracellular calcium ([Ca2+]i) elevation were measured using fluorescence measurement systems after HI.
To evaluate the effects of Glu on primary cultured neurones, the mean maximum change in fluorescence intensity (Fmax) upon addition of Glu was measured and normalized to the baseline fluorescence obtained before Glu application (F0). We measured the mean value of Fmax/F0 (height) calculated from the integration of [Ca2+]i response curves of the surviving neurones. A height ratio (HR) was calculated as follows: the height of the [Ca2+]i response in an experimental culture divided by the height of the [Ca2+]i response in the control culture (normoxia, normothermia, no edaravone).
Data are expressed as the mean ± SEM. The differences between the means were assessed using analyses of variance followed by post-hoc Tukey-Kramer honestly significant difference tests. P-values < 0.05 were considered significant.

Results
HR of hypoxic neurones with hypothermia was significantly higher than those with no neuroprotectants (0.95 ± 0.18 vs 0.41 ± 0.06, P = 0.01), while those with edaravone alone was not so significantly different (0.66 ± 0.13, P = 0.47). HRs of these two individual administrations did not differ significantly from control (P = 1.00; 0.19) or combined administration of edaravone and hypothermia (0.88 ± 0.05, P = 0.99; 0.62). The degree of variability of HR was smaller in combined administration than each individual administration.

Conclusions
Mild hypothermia by itself preserved the response of hypoxic neurones to Glu. Even if edaravone was also co-administrated, there was no significant change.

04 / The neuroprotective effects of individual and combined administration of edaravone and hypothermia against hypoxic insults