冷却对大鼠难治性癫癎模型海马神经细胞存活率的影响

Effect of Cooling on Survival Rate of Hippocampal Neurons from Refractory Epilepsy Models

目的：探讨不同冷却温度和冷却时间对大鼠难治性癫癎模型海马神经细胞存活率的影响。方法：①6μmol.L-1荷包牡丹碱（Bic）＋4-氨基吡啶（4-AP）联合刺激培养的大鼠海马组织切片（OHS）诱发癎样放电,制作难治性癫癎OHS模型;②在36℃和25℃培养1~24 h;③在36℃、30℃、25℃、20℃和15℃培养6 h;④分别测定OHS神经细胞的碘化丙啶（PI）摄取量。结果：Bic＋4-AP联合刺激3 h后,摄取PI的神经细胞首先出现在海马的CA2区。随着刺激时间的延长,摄取PI神经细胞的分布范围扩散到其他区域,以CA3区最为明显。25℃冷却处理可以显著抑制神经细胞的PI摄取,然而,20℃、15℃深低温冷却处理可进使神经细胞的PI摄取。结论：①培养的大鼠海马组织CA2区的神经细胞对Bic＋4-AP联合刺激最敏感,神经细胞的损伤与兴奋性刺激时间和海马的区域有关;②冷却处理神经细胞的保护作用与冷却的温度和时间有关。

Aim： To investigate the effect of different cooling-temperature and cooling-duration on survival rate of hippocampal neurons from the refractory epilepsy models.Methods： A final concentration of 6 μmol.L-1 of bicuculline（Bic） and 4-aminopyridine（4-AP） was applied to culturing organotypic hippocampal slice（OHS） to develop the refractory epilepsy model.The viability marker propidium iodide（PI） was used to monitor the neuronal cell death in the culture on the condition of 36℃ and 25℃ for 24 h,and 36℃,30℃,25℃,20℃ and 15℃ for 6 h.The digital images were analyzed by using a densitometry analysis program.Results： After stimulating by Bic combined with 4-AP for 3 h on rat OHS cultures,the PI uptake appeared in the CA2 sector at the first time.With the time of stimulation increasing,the distribution of damaged neuronal cells was extended to large area,predominantly distributed in the CA3 sector.The damaged neuronal cells’PI uptake were significantly inhibited by cooling treatment at 25℃ for 3 h to 12 h,however,the PI uptake could be promoted by the deep hypothermic cooling treatment at 20℃ and 15℃.Conclusion： ① The neuronal cellsof CA2 sector in hippocampal silce cultures were the most sensitive to BiC combined with 4-AP stimulation.The effect of Bic combined with 4-AP on neuronal cell damage was time and region dependent.② The protective effect of cooling on neuronal cells was temperature and duration dependent.