Dardalas Ioannis, Kosmidis Efstratios K, Lagoudaki Roza, Kimiskidis Vasilios K, Samaras Theodoros, Moysiadis Theodoros, Kouvelas Dimitrios, Pourzitaki Chryssa
Laboratory of Clinical Pharmacology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
Laboratory of Physiology, Department of Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
Biomedicines. 2025 Jul 30;13(8):1857. doi: 10.3390/biomedicines13081857.
Epilepsy is a brain condition that affects millions of people worldwide. Although there are many antiepileptic drugs with different mechanisms of action, many patients still fail to control their agonizing symptoms, a situation that highlights the need for more strategies to address this issue. In this in vitro study, we elucidated and characterized the alterations in intracellular Ca levels in cell cultures where diazepam and repetitive transcranial magnetic stimulation were implemented, alone or in combination. Using the differentiated human-derived neuroblastoma cell line SH-SY5Y, we measured the alterations in intracellular Ca levels under the impact of either low-frequency repetitive transcranial magnetic stimulation (1 Hz), diazepam (14 μM), or their combination. We used the Ca-sensitive fluorescent indicator Fluo-4 acetoxymethyl ester for calcium imaging, while neuronal excitation was achieved with 50 mM KCl. The highest median fluorescence intensity increase (%ΔF/F = 24.80) was observed in control cell cultures, followed by rTMS cultures (%ΔF/F = 16.96) and diazepam cultures (%ΔF/F = 11.46). The lowest median fluorescence intensity value (%ΔF/F =-0.44) was observed when diazepam was used concomitantly with repetitive transcranial magnetic stimulation. Post hoc analysis assessed pairwise differences, showing statistically significant differentiation between the control group and all other groups. Additionally, statistically significant results were observed between repetitive transcranial magnetic stimulation or diazepam and their combination, but not between them. The combination of diazepam and repetitive transcranial magnetic stimulation resulted in the most significant reduction in intracellular Ca levels, as indicated by the lowest fluorescence values compared with the control group. Individually, each treatment produced a notable but less pronounced effect. We conclude that both diazepam and low-frequency repetitive transcranial magnetic stimulation can control epileptiform activity in vitro, while their combination is the most effective treatment.
癫痫是一种影响全球数百万人的脑部疾病。尽管有许多作用机制不同的抗癫痫药物，但许多患者仍无法控制其令人痛苦的症状，这种情况凸显了需要更多策略来解决这一问题。在这项体外研究中，我们阐明并表征了单独或联合使用地西泮和重复经颅磁刺激的细胞培养物中细胞内钙水平的变化。使用分化的人源神经母细胞瘤细胞系SH-SY5Y，我们测量了在低频重复经颅磁刺激（1Hz）、地西泮（14μM）或它们的组合影响下细胞内钙水平的变化。我们使用钙敏感荧光指示剂Fluo-4乙酰氧基甲酯进行钙成像，同时用50mM氯化钾实现神经元兴奋。在对照细胞培养物中观察到最高的中位荧光强度增加（%ΔF/F = 24.80），其次是重复经颅磁刺激培养物（%ΔF/F = 16.96）和地西泮培养物（%ΔF/F = 11.46）。当地西泮与重复经颅磁刺激同时使用时，观察到最低的中位荧光强度值（%ΔF/F = -0.44）。事后分析评估了两两之间的差异，显示对照组与所有其他组之间存在统计学上的显著差异。此外，在重复经颅磁刺激或地西泮及其组合之间观察到统计学上的显著结果，但它们之间没有差异。与对照组相比，地西泮和重复经颅磁刺激的组合导致细胞内钙水平的最显著降低，荧光值最低表明了这一点。单独来看，每种治疗都产生了显著但不太明显的效果。我们得出结论，地西泮和低频重复经颅磁刺激都可以在体外控制癫痫样活动，而它们的组合是最有效的治疗方法。
