Toki-Shakuyaku-San intervenes in apoptosis induced by lowering of potassium chloride concentrations in cultured cerebellar granule cells

Cerebellar granular cells harvested from 8-day-old rats were cultured in a basic medium of Eagle's salt (BME) containing 10% serum and 25 mM KCl. At 7 days in vitro (DIV), the culture medium was switched to serum-free BME containing 5 mM KCL Fluorescein diacetate/propidium iodide staining was used to examine the neuronal survival. Phase-contrast microscopy and DNA fragmentation with agarose gel were used for examination of apoptosis. A progressive neuronal death with nuclear condensation, extensive damage of the neuritis network, and DNA fragmentation were observed following a switch to 5 mM KCl BME. The neuronal survival was 64%, 48% and 30% at 24, 48 and 72 h following a switch to 5 mM KCl BME respectively. An application of 0.05 mg/mL of Toki-Shakuyaku-San (TJ-23, a recipe of Japanese traditional medicine: Kampo medicine) to 5 mM KCl BME at 7 DIV intervened in neuronal death. The neuronal survival was 74% at 24 (p < 0.05), 72% at 48 h (p < 0.001) and 67% at 72 h (p < 0.001) following a switch to 5 mM KCl BME. An application of T J-23 also intervened in nuclear condensation, DNA fragmentation, and damage of the neuritis network caused by a switch to 5 mM KCl BME. This Study provides information that switching to 5 mM KCl BME from 25 mM KCl BME at 7 DIV causes apoptosis in the cultured cerebellar granule cells. Moreover, TJ- 23 intervenes in apoptosis induced by a switch to 5 mM KCl BME in cultured cerebellar granule cells.