Fig. 7.

H₂O₂-mediated activation of SPAK–NKCC1 cascade-mediated Na⁺, K⁺ (Rb⁺) influx and cell death in cultured neurons. a Experiment protocol. b Representative fluorescent images of primary cortical neuron cultures. Cells were stained with Calcein-AM and propidium iodide (PI) at 24 h post-exposure either to control (CTL), 20 µM H₂O₂, 20 µM H₂O₂ + 100 nM Ebselen (H + E), 20 µM H₂O₂ + 1 μM ZT-1a (H + Z), or 20 µM H₂O₂ + 10 μM BMT (H + B). Arrowheads: live neurons were stained with Calcein-AM (green); arrows: dead neurons were stained with PI (Red). c Neuronal survival rates. Values are calculated as the number of live cells divided by the total number of live plus dead cells, multiplied by 100. Data are expressed as mean ± SD (n = 4), **p < 0.01, ***p < 0.001. d Representative intracellular Na⁺ [Na⁺]_i concentration using Na⁺-sensitive dye SBFI/AM at 24 h post-exposure of CTL and H₂O₂ insult ± drug treatment conditions. Values are mean ± SD (n = 4–5), **p < 0.01, ***p < 0.001. e and f Total Rb⁺ (K⁺) influx and NKCC1-mediated Rb⁺ (K⁺) influx. Neuron cells were exposed to each condition for 24 h. Rb⁺ influx into cells under isotonic (310 mOsm, pH 7.4) solutions were assayed for 10 min and identified using ICR 8000. Values are mean ± SD (n = 4), *p < 0.05, **p < 0.01. g Representative immunoblots of primary cortical neuron cultures under conditions as in b. Cell lysates were subjected to SDS-PAGE and incubated with appropriate antibody. h Summary. Values are expressed as mean ± SD (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. i Representative immunoblots of primary cortical neuron cultures under conditions as in b. j Summary. Data are mean ± SD (n = 4), *p < 0.05, **p < 0.01. One-way ANOVA