BK_Ca channel is a molecular target of vitamin C to protect against ischemic brain stroke

Figures & data

Figure 1. Effect of long-term, low-dose vitamin C on MCAO induced infarct size and neurological deficit score. (a) Representative images of TTC stained brain slices obtained at 24 hr post-MCAO from MCAO rats pretreated with vitamin C at a dose of 0, 0.5, 5, 20 and 50 mg.kg⁻¹.day⁻¹ for 3 weeks. (b,c) Bars showing the relative infract size (b) or neurological deficit score (c) of MCAO rats pretreated with vitamin C at a dose of 0, 0.5, 5, 20 and 50 mg.kg⁻¹.day⁻¹ for 3 weeks. *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 2. Application of vitamin C twice a day does not have more beneficial effect on ischemic stroke than that applied once a day. (a) Representative images of brain slices obtained at 24 hr post-MCAO from MCAO rats pretreated with vehicle as well as pretreated with vitamin C at a dose of 5 mg.kg⁻¹ once a day or 2.5 mg.kg⁻¹ twice a day for 3 weeks. (b,c) Bars showing the relative infract size (b) and neurological deficit score (c) of MCAO rats pretreated with vehicle and vitamin C at a dose of 5 mg.kg⁻¹ once a day or 2.5 mg.kg⁻¹ twice a day (2*2.5 mg.kg⁻¹.day⁻¹) for 3 weeks. *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 3. Effect of long-term and low dose pretreatment with vitamin C on the concentration of vitamin C in the serum and brain. Concentration of vitamin C in the serum (a) or right and left brain (b) for the rats treated with vehicle or treated with vitamin C at a dose of 5 mg.kg⁻¹ once a day or 2.5 mg.kg⁻¹ twice a day for three weeks. The concentration of vitamin C was measured 24 hr after the last administration of vitamin C. **p < 0.01, ***p < 0.005. (c–e) Concentration of vitamin C in the serum (c) and contralateral side (d) or ipsilateral side (e) of the brain for sham-operated, vehicle pretreated MCAO rats and vitamin C pretreated MCAO rats. Vitamin C was intraperitoneally administrated at a dose of 5 mg.kg⁻¹ once a day or 2.5 mg.kg⁻¹ twice a day for three weeks prior to MCAO. The concentration of vitamin C was measured 48 hr after the last administration of vitamin C.

Figure 4. A single high dose vitamin C has no effect on MCAO. (a) Representative images of brain slices obtained at 24 hr post-MCAO from rats treated with vehicle or treated with a single high dose vitamin C (500 mg.kg⁻¹) 24 hr before MCAO. (b,c) Bar graphs showing the relative infract size (b) and neurological deficit score (c) for a single high dose vitamin C-treated (500 mg.kg⁻¹) vs vehicle-treated MCAO rats. (d,e) Vitamin C concentration in the serum (d) or brain tissue (e) of rats after 24 hr treament with vehicle or a single high dose vitamin C (500 mg.kg⁻¹).

Figure 5. Vitamin C activates BK_Ca channels at the whole-cell level. (a–c) Representative whole-cell BK_Ca channel currents before (upper panel) and after 10 min (lower panel) application of 0 (a), 200 (b) and 500 (c) μM vitamin C in the bath solution at Ca²⁺ concentration of 1 μM. The currents were evoked by step depolarization from a hold potential of -80 mV to test potentials between −80 and +60 mV for 200 ms in 20 mV increments. (d) Ratio values (I/Io) of the whole-cell BK_Ca channel current at + 60 mV before (Io) and after 10 min (I) application of different concentration of vitamin C in the bath solution. **p < 0.01.

Figure 6. Vitamin C activates BK_Ca channels at the single-channel level. (a,b) Representative single channel current traces of the BK_Ca channels in the inside-out configuration at membrane potentials ranging from −80 mV to +60 mV with 20 mV increment before (a) and after application of 200 μM vitamin C (b) in the bath solution. Arrows indicate the level that all the channels are in their closed state. (c) The relationships between the single channel open probability (Po) of the BK_Ca channels and the membrane potentials in the absence and presence of 200 μM vitamin C are fitted with the Boltzmann equation (see text for detail). (d) Statistical data on Po of the channel at a voltage of +20 mV in the absence and presence of 50, 200, 500 and 1000 μM vitamin C. ***p < 0.001.

Figure 7. BK_Ca channel blocker Penitrem A (PA) suppresses the effect of vitamin C on MCAO rats. (a) Representative images of brain slices for vehicle-treated MCAO rats and vitamin C pretreated (5 mg.kg⁻¹.day⁻¹ for three weeks prior to MCAO) MCAO rats at 24 hr post-MCAO in the absence and presence of PA (0.4 and 4 μg.kg⁻¹). (b,c) Bar graphs showing relative infarct size (b) and neurological deficit score (c) in vehicle-treated and vitamin C pretreated MCAO rats at 24 hr post-MCAO in the absence and presence of PA.

Figure 8. BK_Ca channel blocker paxiiline (Pax) suppresses the effect of vitamin C on MCAO rats. (a) Representative images of brain slices for vehicle-treated MCAO rats and vitamin C pretreated (5 mg.kg⁻¹.day⁻¹ for three weeks prior to MCAO) MCAO rats at 24 hr post-MCAO in the absence and presence of Pax (5 μg.kg⁻¹). (b,c) Bar graphs showing relative infarct size (b) and neurological deficit score (c) in vehicle-treated and vitamin C pretreated MCAO rats at 24 hr post-MCAO in the absence and presence of Pax.