Fig 1.
GM1 (50 mg/kg) treatment improves neurological scores and reduces infarct volume at 72 hours after MCAO.
GM1 was administrated soon after reperfusion and then once daily for 2 days at three doses (25, 50, and 100 mg/kg) by intraperitoneal injection. MCAO rats were injected with the same volume of saline as a control. (A) Experimental design and animal treatment. (B) Neurological scores for the modified Garcia test in sham, MCAO+saline and GM1 (25, 50, 100 mg/kg) treatment groups. n = 10–12 rats per group. (C) Representative images of TTC stained sections and infarct volume in sham, MCAO+saline and GM1 treatment groups. n = 6 per group. *p < 0.05 vs sham; #p < 0.05 vs MCAO+saline.
Fig 2.
Immunofluorescence staining for LC3-II and Beclin-1 expression in sham, MCAO+saline and GM1 (50 mg/kg) treatment groups 72 hours following MCAO injury.
(A) Representative images of LC3-II (red) and Beclin-1 (green) staining in the perihematomal area. Bar = 50μm. (B) Schematic diagram showing examples of the areas (black squares) that were selected for counting of LC3-II and Beclin-1 positive cells in the perihematomal region. (C) Quantification of LC3-II positive cells in the perihematomal region (10 fields/brain). (D) Quantification of Beclin-1 positive cells in the perihematomal region (10 fields/brain). The data show that GM1 treatment significantly reduced the number of LC3-II and Beclin-1 positive cells after MCAO insult. n = 5 per group. *p < 0.05 vs sham; #p < 0.05 vs MCAO.
Fig 3.
GM1 inhibits neuronal autophagic activity following MCAO injury.
(A) Representative Western blotting images of LC3-I, LC3-II, P62, and Beclin-1 in the ipsilateral hemisphere at 72 hours after MCAO injury. β-Actin is shown as a loading control. (B) Quantification of the LC3-II/LC3-I ratio in the ipsilateral hemisphere at 72 hours after MCAO injury. (C) Quantification of P62 in the ipsilateral hemisphere at 72 hours after MCAO injury. (D) Quantification of Beclin-1 in the ipsilateral hemisphere at 72 hours after MCAO injury. n = 6 per group. *p < 0.05 vs sham; #p < 0.05 vs MCAO+saline.
Fig 4.
Enhanced autophagy induced by Tat–Beclin-1 abolishes the neuroprotective effects of GM1.
(A) Representative images of TTC stained sections and quantification of infarct size in sham, MCAO+saline, MCAO+Tat–Beclin-1, MCAO+GM1, and MCAO+Tat–Beclin-1+GM1 groups. n = 6 per group. (B) Neurological scores for the modified Garcia test. n = 6–12 per group. (C) Representative Western blotting images of LC3-I, LC3-II, P62, Beclin-1 and β-Actin in MCAO+saline, MCAO+GM1, MCAO+Tat–Beclin-1, and MCAO+Tat–Beclin-1+GM1 groups. n = 6 per group. (D) Quantification of LC3-II/LC3-I in the ipsilateral hemisphere. (E) Quantification of P62 in the ipsilateral hemisphere. (F) Quantification of Beclin-1 in the ipsilateral hemisphere.*p < 0.05 vs sham; #p < 0.05 vs MCAO+saline; @p < 0.05 vs MCAO+Tat–Beclin-1; & p < 0.05 vs MCAO+GM1.