Figure 9.

Inhibition of AMPK significantly attenuates NGF-induced autophagic activities, myelin clearance and neural regeneration. (A) The ratios of p-AMPK/AMPK, p-p70s6k/p70s6k and p-mTOR/mTOR were evaluated by western blotting in PNI, NGF and NGF+Cpd C rat sciatic nerve tissue lysates at 5 days post-injury. (B) Autophagy related proteins expression (including ATG-7, ATG-5, Beclin-1 P62 and LC3) were detected through western blotting. (C) Representative immunoblots for MBP and MPZ in each group of rats. Quantitative data and statistical analysis of western blotting results in these three groups were showed in table 3. Data are presented as the mean ± SEM; n = 3 or 4 independent experiments. (D, E) Co-immunofluorescence images and quantification showing MPZ (green) and GFAP (red) in injured sciatic nerve at day 5. Nuclei are blue (DAPI). Original scale bar = 100 µm and close-up scale bar = 25 µm. Data are presented as the mean ± SEM; n = 3 rats per group. MPZ F(2, 6) = 18.89, ^**P_{PNI vs NGF} = 0.0053, ^*P_{NGF vs NGF+Cpd C} = 0.015. (F) TEM images and double staining for MBP (red)/NF-200 (green) of sections from the injured sciatic nerve in each rat group at 14 days post-injury. Nuclei are blue (DAPI). (G-J) Analysis of G-ratio, myelin thickness distribution, and NF-200- and MBP- positive staining in each group. Data are presented as the mean ± SEM; n = 3 rats per group. G-ratio F_{(2, 6)} = 8.64, ^*P_{PNI vs NGF} = 0.043, ^*P_{NGF vs NGF+Cpd C} = 0.045; NF-200 F(2, 6) = 11.89, ^**P_{PNI vs NGF} = 0.0078, ^*P_{NGF vs NGF+Cpd C} = 0.034; MBP F(2, 6) = 10.08, ^*P_{PNI vs NGF} = 0.041, ^*P_{NGF vs NGF+Cpd C} = 0.043.