Figure 3. Metformin augmented neurite outgrowth and activated AMPK in adult sensory neurons.

In (A) and (B) are fluorescent images of adult DRG neuron cultures maintained for 24 hours without neurotrophic growth factors. The size marker indicates 100 μm. In both (C) and (D) are fluorescent images of adult rat sensory neurons that were treated with 0.3 mM metformin. In (E) is quantification of total neurite outgrowth in response to a range of metformin doses (0.03 mM, 0.1 mM and 0.3 mM). The pixel density used to quantify neurite outgrowth was adjusted to cell number. See [126] for details on this in vitro culture system. Values are means ± SEM (n = 6 replicate cultures). *P < 0.05 vs control. Statistics were performed by one-way ANOVA with Dunnett’s post hoc test. DRG = dorsal root ganglia; met = metformin. In (F) are presented Western blots derived from total protein samples of DRG cultures exposed to 0.3 mM metformin for 0–60 min and probed for total AMPK (T-AMPK) and phosphorylated AMPK (P-AMPK). (G) reveals the data for P-AMPK normalized to T-AMPK. Values are means ± SEM, n = 3 replicate cultures. *P < 0.01 vs control. Statistics were performed by one-way ANOVA with Dunnett’s post hoc test. Similar effect of metformin was observed when P-AMPK was normalized to total protein on the blot (not shown). We confirm that all ethical standards were followed according to Institutional Animal Care and Use Committee of the University of California, San Diego, and by the University of Manitoba Animal Care Committee following Canadian Council of Animal Care rules.