Figure 9.

Inhibition of the AMPK/eNOS pathway reverses the beneficial effects of metformin on angiogenesis in aged mice following injury.

(A) Representative images of spinal cord blood vessels immunostained with CD31 (green-Alexa Fluor® 488) in aged mice treated with metformin (100 mg/kg) or metformin + compound C. The metformin + compound C group exhibited less vascular density than the metformin only group. Red lines indicate the epicenter of the injury site. (B) Quantification of the CD31-labeled blood vessels. (C) Representative images of blood vessels immunostained with CD31 (green-Alexa Fluor® 488) and Ki67 (red-Alexa Fluor® 594) in the sham group and in aged mice treated with metformin or metformin + compound C at 7 days post-SCI. The metformin + compound C group exhibited less vascular regeneration than the metformin only group. Scale bars: 150 μm in A, 50 μm in C. (D) Quantification of CD31⁺Ki67⁺ cells. The data are presented as the mean ± SEM (n = 6 per group). #P < 0.05 (two-tailed Student’s t-test). AMPK: Adenosine monophosphate-activated protein kinase; CC: Compound C, an AMPK inhibitor; DAPI: 4′,6-diamidino-2-phenylindole; eNOS: endothelial nitric oxide synthase; Met: metformin; SCI: spinal cord injury.