Figure 1. Aged and chronically denervated sciatic nerves show reduced regeneration and higher senescent cell accumulation.

• A
  Schematic representation of the sciatic nerve transection model. The sciatic nerve divides into three branches, the tibial, peroneal and sural nerves (upper panel). The tibial branch is first transected (Tibial Nerve Transection, TN‐T) and the distal stump is sutured to the nearest muscle to prevent reconnection (middle panel). The time of denervation until analysis of the tissue defines acute (Acu‐D) or chronic (Chr‐D) denervation. To evaluate regeneration, the distal tibial nerve is detached from the muscle and reconnected to the freshly transected common peroneal nerve (Tibial Nerve reconnection, TN‐R, bottom panel).
• B
  c‐Jun expression in the nucleus of SC on longitudinal cryostat sections of adult and aged mice sciatic nerves. We compared non‐damaged nerves, and sciatic nerves distal to the nerve cut in acute (7 dpi) and chronic (42 dpi) denervation conditions. N = 3–5 animals per group. One‐way ANOVA and Fisher's LSD multicomparison post‐test.
• C
  Representative western blot against c‐Jun comparing Acu‐D in adult mice (N = 5) to Acu‐D in aged mice (N = 4) and to Chr‐D in adult mice (N = 3).
• D
  The scheme in the upper left show the timeline for transection and reconnection surgeries to evaluate axonal regeneration. Comparison of axonal density and distance 7 days after reconnection surgery in aged (N = 4) and adult mice with Acu‐D (N = 5), or adult mice with Chr‐D (N = 3). Multiple t‐test for each distance point (x‐axis) was performed, comparing the difference among Acu‐D in adult versus aged mice (#), and adult mice with Acu‐D versus Chr‐D (*). Significative differences are shown with symbols, and the exact P‐values can be seen in Appendix Table S1. Data is presented as mean ± SEM. Representative IF images of reconnected sciatic nerves are shown to the right using SCG10 marker in green. Arrowheads indicate the reconnection site (see Materials and Methods for details). The dataset used for this panel (D) corresponds to control conditions of the experiment shown in Fig 5B–D. Scale bar, 500 μm.
• E, G
  Brightfield and fluorescence confocal acquisition of β‐galactosidase activity in adult and aged animals, measured on longitudinal sections from non‐damaged and Chr‐D damaged nerves, distal to the injury. Scale bar, 42 μm. N = 5 animals per group. Complete nerves in brightfield can be seen in Fig EV1.
• F, H
  Immunofluorescence against p16INK4a and SOX10 in contralateral non‐injured nerves and chronically transected sciatic nerves from adult and aged mice. Scale bar, 50 μM. Quantification of p16INK4a‐positive SC correspond to N = 3 mice per condition. One‐way ANOVA and Fisher's LSD multicomparison post‐test.
• I
  Quantification of total p16INK4a‐positive cells in contralateral non injured nerves and damaged nerves from adult animals (N = 3 mice per condition with 4 micrographs per animal each).
• J
  Proportion of different p16INK4a‐positive cell types in uninjured and injured nerves from adult and aged mice (N = 3). The total 100% percent of each condition corresponds to total p16INK4a⁺ cells quantified in (I). Detailed quantification and immunofluorescence for each cell type can be seen in Fig EV2.
• K, L
  Fluorescence confocal acquisition and quantification of β‐galactosidase activity assay on longitudinal sections of injured sciatic nerves, distal to the nerve cut, after acute denervation in adult wild type and c‐Jun OE animals. N = 3 or 4 mice. Scale bar, 40 μM.
• M, N
  Immunofluorescent staining against p16INK4a in distal sciatic nerves after chronic denervation in adult wild type and c‐Jun OE animals. Scale bar, 40 μM. Quantification of p16INK4a‐positice Schwann cells shown in (M) are quantified in (N). N = 3 animals per group.

Data information: For all (A–N), when two groups were compared, a non‐paired, one‐tailed Student's t‐test was performed; when more than two groups were compared, One‐way ANOVA and Fisher's LSD multicomparison post‐test. Data is presented as mean ± SEM.

Source data are available online for this figure.