Figure 6.

Demyelination is decreased after treatment with TRO19622 and spontaneously induced after treatment with MJ. (a) Upper panels: Western blot analysis for phospho-ERK1/2 (P-ERK1/2), phospho-p38 (P-p38), phospho-JNK (P-JNK), phospho-cJun (P-cJun), activated cleaved-caspase3 (a-caspase3) and phospho-Bcl2 (P-Bcl2) in the sciatic nerve of mice without injury (No crush), 4 h after injury (Crush), 4 h after injury with TRO19622 treatment 30 min before injury (Crush + TRO), or without injury but 4 h after methyl jasmonate treatment (No crush + MJ). GAPDH was used as loading control. Blots were stripped and re-hybridized with different antibodies against proteins of interest, so GAPDH is a loading control for different immunoblots. n = 3 independent experiments. Lower panels: WB results analyzed by densitometry and normalized on GAPDH respective values on the same blot. As similar changes were observed at 4 and 12 h after injury (see Figure S3 for 12 h representative images), data from both experiments were pooled except for P-cJun, for which changes were consistent but much higher at 12 h. So, only 4 h data are presented. All data are presented as fold over No crush condition. Error bars show SD. Statistical tests are one-way ANOVA followed by a Dunnett’s multiple comparison post hoc tests for each marker. n = 2 (P-cJun) to 4 mice. ns= non-significant. (b) Immunohistochemistry for phospho-cJun (P-cJun, red) and nuclear TOPRO3 (blue) on teased fibers of mouse nerve transduced with virus expressing control shRNA or shRNA 2 or 3 targeting VDAC1 in addition to GFP (green). Without injury (No crush) non infected cells or cells expressing control shRNA and GFP show no P-cJun in their nucleus. Four days after injury (Crush), non-infected cells and cells expressing control shRNA and GFP express P-cJun in their nucleus, while cells expressing shRNA2 or 3 and GFP show low amount of P-cJun in their nucleus (arrowheads). Scale bars = 10 μm. Same magnification for all pictures. (c)—Immunohistochemistry for phospho-cJun (P-cJun, red) and nuclear TOPRO3 (white) on teased fibers of mouse nerve without injury (No crush) or 4 days after injury (Crush). In both cases, animals were treated with TRO19622 (TRO) or vehicle, once intraperitoneally (3 mg/kg) 10 h before injury, once intrasciatically (2 µL 20 µM) 30 min before injury, and then intraperitoneally for 4 consecutive days. Scale bars = 10 μm. Same magnification for all pictures. (d) Immunohistochemistry for phospho-cJun (P-cJun, red) and nuclear TOPRO3 (white) on teased fibers of mouse nerve without injury (No crush) 4 days after treatment with methyl jasmonate (MJ) or vehicle. Scale bars = 10 μm. Same magnification for all pictures. (e) Representative images of the morphological features occurring in myelinating SC during demyelination 4 days after nerve injury. These cells express virally-delivered GFP (Green). Arrowheads show myelinating SC and arrows non-myelin-forming SC. Scale bars = 100 μm. (f) Quantification of myelinating and non-myelinating SC frequency in nerves transduced with AAV- expressing Control shRNA (Cont sh) or shRNAs 2 or 3 targeting VDAC1 (VDAC1 sh) in addition to GFP with (Crush) or without nerve injury (No crush). Nerves were collected 4 days after the nerve injury and GFP-positive cells were counted as myelinating or non-myelin forming as shown in Figure 6e. Data are represented as mean ±SD. Statistical significances were determined using a two-tailed Student’s t-test. n= 5 (No Crush, Cont sh), 4 (Crush, Cont sh) and 4 (Crush, VDAC1 sh) mice. (g) Representative CARS images of sciatic nerves freshly collected in non-injured control animals (Control), in animals 4 days after crush nerve injury and after vehicle (Crush + vehicle) or TRO19622 (Crush + TRO, 3 mg/kg) treatment or in animals 4 days after injection of MJ in the sciatic nerve (No Crush + MJ). Scale bars= 20 μm. (h) The number of ovoid per surface unit (left) and the percentage of demyelinated fibers (right) were measured on the CARS images. Data are presented as mean ± SEM. n = 6 (Crush + vehicle), 6 (Crush + TRO) and 4 (No Crush + MJ) animals. Statistical analysis shows one-way ANOVA Sidak’s post hoc test. * p < 0.05, ** p < 0.01, or *** p < 0.001.