Fig 2: The involvement of BDNF in SCF+G-CSF-induced enhancement of neurite outgrowth.(A) The role of BDNF and NGF on SCF+G-CSF-induced enhancement of neurite outgrowth. Neurite outgrowth was quantified with a Neurite Outgrowth Quantification kit 72h after introducing siRNAs to neurons. SCF and G-CSF were added to neurons one hour after siRNA transfection. Note that SCF+G-CSF treatment results in a significant enhancement of neurite outgrowth. In addition, SCF+G-CSF-induced neurite outgrowth is significantly blocked by BDNF siRNA or BDNF siRNA+NGF siRNA, whereas BDNF siRNA+NGF siRNA treatment has no significant difference as compared to the BDNF siRNA treatment. NGF siRNA shows a trend towards decreasing SCF+G-CSF-induced neurite outgrowth. Negative siRNAs do not affect the SCF+G-CSF-induced the enhancement of neurite outgrowth as the neurite outgrowth in the negative siRNA-treated neurons still remains significantly greater than those of medium controls and BDNF siRNA+NGF siRNA. Neg: negative controls for siRNA. **p < 0.01, *p < 0.05. Mean ± SE, n=8. (B) SCF+G-CSF-induced enhancement of neurite outgrowth is mediated by BDNF receptor, TrkB. The neurite outgrowth was tested 72 h after treatment. Note that SCF+G-CSF-induced the increase in neurite extension (SCF+G-CSF vs. control, *p < 0.05) is significantly blocked by an anti-TrkB antibody (SCF+G-CSF vs. SCF+G-CSF+TrkB antibody, **p < 0.01). Scale bars, 50µm. Mean ± SE, n=3.

Fig 3: Effects of RES on inflammatory pathways. mRNA levels of (A and B) IL-1β, (C and D) IL-6, (E and F) NGF and (G and H) IGF-1 at 7 and 28 days post-AMI, respectively were measured by RT-qPCR. Data are expressed as the fold change relative to the sham group (n=8 per group). *P<0.05, **P<0.01, ***P<0.001; ns, not significant. RES, resveratrol; AMI, acute myocardial infarction; IL, interleukin; NGF, nerve growth factor; IGF-1, insulin-like growth factor-1.

Fig 4: Effects of mechano-electrical stimulation (MES) on Schwann cell behaviors, including NGF secretion and cell maturation.a Immunofluorescence images showing NGF expression of RSC96 cells, a Schwann cell line, cultured statically or subjected to MES on P(VDF-TrFE) scaffolds (scale bar: 100 µm) and (b) their quantification of the fluorescent intensities. c The relative gene expression of neurotrophic factor (Ngf), myelination markers (Krox20, Pmp22), and early Schwann cell marker Ncam-1 in RSC96 subjected to MES for 7 days as compared to that of the statically cultured cells, determined by qRT-PCR. Error bars represent standard error of means (n = 6). * and ** denote statistical significance of p < 0.05 and p < 0.01, respectively. Six images or samples were used for quantification in each graph.

Fig 5: SCF+G-CSF upregulates BDNF and NGF at both the levels of transcription (mRNA) and translation (protein).(A and B) BDNF and NGF gene expression after SCF, G-CSF, or SCF+G-CSF treatment. Quantification of BDNF and NGF gene expression was performed with Real-Time PCR. (A) Quantification data for BDNF gene expression. Note that SCF or SCF+G-CSF induces a significant increase in BDNF gene expression at both 6 and 24h after treatment as compared to medium controls (**p < 0.01). The levels of BDNF gene expression are much higher in SCF+G-CSF-treated neurons than those in SCF or G-CSF-treated at both 6 and 24h after treatment (◆◆p<0.01). SCF also causes a significantly higher level of BDNF gene expression than those of G-CSF at 6 h post-treatment (◆◆p<0.01). G-CSF only induces an increase in BDNF gene expression at 24h (**p < 0.01) but not at 6h post-treatment as compared to medium controls. SCF-, G-CSF- (24h only), or SCF+G-CSF-induced upregulation of BDNF gene expression is significantly blocked by BAY 11-7082 (●● p < 0.01). (B) Quantification data for NGF gene expression. Note that both SCF and SCF+G-CSF treatments result in a significant upregulation of NGF gene expression 6 and 24 h post-treatment when compared to medium controls (** P < 0.01) or G-CSF (◆◆p<0.01). BAY 11-7082 significantly prevents SCF- or SCF+G-CSF-induced upregulation of NGF gene expression (●● p < 0.01). Mean ± SD, n=3-4. (C and D) SCF+G-CSF-induced the upregulation of BDNF and NGF gene expression is prevented by siRNAs against BDNF (C) or NGF (D). Gene expression was examined 36h after adding siRNAs. Neg: negative controls for siRNA. **p < 0.01. Mean ± SE. N=3-4. (E-H) SCF+G-CSF-induced increases of BDNF and NGF protein production are inhibited by siRNAs against BDNF (E, G) or NGF (F,H) 72h after adding siRNAs to neurons. (E, F) Representative Western-Blot bands for BDNF (E) and NGF (F) proteins after different treatments. S: SCF. G: G-CSF. S+G: SCF+G-CSF. Neg: negative controls for siRNAs. (G and H) Semi-quantification of Western-blot data for BDNF (G) and NGF (H) proteins. *p < 0.05. Mean ± SE, n=3-4.