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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Brain Behav Immun. 2017 Nov 4;69:91–112. doi: 10.1016/j.bbi.2017.11.004

Figure 4. Pro-inflammatory characterization of the CCI-damaged sciatic nerve following i.t. non-viral IL-10 gene therapy in IL-10 KO mice.

Figure 4

mRNA was isolated from ipsilateral sciatic nerve (ipsi SCN) collected on Day 12 post-injection (Day 17 post-surgery) from previously behaviorally verified animals (Fig 3). (A–F) Baseline transcription levels are not significantly different between WT Naïve and IL-10 KO Naïve groups (N = 2–3 mice/group) for all targets (P > 0.05): Tnf, Il1b, Tgfb1, Ccl2, Itgam, and Cd3e. (A–B) Post-surgical mRNA levels of pro-inflammatory cytokines TNF (Tnf) and IL-1β (Il1b) are greatly increased in ipsi SCN from all CCI conditions compared to Sham+DM/pDNA-Control treated mice (F(4, 31) = 5.07, P < 0.01; F(4, 29) = 3.769, P = 0.0138, respectively). (C) mRNA levels of pro-inflammatory chemokine CCL2 (Ccl2) are greatly increased across all CCI conditions compared to Sham+DM/pDNA-Ctrl treated mice (F(4, 30) = 5.92, P < 0.01). (D) Following CCI surgery, the macrophage activation marker CD11b (itgam) is elevated in ipsi SCN, with CD11b mRNA levels significantly increased across all CCI conditions compared to Sham+DM/pDNA-Ctrl treated mice (F(4, 29) = 3.77, P < 0.05). (E) The general T cell marker CD3e (Cd3e) is elevated in sciatic tissue following CCI surgery, with CD3e mRNA expression significantly increased across all CCI conditions compared to Sham+DM/pDNA-Ctrl treated mice (F(4, 29) = 3.77, P < 0.05). (F) Post-surgical mRNA transcript levels of anti-inflammatory cytokine TGF-β1 are greatly increased across all CCI conditions compared to Sham+DM/pDNA-Ctrl treated mice (F(4, 31) = 11.13, P < 0.0001). N = 5–9 mice/group for treatment groups, as indicated on graphs. (G–L) Complementary protein analysis for ipsi SCN from the same animals previously assessed for mRNA analysis (A–F). Baseline protein levels are not significantly different between WT Naïve and IL-10 KO Naïve groups (N = 2–3 mice/group) for all targets (data not shown; P > 0.05): TNF, IL-1β, IFN-γ, IL-6, CXCL1, and IL-12p70. No significant differences were observed between Sham conditions (Sham+DM/pDNA-Ctrl and Sham+DM/pDNA-IL-10) for all protein targets (P > 0.05). (G) Pro-inflammatory cytokine TNF protein expression levels were elevated across all CCI conditions compared to Sham+DM/pDNA-IL-10 (F(4, 28) = 15.54, P < 0.0001). (H) In most CCI conditions, protein levels for the pro-inflammatory cytokine IL-1β are significantly increased in ipsi SCN compared to Sham+DM/pDNA-IL-10 (F(4, 28) = 4.33, P < 0.01). (I) Protein levels for the pro-inflammatory cytokine IFN-γ are greater in most CCI conditions compared to Sham+DM/pDNA-IL-10 (F(4, 28) = 5.97, P < 0.01). (J) IL-6, a pro-inflammatory cytokine, is significantly increased in ipsi SCN for most CCI conditions compared to Sham+DM/pDNA-IL-10 (F(4, 27) = 3.47, P < 0.05). (K) CXCL1 (a.k.a. KC/GRO), a neutrophil chemoattractant, is significantly elevated following CCI surgery compared to Sham+DM/pDNA-IL-10 (F(4, 27) = 6.45, P < 0.001). (L) No significant increases in IL-12p70, a pro-inflammatory cytokine downstream of CpG-activated TLR-9, were observed for any condition (F(4, 28) = 1.46, P > 0.05). For protein analyses, N = 6–8 mice/group as indicated, and protein levels are presented as mean ± SEM. For mRNA analyses, N = 5–9 mice/group as indicated, and mRNA levels (mean ± SEM) are normalized to 18S rRNA and graphically presented relative to mean WT Naïve levels. Post-hoc multiple comparisons via Fisher’s LSD (α = 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).