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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 2. I.t. non-viral IL-10 gene therapy in IL-10 KO mice leads to expression of IL-10 transgene in pain-relevant lumbar dorsal root ganglia.

Figure 2

Absolute threshold behavioral responses for (A) ipsilateral and (B) contralateral hindpaws are shown. At baseline (BL), responses to low threshold mechanical stimuli were similar between all groups (ipsilateral, F(4, 39) = 1.39, P > 0.05; contralateral, F(4, 39) = 1.04, P > 0.05). For data collected between BL through Day 5 post-surgery a main effect of time (ipsilateral, F(1.7, 66.1) = 184.69, P < 0.001; contralateral, F(2, 78) = 186.41, P < 0.001) is observed. After behavioral assessment on Day 5 post-surgery, Sham- or CCI-treated mice received an i.t. co-injection of DM (25μg in 3μL) with either pDNA-IL-10 (3μg in 7.5μL) or pDNA-Ctrl (N = 6–12 mice/group). An additional control group consisted of CCI-treated mice that received i.t. saline with pDNA-IL-10 (N = 7). Following injection, a main effect of time (ipsilateral, F(6.0, 233.1) = 49.09, P < 0.001; contralateral, F(8, 312) = 46.2, P < 0.001) and an interaction of time and treatment (ipsilateral, F(23.9, 233.1) = 29.11, P < 0.001; contralateral, F(32, 312) = 25.13, P < 0.001) were observed. Comparisons between groups reveal that on Day 2 post-injection, CCI+DM/pDNA-IL-10-treated mice exhibit bilateral reversal from allodynia compared to mice lacking DM treatment (CCI+Saline/pDNA-IL-10) (ipsilateral and contralateral, P < 0.001). Surprisingly, on Day 3 post-injection, mice given DM and the control plasmid (CCI+DM/pDNA-Ctrl) reach levels of hindpaw sensitivity similar to Sham+DM/pDNA-IL-10-treated controls (ipsilateral and contralateral, P > 0.05). Beginning on Day 6 post-injection, CCI+DM/pDNA-Ctrl mice begin to return to bilateral allodynia (ipsilateral, F(3, 117) = 19.67, P < 0.001; contralateral, F(3, 117) = 9.73, P < 0.001), while mice treated with DM/pDNA-IL-10 never return to allodynia. (C–E) mRNA isolated from DRG and cauda equina tissues collected on Day 12 post-injection (Day 17 post-surgery) reveal IL-10 expression. IL-10 (Il10) mRNA levels were observed in WT Naïve positive controls (N = 3) but not in IL-10 KO Naïves (N = 2) for (C) ipsilateral, (D) contralateral lumbar DRG, and (F) lumbar spinal cord tissues (*P < 0.05), but not (E) cauda equina, despite a strong trend for positive expression (P = 0.062). IL-10 mRNA expression was not significantly different between Sham-treated conditions for DRG, cauda equina, and lumbar spinal cord (P > 0.05). (C) IL-10 transgene mRNA expression was significantly elevated in ipsilateral DRG of pain-relieved CCI+DM/pDNA-IL-10-treated IL-10 KO mice compared to IL-10 KO mice given either CCI+DM/pDNA-Ctrl or Sham+DM/pDNA-Ctrl (F(4, 28) = 7.72, P < 0.001). (D) No significant increase in IL-10 transgene mRNA levels were detected in contralateral DRG following i.t. injection (F(4, 29) = 2.20, P = 0.093). However, a priori comparison revealed that contralateral DRG from CCI-treated mice given DM co-treated with the IL-10 transgene contained significantly greater IL-10 mRNA levels than DRG from CCI-treated mice given DM with the control plasmid (P < 0.05) or Sham-treated mice with DM and the control plasmid (P < 0.05). (E) In the cauda equina, IL-10 transgene mRNA expression was greatly elevated in pain-relieved CCI+DM/pDNA-IL-10 KO mice compared to negative control conditions CCI+DM/pDNA-Ctrl and Sham/DM+pDNA-Ctrl (F(4, 23) = 4.19, P = 0.011). (G) IL-10 protein was detected in ipsilateral sciatic nerve (collected Day 12 post-injection) of WT Naïve but not IL-10 KO Naïve mice (P < 0.05), but no significant increase in IL-10 expression was detected in any i.t. injection condition (F(4, 27) = 0.65, P > 0.05). For mRNA and protein analyses, N = 5–7 mice/group. mRNA levels (mean ± SEM) are normalized to 18S rRNA and graphically presented relative to mean WT Naïve levels. For protein analyses, protein levels are presented as mean ± SEM. Post-hoc multiple comparisons via Fisher’s LSD (α = 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).