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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 1. Intrathecal non-viral IL-10 gene therapy provides stable long-lasting relief of allodynia in mice and is independent of endogenous IL-10.

Figure 1

Absolute threshold behavioral responses for (A) ipsilateral and (B) contralateral hindpaws for WT and IL-10 KO mice are shown (N = 6–8 mice/group as indicated in figure legend). At baseline (BL), responses to low threshold mechanical stimuli were similar between all groups (ipsilateral, F(5, 33) = 1.01, P > 0.05; contralateral, F(5, 33) =1.31, P > 0.05). For data collected between BL through Day 5 post-surgery, a main effect of time (ipsilateral, F(2, 66) = 170.08, P < 0.001; contralateral, F(2, 66) = 134.35, P < 0.001) and an interaction between time and surgical manipulation (ipsilateral, F(2, 66) = 79.76, P < 0.001; contralateral, F(2, 66) = 64.04, P < 0.001) were observed. After behavioral assessment on Day 5 post-surgery, all mice received an intrathecal (i.t.) co-injection of plasmid DNA encoding interleukin-10 (pDNA-IL-10; 3μg in 7.5μL) with D-mannose (DM; 25μg in 3μL) vs. pDNA-Control (Ctrl; 3μg in 7.5μL) with isotonic sterile saline (saline; 3μL). Following injection, both WT and IL-10 KO CCI-mice given i.t. DM/pDNA-IL-10 reveal bilateral reversal to normal levels of hindpaw sensitivity (ipsilateral, F(2, 66) = 54.49, P < 0.001; contralateral, F(2, 66) = 34.34, P < 0.001). This reversal remains stable through Day 30 post-injection (ipsilateral, F(4.9, 162.5) = 0.432, P > 0.05; contralateral, F(4.9, 163.1) = 0.871, P > 0.05). Not surprisingly, control injected CCI-mice demonstrate bilateral spontaneous reversal from allodynia between Days 30–40 post-injection (ipsilateral, F(2, 66) = 141.30, P < 0.001; contralateral, F(1.5, 48.5) = 48.54, P < 0.001), with all groups experiencing similar normal levels of light touch sensitivity by Day 40 post-injection (ipsilateral, F(5, 33) = 0.81, P > 0.05; contralateral, F(5, 33) = 0.193, P > 0.05). (C) Plasmid map of the pDNA-IL-10 construct. The modified plasmid was derived from the expression cassette previously used for adeno-associated virus-2. pDNA-IL-10 is a 5.9 Kb plasmid with gene transcription driven by a chicken β-actin promoter (CB pro) hybridized with a cytomegalovirus enhancer (CMV enh). The expression cassette contains the transgene for mouse IL-10 with a point mutation (serine substitution for phenylalanine at amino acid 129) (IL-10F129S), and a viral SV40 polyadenylation signal (SV40 poly(A)), and is flanked by two inverted terminal repeat (ITR) sequences. The plasmid vector also contains an intervening sequence (IVS; intron) and an ampicillin-resistance (Ampr) gene.