Article Figures & Data
Figures
- Figure 1.
Chemogenetic inhibition of TRPV1+ afferent terminals attenuates mechanical hyperalgesia and ongoing pain in mice with ION-CCI. A, Effects of CNO on mechanical hyperalgesia following ION-CCI in mice expressing inhibitory DREADD in TRPV1-LN neurons (TRPV1Cre;R26LSL-hM4Di; hM4Di) or TRPV1Cre mice (Cre). CNO was repeatedly injected intraperitoneally (40 µg/20 µl), to ipsilateral (ipsi; 20 µg/10 µl) or contralateral (contra; 20 µg/10 µl) V2 skin or intra-Vc (Vc; 0.1 µg/1 µl) at indicated time points. Mechanical sensitivity is presented as EF50 (the mechanical force that produced a 50% response frequency) in the ipsilateral V2 skin; *p < 0.05, **p < 0.01, ***p < 0.001 in Bonferroni post hoc test following two-way ANOVA. Throughout the figures, numbers in parenthesis represent the number of mice in each group. B, Preconditioning habituation started at four to five weeks after CCI or sham surgery. Following the habituation period, the mice received intra-Vc microinjection of vehicle (0.5-µl saline) in the ipsilateral side and paired with a chamber for 30 min. Four hours later, the same mouse received 0.5-µl CNO paired with the other conditioning chamber for 30 min. On the following test day, the mice were placed in the CPP chambers with free access to all chambers to measure time spent in both chambers. Mice were recorded for 15 min, and difference scores were calculated by subtracting time spent in CNO-paired chambers before conditioning from the time after conditioning; *p < 0.05, **p < 0.01 in Bonferroni post hoc test following two-way ANOVA. C, The effects of AMG9810 (100 nmol/10 µl), a TRPV1 antagonist, or vehicle injected into the ipsilateral V2 skin on mechanical sensitivity in the ipsilateral V2 skin of mice with ION-CCI.
- Figure 2.
Single subcutaneous injection of capsaicin attenuates mechanical hyperalgesia in mice with ION-CCI. A, B, Mechanical sensitivity in the ipsilateral V2 skin (A) or in the ipsilateral V3 skin (B). Vehicle or capsaicin (10 µg) was subcutaneously injected into the ipsilateral V2 skin area 14 d after CCI or sham surgery. C, D, Percent response frequency in response to VF hair 3.61 (C) or 4.08 (D). Symbols and n numbers within parenthesis in A, B are also applied to C, D. Two-way ANOVA followed by Bonferroni post hoc test (CCI/Veh vs CCI/Cap); *p < 0.05, **p < 0.01, ****p < 0.0001.
- Figure 3.
CPP test following capsaicin or vehicle injection in mice with CCI-ION or sham surgery. A, Time course of CPP experiment. Preconditioning habituation started at four weeks after CCI or sham surgery (two weeks after cap or vehicle injection). Time spent in two chambers were recorded for 15 min (preconditioning trial). Following the habituation period, the mice received intra-Vc microinjection of vehicle (0.5-µl saline) in the ipsilateral side and paired with a chamber for 30 min. Four hours later, the same mouse received 0.5-µl 2% lidocaine paired with the other conditioning chamber for 30 min. On the following test day, the mice were placed in the CPP chambers with free access to all chambers to measure time spent in both chambers (testing trial). Mice were recorded for 15 min, and chamber preference in comparison with preconditioning time was analyzed. B, Difference scores were calculated by subtracting time spent in lidocaine-paired chambers during preconditioning trial from the time during testing trial; ***p < 0.001 in Bonferroni post hoc test following one-way ANOVA.
- Figure 4.
Properties of capsaicin-induced analgesia. A, In mice with ION-CCI on the left side, the effects of capsaicin (red) or vehicle (black) injection into V2 skin on the right contralateral side. Mechanical sensitivity was measured from V2 skin in the contralateral side (open circles; capsaicin-injected right side) and ipsilateral side (filled circles; left side with ION-CCI). B, The effects of capsaicin injection into the ipsilateral V2 skin on mechanical sensitivity in the ipsilateral V2 skin of mice with ION-CCI (red) or sham (blue) at indicated time points. Capsaicin-induced analgesia was not initiated at 6 h following capsaicin injection. One-way RM ANOVA followed by Sidak multiple comparison test; *p < 0.05 (vs Cap 0 d in CCI/Cap group), NS, not significant. C, The effects of repeated injection of capsaicin into the ipsilateral V2 skin on mechanical sensitivity in the ipsilateral V2 skin of mice with ION-CCI at indicated time points. Analgesia by first capsaicin disappeared after seven weeks following injection. Second capsaicin produced analgesia again; *p < 0.05 (vs CCI/Veh group in each time point) in post hoc test following two-way RM ANOVA.
- Figure 5.
Capsaicin-induced ablation of TRPV1-LN afferent terminals is reversible and repeatable. Longitudinal monitoring of tdTomato+ afferent terminals in intact hindpaw skin of a mouse expressing tdTomato from the locus of TRPV1 (TRPV1tdTomato) using two-photon microscopy. To monitor capsaicin-mediated changes of tdTomato+ afferent terminals, the same site was imaged repeatedly before (A) and 3 d after first intraplantar injection of capsaicin (10 µg; B). Six weeks after first injection of capsaicin (C), the fibers were regenerated. Second injection of capsaicin reproduced ablation of the fibers (D). Projected image stacks across ∼680 µm (340 z-axis sections at 2 µm). Scale bar: 100 µm.
- Figure 6.
Inhibition of calpain attenuates capsaicin-induced analgesia for TNP. Mechanical hyperalgesia in V2 (A) or V3 (B) skin following ION-CCI was attenuated by injection of capsaicin (Cap; 10 µg; blue). Co-administration of capsaicin with calpain inhibitor MDL28170 (MD; 10 μm in 20 µl) into whisker pad area abolished capsaicin-induced analgesia for TNP compared with the injection of vehicle (Veh). Two-way RM ANOVA followed by Bonferroni post hoc test (CCI/Cap/Veh vs CCI/Cap/MDL); *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001.
- Figure 7.
Inhibition of calpain attenuates capsaicin-induced ablation of TRPV1-LN afferent terminals in hindpaw. A, Longitudinal in vivo two-photon imaging of GFP+ afferent terminals in intact hindpaw of a mouse expressing mGFP from the locus of TRPV1 (TRPV1mGFP). To assess changes of GFP+ afferent terminals, the same site was imaged repeatedly before (baseline) and 3 d after intraplantar injection of vehicle or MDL28170 (10 μm in 10 µl) followed by capsaicin (10 µg). The images represent projected image stacks across 100–150 µm (200–300 z-axis sections at 0.5 µm). Scale bar: 50 µm. B, In each stacked image, the proportion of GFP+ pixels/total pixels in the imaged area was calculated. N = 7 hindpaws from 5 mice per group; **p < 0.01 in Sidak post hoc assay following two-way ANOVA.
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