Research ArticlePain

Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice

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Science Translational Medicine  18 Jul 2018:
Vol. 10, Issue 450, eaar7384
DOI: 10.1126/scitranslmed.aar7384
  • Fig. 1 SP-BOT administered intrathecally reduced the mechanical hypersensitivity that developed in long-term inflammatory and neuropathic pain states.

    (A) Mechanical threshold assessed using von Frey filaments in naïve mice before (B1) and 1 to 7 days (D1 to D7) after intrathecal injection of SP-BOT (100 ng/3 μl; n = 4 per group). (B) Time on rotarod apparatus after SP-BOT intrathecal injection (n = 4 per group). (C) von Frey filaments were used to measure mechanical hypersensitivity in mice injected with 5 μl of CFA in the ankle joint and injected 3 days later with intrathecal SP-BOT (100 ng/3 μl). Mice were tested at baseline and up to 21 days after CFA injection (n = 5 to 6 per group). (D) CFA (20 μl) was also injected into the plantar surface of the hind paw, and 4 days later, mice received intrathecal SP-BOT (100 ng/3 μl; n = 7 per group). (E) SP-BOT was injected intrathecally 5 days after SNI and alleviated the neuropathic mechanical sensitivity that had developed (n = 8 per group). (F) NK1R−/− mice and their WT littermates received intrathecal SP-BOT 5 days after SNI (n = 8 per group). Data show means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Difference in sensitivity was assessed using repeated measures two-way followed by one-way analysis of variance (ANOVA). For complete statistical analyses, please refer to table S1, and for maximum possible effect (%MPE), please refer to table S2.

  • Fig. 2 SP-BOT was internalized by NK1R-positive neurons without toxicity.

    (A) Images of NK1R and cSNAP25 immunoreactivity in the superficial dorsal horn of mice 14 days after intrathecal injection of SP-BOT. Green, cSNAP25; red, NK1R. Scale bars, 100 μm. (B) Images of selective targeting of NK1R-expressing neurons in the superficial dorsal horn 96 hours (top) or 14 days (bottom) after intrathecal injection of SP-BOT. Green, cSNAP25; red, NK1R. Scale bars, 20 μm (top) and 10 μm (bottom). (C) Schematic illustration and images of the lateral parabrachial (LPb) area of mice 25 days after intrathecal injection of SP-BOT or saline. Green, cSNAP25 in spinoparabrachial axons. Scale bar, 80 μm. DRG, dorsal root ganglia. (D) Bar graph illustrating the number of c-Fos–immunostained nuclei in the PB from both saline and SP-BOT–injected mice. Mice received intrathecal SP-BOT, and 3 days later, they were injected with CFA into the plantar surface of the hind paw. Tissue was taken 6 hours later. Values reported are the mean number of c-Fos+ nuclei (±SEM) normalized to the mean of c-Fos+ nuclei in naïve control mice (n = 4 per group). (E) Quantification of NK1R fluorescence intensity in the contralateral superficial dorsal horn of mice 18 days after intraplantar CFA injection and 14 days after intrathecal injection of SP-BOT or saline. All data were normalized to laminae I/II saline-treated mice (n = 4 per group). *P < 0.05. The comparison of three groups was determined using one-way ANOVA.

  • Fig. 3 Derm-BOT reduced the mechanical hypersensitivity in inflammatory and neuropathic pain models in mice.

    (A) Mechanical threshold assessed using von Frey filaments in naïve mice before (B1) and after (D1 to D7) intrathecal injection of Derm-BOT (100 ng/3 μl; n = 4 per group). (B) Mechanical threshold was measured in mice before and after CFA injection (5 μl) in the ankle joint. Four days later, mice were injected intrathecal with Derm-BOT (100 ng/3 μl). Mice were tested at baseline and up to 14 days after CFA injection (n = 5 per group). (C) CFA (20 μl) was injected into the plantar surface of the hind paw, and 4 days later, mice received intrathecal Derm-BOT (100 ng/3 μl; n = 8 per group). (D) Mechanical threshold measured using von Frey filaments in mice injected with CFA (5 μl) in the ankle joint and injected 3 days later with intrathecal SP-BOT (100 ng/3 μl). Two weeks later, mice injected with SP-BOT were reinjected with intrathecal Derm-BOT (n = 4 per group). (E) Derm-BOT was injected intrathecally in mice 5 days after SNI surgery (n = 9 per group). Data show means ± SEM. *P < 0.05, **P < 0.01, ***P ≤ 0.001. Difference in sensitivity was assessed using repeated-measures two-way, followed by one-way ANOVA.

  • Fig. 4 Derm-BOT was internalized by MOR-expressing neurons.

    (A) Images of cSNAP25 and MOR immunoreactivity in the superficial dorsal horn of mice 14 days after injection of intrathecal Derm-BOT. Green, cSNAP25; red, MOR. Scale bar, 100 μm. (B) Images of selective targeting of cSNAP25 to MOR-expressing neurons in the superficial dorsal horn 96 hours (top and bottom) or 14 days after intrathecal injection of Derm-BOT. Green, cSNAP25; red, MOR. Scale bars, 20 μm (top) and 10 μm (middle and bottom).

  • Fig. 5 Derm-BOT precludes the effect of morphine and retains efficacy in NK1R−/− mice.

    (A) Mechanical threshold using von Frey filaments in mice injected intrathecally with Derm-BOT 5 days after SNI surgery. Twenty-nine days later, mice were injected with intrathecal morphine (5 nM; n = 9 per group). (B) Mechanical threshold measured using von Frey filaments in NK1R−/− mice before and after SNI surgery. Five days after surgery, mice were injected with intrathecal SP-BOT and were injected with intrathecal Derm-BOT 2 weeks later (n = 8 per group). Data show means ± SEM. */#P < 0.05, **P < 0.01, ***/###P ≤ 0.001. Difference in sensitivity was assessed using repeated-measures two-way followed by one-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/450/eaar7384/DC1

    Fig. S1. Synthesis of botulinum peptide conjugates using a stapling bridge.

    Fig. S2. SP-BOT has no effect on mechanical threshold in the contralateral paw.

    Fig. S3. Effect of different doses of intrathecal SP-BOT or Derm-BOT on CFA-induced hypersensitivity.

    Fig. S4. Intrathecal injection of unconjugated BOT LcTd (Neg-BITOX) without a receptor binding domain had no effect on inflammatory hyperalgesia.

    Fig. S5. cSNAP25-positive neurons after SP-BOT or Derm-BOT intrathecal injection.

    Fig. S6. SP-BOT or Derm-BOT intrathecal injection does not induce glial activation in the dorsal horn.

    Fig. S7. Quantification of MOR fluorescence intensity.

    Fig. S8. Effect of SP-BOT injection on withdrawal threshold plotted as force.

    Fig. S9. Effect of Derm-BOT injection on withdrawal threshold plotted as force.

    Table S1. Statistical analysis for Figs. 1, 2, 3, and 5 and figs. S2 and S7.

    Table S2. Maximum possible effect.

    Table S3. Raw data (Excel file).

  • The PDF file includes:

    • Fig. S1. Synthesis of botulinum peptide conjugates using a stapling bridge.
    • Fig. S2. SP-BOT has no effect on mechanical threshold in the contralateral paw.
    • Fig. S3. Effect of different doses of intrathecal SP-BOT or Derm-BOT on CFA-induced hypersensitivity.
    • Fig. S4. Intrathecal injection of unconjugated BOT LcTd (Neg-BITOX) without a receptor binding domain had no effect on inflammatory hyperalgesia.
    • Fig. S5. cSNAP25-positive neurons after SP-BOT or Derm-BOT intrathecal injection.
    • Fig. S6. SP-BOT or Derm-BOT intrathecal injection does not induce glial activation in the dorsal horn.
    • Fig. S7. Quantification of MOR fluorescence intensity.
    • Fig. S8. Effect of SP-BOT injection on withdrawal threshold plotted as force.
    • Fig. S9. Effect of Derm-BOT injection on withdrawal threshold plotted as force.
    • Table S1. Statistical analysis for Figs. 1, 2, 3, and 5 and figs. S2 and S7.
    • Table S2. Maximum possible effect.
    • Legend for Table S3

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    Other Supplementary Material for this manuscript includes the following:

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