Research ArticlePain

The mechanosensitive ion channel Piezo2 mediates sensitivity to mechanical pain in mice

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Science Translational Medicine  10 Oct 2018:
Vol. 10, Issue 462, eaat9897
DOI: 10.1126/scitranslmed.aat9897
  • Fig. 1 Activation of Piezo2-expressing neurons evoke nocifensive behavior.

    (A) Schematic describing genetic strategy for generating constitutive and postnatal Piezo2-ChR2–positive mice (P2-ChR2+). i.p., intraperitoneal. (B) Percent behavioral response (paw withdrawal, paw licking, paw guarding, flinching, jumping, and vocalization) in constitutive Piezo2-ChR2+ (n = 8) and postnatal Piezo2-ChR2+ (n = 12) mice evoked by blue light (462 nm) stimulation (2 Hz, 100-ms pulses) of the plantar surface of the hindpaws [% withdrawal: ***P = 0.0006; % licking: **P = 0.0092 and ****P < 0.0001; % guarding: ***P = 0.0009 (0.151 mW/mm2) and ***P = 0.0002 (0.516 mW/mm2); % flinching: ****P < 0.0001; % jumping: ****P < 0.0001; % vocalization: ****P < 0.0001; two-way analysis of variance (ANOVA) Sidak’s multiple comparison test]. (C and D) Bar graphs representing frequency distribution of DRG neurons characterized by the presence (responders) or absence (nonresponders) of mechanically activated currents; DRG neurons cultured from CGRP-GFP (C) and MRGPRD-GFP (D) mice. Mechanically activated currents were recorded from green fluorescent protein (GFP)–positive neurons transfected with scrambled siRNA (CGRP, n = 32; MRGPRD, n = 50) or Piezo2 siRNA (CGRP, n = 33; MRGPRD, n = 43). Data from two to three independent experiments [*P = 0.006, **P = 0.003 (responder), and **P = 0.002 (nonresponder), nonparametric t test].

  • Fig. 2 Mechanical nociception is partially Piezo2 dependent.

    (A) Representative images of Piezo2 in situ hybridization in lumbar DRG sections from wild-type (Piezo2WT; n = 3), AdvillinCreERT2 (Piezo2iAdv; n = 4), and HoxB8Cre (Piezo2HoxB8; n = 2) mice. White, Piezo2 transcript; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 25 μm. Right: Quantification of Piezo2 transcript labeling area and mean intensity. Each dot represents mean values from one mouse. Bars represent population mean. (B) Percent paw withdrawal (four trials) in response to von Frey filament at different forces in Piezo2iAdv mice [left; Piezo2WT, n = 16; Piezo2iAdv, n = 18; **P = 0.002 (0.4g), **P = 0.008 (0.6g), ***P < 0.0001 (1.0g), and **P = 0.008 (1.4g), one-way ANOVA with Holm-Sidak’s multiple comparison test] and Piezo2HoxB8 mice (right; Piezo2WT, n = 14; Piezo2HoxB8, n = 15) (data at each force are means ± SEM; one-way ANOVA with Holm-Sidak’s multiple comparison test, ***P < 0.0001). (C) Thermal paw withdrawal threshold measured with a thermal probe in Piezo2WT (n = 16) and Piezo2HoxB8 (n = 9) mice. (D) Percent response (five trials) to cotton swab stroke on the hindpaw in Piezo2WT (n = 15) and Piezo2HoxB8 (n = 13) mice. (E) Number of bouts observed in response to an adhesive tape applied to the lower back of Piezo2WT (n = 11) and Piezo2HoxB8 (n = 11) mice. (F) Mechanical threshold measured in the range of 0.04g to 6g in Piezo2WT (n = 18) and Piezo2HoxB8 (n = 17) mice. (G) Percent response (10 trials) to pinprick on hindpaw in Piezo2WT (n = 16) and Piezo2HoxB8 (n = 15) mice. (H) Latency to response when an alligator clip (500g) is placed on the base of the tail in Piezo2WT (n = 16) and Piezo2HoxB8 (n = 14) mice. Data for all experiments were collected from at least three separate cohorts of both male and female littermate wild-type mice. (A and C to H) Bars represent means ± SEM; *P = 0.011 and ***P < 0.0001, n.s., not significant, Mann-Whitney nonparametric analysis.

  • Fig. 3 Mechanical stimuli–induced nociceptor firing is impaired in Piezo2HoxB8 mice.

    (A) Schematic of ex vivo saphenous skin-nerve preparation. (B) Percentage of mechanically sensitive Aβ-, Aδ-, and C-fibers in wild-type and Piezo2HoxB8 mice. (C) Left: Representative example of Aδ-fiber firing activity during 400-mN mechanical stimulation in Piezo2WT and Piezo2HoxB8 mice. Responses to the ramp phase of the stimulus are enlarged and shown on the right. Right: Mean Aδ-fiber firing activity during the 400-mN stimulation in Piezo2WT and Piezo2HoxB8 mice (repeated-measures two-way ANOVA with Bonferroni post hoc analysis, ***P < 0.001). (D) Left: Representative example of C-fiber firing activity during 400-mN mechanical stimulation in Piezo2WT and Piezo2HoxB8 mice. Right: Mean C-fiber firing activity during the 400-mN stimulation in Piezo2WT and Piezo2HoxB8 mice (***P < 0.001, repeated-measures two-way ANOVA with Bonferroni post hoc analysis). (E and F) Spike activity during ramp phase of high-force mechanical stimulation in Piezo2WT and Piezo2HoxB8 Aδ-fibers (E) (repeated-measures two-way ANOVA with Bonferroni post hoc analysis, **P < 0.01 and ***P < 0.001) and in C-fibers (F) (repeated-measures two-way ANOVA with Bonferroni post hoc analysis, ***P < 0.001). Data are represented as means ± SEM. (B to F) n values indicate number of fibers recorded from. Data were collected across multiple mice for each genotype. Piezo2WT, n = 10 mice; Piezo2HoxB8, n = 8 mice.

  • Fig. 4 Capsaicin- and nerve injury–induced mechanical allodynia is mediated by Piezo2.

    (A) Mechanical threshold at baseline (before capsaicin injection) and at 5, 15, and 30 min after capsaicin injection in Piezo2WT (n = 9) and Piezo2HoxB8 (n = 9) mice. (B) Dynamic allodynia score measured in response to a paintbrush stroke on the hindpaw before (baseline) and 5, 15, and 30 min after capsaicin injection in Piezo2WT (n = 9) and Piezo2HoxB8 (n = 9) mice. (C) Mechanical threshold (Piezo2WT, n = 13; Piezo2HoxB8, n = 13), (D) dynamic allodynia (Piezo2WT, n = 17; Piezo2HoxB8, n = 15), and (E) pinprick hyperalgesia (Piezo2WT, n = 7; Piezo2HoxB8, n = 7) assessed in Piezo2WT and Piezo2HoxB8 mice on days 0 (before injury), 7, 14, and 21 after performing SNI. In (B), (D), and (E), higher score (see Materials and Methods for scoring details) indicates nocifensive behavior. (A to E) ****P < 0.0001, two-way ANOVA with Holm-Sidak’s multiple comparison test. (F) Representative images of pinprick-induced c-Fos immunostaining in dorsal horn of spinal cord of Piezo2WT and Piezo2HoxB8 mice on day 21 after SNI, ipsilateral (left) and contralateral (right) to the injury. Scale bars, 50 μm. (G) Quantification of number of c-Fos–positive cells in lamina I of the spinal cord (Piezo2WT ipsilateral versus Piezo2HoxB8 ipsilateral, *P = 0.0202, one-way ANOVA with Tukey’s multiple comparison test; Piezo2WT: ipsilateral versus contralateral, *P = 0.028; Piezo2HoxB8: ipsilateral versus contralateral, *P = 0.026, Mann-Whitney nonparametric analysis).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/462/eaat9897/DC1

    Fig. S1. Genetic strategy to generate Piezo2-ChR2 mice.

    Fig. S2. Mechanically activated currents in nociceptor subtypes of DRG neurons are Piezo2 dependent.

    Fig. S3. Response to mechanosensory stimulation of the eye (the blink reflex) is normal in Piezo2HoxB8 mice.

    Fig. S4. Response to innocuous and noxious stimuli in Piezo2HoxB8 mice is not dependent on the sex of the animal.

    Fig. S5. Aδ- and C-fiber mechanical and thermal thresholds in Piezo2HoxB8 mice.

    Fig. S6. Capsaicin-induced thermal hyperalgesia is unaffected in Piezo2HoxB8 mice.

    Fig. S7. Capsaicin-induced mechanical sensitization in Piezo2WT and Piezo2HoxB8 mice.

    Fig. S8. Capsaicin-induced mechanical allodynia is compromised in Piezo2iAdv mice.

    Fig. S9. SNI-induced mechanical sensitization in Piezo2WT and Piezo2HoxB8 mice.

    Table S1. Source data for Figs. 1, 2, and 4 and figs. S1, S3, S5, S6, and S8.

  • The PDF file includes:

    • Fig. S1. Genetic strategy to generate Piezo2-ChR2 mice.
    • Fig. S2. Mechanically activated currents in nociceptor subtypes of DRG neurons are Piezo2 dependent.
    • Fig. S3. Response to mechanosensory stimulation of the eye (the blink reflex) is normal in Piezo2HoxB8 mice.
    • Fig. S4. Response to innocuous and noxious stimuli in Piezo2HoxB8 mice is not dependent on the sex of the animal.
    • Fig. S5. Aδ- and C-fiber mechanical and thermal thresholds in Piezo2HoxB8 mice.
    • Fig. S6. Capsaicin-induced thermal hyperalgesia is unaffected in Piezo2HoxB8 mice.
    • Fig. S7. Capsaicin-induced mechanical sensitization in Piezo2WT and Piezo2HoxB8 mice.
    • Fig. S8. Capsaicin-induced mechanical allodynia is compromised in Piezo2iAdv mice.
    • Fig. S9. SNI-induced mechanical sensitization in Piezo2WT and Piezo2HoxB8 mice.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Source data for Figs. 1, 2, and 4 and figs. S1, S3, S5, S6, and S8.

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