Research ArticlePain

PIEZO2 mediates injury-induced tactile pain in mice and humans

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Science Translational Medicine  10 Oct 2018:
Vol. 10, Issue 462, eaat9892
DOI: 10.1126/scitranslmed.aat9892
  • Fig. 1 A viral strategy efficiently generates viable Piezo2 mosaic knockouts.

    (A) Cartoon depicting our strategy for simultaneously knocking out Piezo2 and marking neurons: A viral vector encoding Cre recombinase was introduced to new born (P0-P2) Piezo2cKO (Piezo2fl/fl/GCaMP+/+) mice; controls were GCaMP6f+/+. Mice were tested at > 8 weeks. (B and C) Representative confocal images of dorsal root ganglia from Piezo2cKO. (B) Green fluorescent protein antibody staining to reveal the GCaMP6f-positive cells (green) and the isolectin IB4 (magenta) to mark putative mechanonociceptors. (C) Representative confocal images of multilabel in situ hybridization; arrowheads highlight multilabeled cells. (D) Quantification of marker positive cells expressing GCaMP6f (N = 5 mice). Scale bars, 50 μm (B) and 25 μm (C).

  • Fig. 2 Functional imaging reveals the role of Piezo2 in detecting mechanical stimuli in living mice.

    In vivo functional imaging of the trigeminal ganglion neurons. Top: Typical imaging fields (grayscale) with responding neurons pseudocolored (green, brush; magenta, pinch) to highlight the magnitude of fluorescence changes. (A) Control mice; magnified boxed region: three responding cells, two activated by both stimuli with soma sizes of C-LTMRs (C–fiber low threshold mechanoreceptors) (small diameter) and fast-conducting touch neurons (large diameter), and one neuron that only responded to pinching (small diameter). Bottom: Examples of changes in fluorescence (Δf/F) for five cells that responded to both brushing and pinch. Stimuli were applied with a cotton swab (brush) or forceps (pinch) with integrated load cells (see fig. S3) that recorded the applied stimulus intensity (stimulus). (B) Piezo2cKO mice; as no neurons responded to brushing, bottom panel shows examples of changes in fluorescence (Δf/F) for five pinch-sensitive cells. Scale bars, 100 μm.

  • Fig. 3 Piezo2 is essential for neural responses to gentle touch during chronic inflammation.

    (A) Representative imaging fields from control mice with responding neurons pseudocolored to reflect fluorescence changes (SD projections; top) illustrate the numbers of neurons responding under normal (untreated) and inflamed (CFA) conditions. In the course of an experiment, multiple applications of each stimulus type were recorded, and the maximum-evoked response for every responding cell was identified, each of these is shown as a line in the heat maps (bottom); n = 364 untreated neurons and n = 174 CFA-treated neurons. TG, trigeminal ganglion. (B) Quantification of responses; P = 0.2537, Fisher’s exact test. (C) Representative fields and (D) quantitation of responses of Piezo2cKO neurons; the representation of pinch responses was not significantly altered by CFA treatment (P > 0.9999; N = 3 mice per group). Scale bars, 50 μm (top) and 1 s (bottom).

  • Fig. 4 Piezo2 is required for gentle touch responses before and after neurogenic inflammation by capsaicin.

    (A) Schematic representation of the experimental strategy. (B and C) Heatmaps representing Δf/F for mechanically responsive cells in Piezo2cKO mice (top; n = 176 neurons, N = 3 mice) and controls (bottom; n = 231 neurons, N = 3 mice), before (t0) and after inflammation (post cap). Each line represents the full stimulation session for a given cell; cell responses were sorted by response maxima for the two imaging sessions (see fig. S7 for analysis of response stability and controls). (B) Pinch responses. (C) Brush responses. (D) Quantitation of the proportion of mechanosensitive neurons responding to brush before and after neurogenic inflammation; capsaicin treatment had no effect on the percentage of mechanosensitive neurons responding to brush in either control (P > 0.9999) or Piezo2cKO neurons (P = 0.6212; Fisher’s exact test). WT, wild type.

  • Fig. 5 PIEZO2 is required for the detection of gentle but not noxious mechanical stimuli in human participants.

    Quantitative sensory evaluation of age- and gender-matched volunteers (black circles) and PIEZO2LOF individuals (red circles). (A) Detection thresholds for von Frey filaments applied to glabrous skin (palm). Mean thresholds for PIEZO2LOF individuals (N = 4) were significantly different from controls (N = 7; *P = 0.0061, Mann-Whitney). (B) Pinprick pain thresholds were no different between groups (P = 0.7619, Mann-Whitney); for controls (N = 7), pinprick pain required higher force than von Frey detection (P = 0.0006, Mann-Whitney), but for PIEZO2LOF individuals (N = 3), there was no difference between these thresholds (P = 0.2286, Mann-Whitney). (C and D) Pinch responses were tested using custom forceps and an integrated feedback system until the individual reported feeling pain. (C) Illustrative force traces for a control (black line) and PIEZO2LOF (red line; median ± SD, N = 3 trials). (D) Quantitation of threshold for controls (N = 7) and PIEZO2LOF individuals (N = 4); difference between groups is not significant (P = 0.2571, Mann-Whitney).

  • Fig. 6 PIEZO2 mediates touch-evoked pain in human participants.

    (A) Model for testing touch allodynia after neurogenic inflammation based on application of topical capsaicin; as illustrated, capsaicin cream was applied to a 3 cm by 3 cm square of the forearm for 30 min. The cream was removed, and inflammation was confirmed using laser doppler imaging, illustrated in (B). Participants were then asked to report whether touching the treated area or a nearby unaffected spot was more painful or intense. (C) Quantitation of forced choice testing; the dotted line represents chance. PIEZO2LOF and control individuals had significantly different responses (*P = 0.0048, Mann-Whitney).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/462/eaat9892/DC1

    Materials and Methods

    Fig. S1. In vitro responses of cultured neurons to touch demonstrate the efficiency of the mosaic knockout strategy.

    Fig. S2. Mechanosensory phenotypes in adult PiezocKO mice.

    Fig. S3. Custom-built devices for mechanical stimulation of the mouse cheek.

    Fig. S4. Piezo2 is required for trigeminal responses to vibration but not heat.

    Fig. S5. CFA induces chronic inflammation and sensitization to heat but not pinch.

    Fig. S6. Responses to gentle mechanical stimuli require Piezo2 in the CFA inflammatory pain model.

    Fig. S7. Controls for brush responses after capsaicin-induced inflammation.

    Fig. S8. Responses to gentle mechanical stimuli require Piezo2 in the spared nerve injury neuropathic pain model.

    Fig. S9. Four individuals with loss-of-function mutations in PIEZO2.

    Fig. S10. Psychophysical evaluation of PIEZO2LOF individuals reveals diagnostic mechanosensory deficits.

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. In vitro responses of cultured neurons to touch demonstrate the efficiency of the mosaic knockout strategy.
    • Fig. S2. Mechanosensory phenotypes in adult PiezocKO mice.
    • Fig. S3. Custom-built devices for mechanical stimulation of the mouse cheek.
    • Fig. S4. Piezo2 is required for trigeminal responses to vibration but not heat.
    • Fig. S5. CFA induces chronic inflammation and sensitization to heat but not pinch.
    • Fig. S6. Responses to gentle mechanical stimuli require Piezo2 in the CFA inflammatory pain model.
    • Fig. S7. Controls for brush responses after capsaicin-induced inflammation.
    • Fig. S8. Responses to gentle mechanical stimuli require Piezo2 in the spared nerve injury neuropathic pain model.
    • Fig. S9. Four individuals with loss-of-function mutations in PIEZO2.
    • Fig. S10. Psychophysical evaluation of PIEZO2LOF individuals reveals diagnostic mechanosensory deficits.

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