Fig 1: Piezo2 knockdown alters the response of Aδ bone afferent neurons to noxious mechanical stimulation. (A,B) Examples of whole-nerve recordings, and rasters of single unit activity, from Piezo2 mismatch (A) and antisense (B) ODN treated animals. (C) There was a significantly reduced total discharge frequency in Aδ bone afferent neurons recorded from Piezo2 antisense treated animals compared to mismatch control animals [mixed model, F (3.278), DFn (2), Dfd (43), *Dunnett’s P < 0.05; n = 23 naïve/31 mismatch/35 antisense, N = 10 naïve/15 mismatch/21 antisense]. The reduction in discharge frequency was observed during both the (D) ramp [F (3.641), DFn (2), Dfd (43), Dunnett’s P < 0.05; n = 23 naïve/31 mismatch/35 antisense, N = 10 naïve/15 mismatch/21 antisense] and the (E) hold phase of the pressure stimulus [F (5.757), DFn (2), Dfd (43), Dunnett’s P < 0.05; n = 23 naïve/31 mismatch/35 antisense, N = 10 naïve/15 mismatch/21 antisense]. (F) There was no difference in the number of Aδ bone afferent neurons isolated per experiment in recordings made from Piezo2 knockdown compared to mismatch control animals (P > 0.05, unpaired t-test, n = 33 mismatch/38 antisense, N = 16 mismatch/21 antisense). (G) There was no difference in the threshold for mechanical activation of Aδ bone afferent neurons in recordings made from Piezo2 knockdown compared to mismatch control animals [mixed model, F (6.139), DFn (2), Dfd (45), Dunnett’s P > 0.05; n = 23 naïve/31 mismatch/35 antisense, N = 10 naïve/15 mismatch/21 antisense].
Fig 2: Piezo2 knockdown prolongs stimulus evoked fatigue. (A) Schematic representation of the repetitive stimulation experimental protocol. The response to repetitive stimulation was measured at 15 and 30 min interstimulus intervals (ISIs) and compared between Piezo2 antisense and mismatch control treated animals. (B,C) There was a significant decrease in the discharge frequency of Aδ bone afferent neurons recorded from animals treated with Piezo2 antisense, relative to those treated with mismatch control, at 15 min (B, *P < 0.05, unpaired t-test, n = 7 mismatch/7 antisense, N = 3 mismatch/4 antisense) but not 30 min (C, P > 0.05, unpaired t-test, n = 6 mismatch/7 antisense, N = 3 mismatch/4 antisense) ISIs. (D,E) There were no differences in the threshold for activation of Aδ bone afferent neurons recorded from animals treated with Piezo2 antisense, relative to those treated with mismatch control, at either 15 min (D, P > 0.05, unpaired t-test, n = 4 mismatch/6 antisense, N = 3 mismatch/4 antisense) or 30 min (E, P > 0.05, unpaired t-test, n = 5 mismatch/7 antisense, N = 3 mismatch/4 antisense) ISIs.
Fig 3: Piezo2 knockdown approach and confirmation of protein knockdown. (A) Piezo2 antisense or mismatch oligodeoxynucleotides (ODNs) were administered by intrathecal injection. (B) Western blot analysis revealed that the intensity of the bands >250 kDa, and at 80 kDa, are significantly reduced by administration of Piezo2 antisense, relative to mismatch ODNs. Pre-adsorption of the Piezo2 antibody with the manufacturer’s peptide (Piezo2 antibody blocking peptide, Novus Biologicals, #NBP1-78624PEP) at 5 μg/ml completely abolishes Piezo2 protein bands at >250 kDa, and at 80 kDa, in rat DRG. (C) Densitometry analysis revealed a significant reduction in the ratio of Piezo2/β-Actin in the DRG of animals injected with Piezo2 antisense ODNs (n = 6) relative to mismatch control ODNs (n = 6). Data represents mean ± SEM, *P < 0.05, unpaired t-test.
Fig 4: Piezo2 knockdown prevents NGF-induced sensitization of Aδ bone afferent neurons. (A) Retrograde labeling of bone afferent neurons (FB, blue, arrowheads) revealed that a substantial proportion of neurons that innervate bone express Piezo2 (red, hashes) and the NGF receptor TrkA (green, asterisks). Scale bars = 100 μm. (B) Frequency histogram showing that the majority of medium-sized bone afferent neurons expressed Piezo2, and that most Piezo2 expressing bone afferent neurons also expressed TrkA. (C) A significantly lower proportion of Aδ bone afferent neurons were sensitized (increased discharge frequencies) by NGF in Piezo2 antisense treated animals compared to mismatch control animals [chi-square test, X2 (1, N = 32) = 10.86,*P < 0.05, n = 15 mismatch/17 antisense, N = 8 mismatch/9 antisense]. (D) A significantly lower proportion of Aδ bone afferent neurons were sensitized (lower thresholds for activation) by NGF in Piezo2 antisense treated animals compared to mismatch control animals (chi-square test, X2 (1, N = 32) = 7.069,*P < 0.05, n = 15 mismatch/17 antisense, N = 8 mismatch/9 antisense).
Fig 5: Successful knockdown of Piezo 2 in L6-S1 DRG neurons with antisense-ODNs. (A) The schedule for the experiments. Prior to CYP injection, Piezo2 mismatch ODNs or antisense ODNs were intrathecally injected once daily for three consecutive days and then every other day thereafter before DRG collection and behavior study. (B-C) FISH staining revealed that intrathecally injection of Piezo2 antisense ODNs significantly reduced Piezo2 mRNA expression in DRG neurons relative to mismatch ODNs. Summarized data from 4 rats in each group were shown in (C). (D-E) Western blot analysis revealed that the intensity of the band at 80 kDa was significantly reduced by Piezo2 antisense ODNs relative to mismatch ODNs (D, left). Pre-adsorption of the Piezo2 antibody with the manufacturer’s peptide (Piezo2 antibody blocking peptide, Novus Biologicals, #NBP1-78624PEP) completely abolished Piezo2 protein bands at 80 kDa (D, right). Summary data (E) revealed a significant reduction in the ratio of Piezo2/β-Actin in L6-S1 DRGs of rats injected with Piezo2 antisense ODNs (n = 4 rats) relative to mismatch ODNs (n = 4 rats). Data are mean ± SEM, ***P < 0.001 (unpaired t-test)
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