Research ArticleNeuroscience

Selective inhibition of CaV3.2 channels reverses hyperexcitability of peripheral nociceptors and alleviates postsurgical pain

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Science Signaling  28 Aug 2018:
Vol. 11, Issue 545, eaao4425
DOI: 10.1126/scisignal.aao4425
  • Fig. 1 Effects of plantar skin incision on T-current kinetics and density in rat DRG neurons.

    (A) Families of T-currents evoked in representative DRG neurons from sham and incised rats were used to generate I-V relationships. (B) Average peak T-current densities calculated from I-V curves. Sham group: n = 14 cells, 10 rats. Postincision group: n = 20 cells, 10 rats. Treatment: F(1,32) = 5.52, P = 0.02; interaction: F(9,288) = 6.72; post hoc: *P = 0.017 at −40 mV and ***P < 0.001 at −35 and −30 mV, two-way repeated-measures (RM) analysis of variance (ANOVA) followed by Sidak’s post hoc test. (C) Time-dependent activation (10 to 90% rise time) measured from I-V curves in DRG neurons. Sham group: n = 14 cells, 10 rats. Postincision group: n = 20 cells, 10 rats. Two-way RM ANOVA: P = 0.3. (D) Inactivation time constant measured from I-V curves. Sham group: n = 14 cells, 10 rats. Postincision group: n = 20 cells, 10 rats. Interaction: F(3,96) = 2.36, P = 0.08; treatment: F(1,32) = 4.33, *P = 0.04, two-way RM ANOVA. (E) Families of T-currents evoked in representative DRG neurons by steady-state inactivation protocols in sham (black traces) and incised (red traces) groups. (F) T-current densities from steady-state inactivation curves. Sham: n = 14 cells, 10 rats. Postincision group: n = 21 cells, 10 rats. Interaction: F(10,330) = 6.78, P < 0.0001; post hoc: *P = 0.026 at −110 mV and *P = 0.041 at −105 mV; treatment: F(1,33) = 4.15, P = 0.049, two-way RM ANOVA followed by Sidak’s post hoc test.

  • Fig. 2 T-channels contribute to incision-induced hyperexcitability of sensory neurons.

    (A) Representative traces of APs in a DRG neuron treated with a T-type channel blocker (TTA-P2) from the postincision group (current injection of 50 pA) before and after application of 3 μM TTA-P2. (B) Number of APs after incision before and after application of TTA-P2. n = 8 cells per group from 6 rats; each data point represents mean ± SEM. Interaction: F(5,35) = 6.69, P < 0.001; post hoc: **P = 0.002, ***P < 0.001, ***P < 0.001, and ***P < 0.001 for current injections 20, 30, 40, and 50 pA, respectively; treatment: F(1,7) = 14.72, P = 0.006, two-way RM ANOVA followed by Sidak’s post hoc. (C) Representative traces from single-spiking, multiple-spiking, and high-frequency–spiking DRG neurons. (D) The pie charts show the distribution of different types of firing patterns of DRG neurons in the postincision group and in the sham group. Sham group: n = 28 cells, 8 rats. Postincision group: n = 29 cells, 8 rats. Fisher’s exact test *P = 0.04. (E) Bar graphs represent frequency of AP firing in the sham group (white bar), untreated postincision group (black bar), and postincision group incubated with TTA-P2 for 5 to 10 min (red bar). n = 13 cells from five rats in the sham group, 15 cells from seven rats in the untreated postincision group, and 8 cells from two rats in the postincision + TTA-P2 group. Unpaired t test; *P = 0.02. The data are expressed as means ± SEM. (F) Experimental time course. i.t., intrathecal. (G) Thermal PWL. n = 8 rats per group. Interaction: F(4,56) = 1.16; P = 0.3; treatment: F(1,14) = 17.61, **P = 0.001, two-way RM ANOVA followed by Sidak’s post hoc test. The data are expressed as means ± SEM.

  • Fig. 3 Repeated application of TTA-P2 alleviates thermal and mechanical hypersensitivity after incision.

    (A) Time course showing presurgical baseline measurements to heat and mechanical stimuli determined 2 days before surgery, 2 hours after incision, and on two consecutive days before intrathecal injections. Heat and mechanical hypersensitivity was assessed 30, 60, and 120 min after intrathecal TTA-P2 or vehicle application in a single group of rats. (B) Analysis of the effect of the first dose of TTA-P2 applied 2 hours after plantar skin incision on heat nociception. n = 6 rats in the vehicle group and n = 10 rats in the treatment group. Two-way RM ANOVA. (C and D) Analysis of the effects of consecutive doses of TTA-P2 applied on day 1 (C) and on day 2 after incision (D) on heat nociception. n = 6 rats in vehicle groups and n = 9 rats in treatment groups. Repeated treatment day 1 after incision: **P = 0.003 and *P = 0.026, Mann-Whitney test; repeated treatment day 2 after incision: interaction: F(2,26) = 0.16, P = 0.853; treatment: F(1,13) = 9.75, **P = 0.008, two-way RM ANOVA. (E to G) Time course of the effect of repeated intrathecal application of TTA-P2 on mechanical hypersensitivity at 2 hours and on days 1 and 2 after incision. n = 6 rats in the vehicle group and n = 5 to 6 rats in the postincision group. Repeated treatment 2 hours after incision: F(1,10) = 19.70, **P = 0.001; repeated treatment day 1 after incision: F(1,10) = 8.73, *P = 0.014; repeated treatment day 2 after incision: F(1,9) = 7.08, *P = 0.026, two-way RM ANOVA; no significant interaction. Each data point represents the mean ± SEM for the incised paw.

  • Fig. 4 Membrane expression of CaV3.2 channels in rat DRG neurons is increased after plantar skin incision.

    (A) Levels of mRNAs encoding three isoforms of T-channels (CaV3.1, CaV3.2, and CaV3.3). Postincision group (pooled samples on days 1 and 2 after the surgery): n = 4 samples, each containing pooled L4 to L6 unilaterally harvested DRGs from 7 rats for 28 rats in total. Sham group: n = 4 samples, each containing pooled L4 to L6 DRGs harvested from 4 rats for 16 rats in total. The data were obtained from at least three independent experiments and presented as means ± SEM. Surgery treatment: F(5,66) = 83.04, **P = 0.002, one-way ANOVA. (B) Representative blot for the CaV3.2 isoform of the T-channel in DRG homogenate. Postincision group: n = 4 samples of L4 to L6 DRG tissue pooled from 12 rats. Sham group: n = 4 samples of L4 to L6 DRG tissue pooled from eight rats. (C) Bar graph shows total protein levels of the CaV3.2 isoform of the T-channel in DRG homogenates from the sham and postincision groups. The data were obtained from four samples in the postincision group, where each sample contains L4 to L6 DRGs from three rats, and from four samples in the sham group, where each sample contains L4 to L6 DRGs from two rats. The data are expressed as means ± SEM. No significant difference was found between the groups (Mann-Whitney test). (D) Relative CaV3.2 immunofluorescence normalized to pan-cadherin (CADH) immunofluorescence 48 hours after incision in ipsilateral (ipsi) and contralateral (contra) rat DRG sensory neurons. n = 32 to 33 cells per group, harvested from four rats in four independent experiments. *P = 0.022, unpaired t test. (E) Representative images of ipsilateral and contralateral rat DRG sensory neurons 48 hours after incision: The images show membrane fluorescence of CaV3.2 (green) and pan-cadherin (red) antibodies. 4′,6-Diamidino-2-phenylindole (DAPI; blue color) shows the nuclei of cells. Scale bars, 50 μm.

  • Fig. 5 Selective targeting of deubiquitination of CaV3.2 channels in nociceptors prevents the development of mechanical hypersensitivity in mice after plantar skin incision.

    (A) Representative Western blotting (IB, immunoblotting) of three independent experiments of immunoprecipitated (IP) USP5 from DRG homogenates from ipsilateral and contralateral L4 to L6 DRGs from WT mice after incision and a bar graph of the relative expression levels of USP5 48 hours after incision. n = 3 mice: Tissue homogenates of L4 to L6 DRGs were harvested separately from ipsilateral and contralateral sides of the spinal column, and Western blotting was performed on the tissue from each animal separately. *P = 0.047, unpaired t test. (B) Experimental time course: Day 2 after surgery, WT and CaV3.2 KO mice were injected intrathecally with the tat III-IV peptide, and PWRs were measured 15, 30, 60, and 90 min after injection. (C) PWR thresholds for incised paws in WT mice as measured 15, 30, 60, and 90 min after injection with the tat III-IV peptide. n = 6 animals per group. *P = 0.040, one-way RM ANOVA with Dunnett’s post hoc test. (D) The effect of the injected tat III-IV peptide on PWR thresholds for incised paws in WT mice was normalized to the preinjection baseline [the same data as in (C)]. (E) PWR thresholds for incised paws of the CaV3.2 KO mice after injection with the tat III-IV peptide. n = 6 mice per group. P = 0.754, one-way RM ANOVA. (F) The effect of the injected tat III-IV peptide on PWR thresholds for incised paws in CaV3.2 KO mice was normalized to the preinjection baseline [the same data as in (E)]. (G) The difference between mechanical sensitivity thresholds recorded on day 2 after incision in WT (black bar) and CaV3.2 KO (red bar) mice. n = 6 mice per group; *P = 0.04, unpaired t test. The data are expressed as means ± SEM.

  • Fig. 6 In vivo silencing of the deubiquitinating enzyme USP5 reduces T-current densities and mechanical hyperalgesia in rats after incision.

    (A) T-currents (Vh, −110 mV; Vt, −30 mV) in representative DRG neurons from the sham (black trace), incised untreated (sham; red trace), and incised rats that were pretreated with USP5-shRNA (gray trace) groups. (B) T-current densities in sham rats (black bar), day 2 postincision untreated group (sham; red bar), and day 2 postincision group that received intrathecal injection of USP5-shRNA 24 hours before plantar incision (gray bar). n = 25 cells from 5 rats in the USP5 shRNA group, n = 21 cells from 10 rats in the untreated (sham) incised group, n = 14 cells from 10 rats in the sham group. *P = 0.012 and **P = 0.007, one-way ANOVA followed by Tukey’s post hoc test, respectively. Each data point represents mean ± SEM. (C) Experimental time course: Baseline PWRs were measured during 2 days before surgery. One day before surgery, shRNA-USP5 or vehicle was injected intrathecally in rats. After surgery, baseline paw responses to mechanical stimulus were measured at 2 hours and on days 1 and 2 after incision. (D) Mechanical hypersensitivity to a punctate stimulus after plantar incision in rats with selective knockdown of USP5 and in the vehicle group. n = 7 to 8 rats per group. Two hours after incision: *P = 0.014, 1 day after incision: ***P < 0.001, 2 days after incision: **P = 0.004, two-way RM ANOVA followed by Sidak’s post hoc test. Each data point represents the average thresholds for PWR ± SEM.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/545/eaao4425/DC1

    Fig S1. Development of postsurgical thermal and mechanical hypersensitivity in rats.

    Fig. S2. The lack of effect of plantar skin incision on voltage-dependent activation, voltage-dependent inactivation, and deactivation kinetics of T-currents in rat DRG neurons.

    Fig. S3. Resting membrane potential and input resistance in rat DRG neurons from sham and postincision groups.

    Fig. S4. Antinociceptive effect of intrathecally applied TTA-P2 in unoperated rats.

    Fig. S5. Preemptive intrathecal application of USP5 shRNA reduces mechanical hypersensitivity after incision in WT mice, but not in CaV3.2 KO mice.

  • This PDF file includes:

    • Fig. S1. Development of postsurgical thermal and mechanical hypersensitivity in rats.
    • Fig. S2. The lack of effect of plantar skin incision on voltage-dependent activation, voltage-dependent inactivation, and deactivation kinetics of T-currents in rat DRG neurons.
    • Fig. S3. Resting membrane potential and input resistance in rat DRG neurons from sham and postincision groups.
    • Fig. S4. Antinociceptive effect of intrathecally applied TTA-P2 in unoperated rats.
    • Fig. S5. Preemptive intrathecal application of USP5 shRNA reduces mechanical hypersensitivity after incision in WT mice, but not in CaV3.2 KO mice.

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