Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade

Abstract

Mechanical allodynia, induced by normally innocuous low-threshold mechanical stimulation, represents a cardinal feature of neuropathic pain. Blockade or ablation of high-threshold, small-diameter unmyelinated group C nerve fibers (C-fibers) has limited effects on mechanical allodynia. Although large, myelinated group A fibers, in particular Aβ-fibers, have previously been implicated in mechanical allodynia, an A-fiber-selective pharmacological blocker is still lacking. Here we report a new method for targeted silencing of A-fibers in neuropathic pain. We found that Toll-like receptor 5 (TLR5) is co-expressed with neurofilament-200 in large-diameter A-fiber neurons in the dorsal root ganglion (DRG). Activation of TLR5 with its ligand flagellin results in neuronal entry of the membrane-impermeable lidocaine derivative QX-314, leading to TLR5-dependent blockade of sodium currents, predominantly in A-fiber neurons of mouse DRGs. Intraplantar co-application of flagellin and QX-314 (flagellin/QX-314) dose-dependently suppresses mechanical allodynia after chemotherapy, nerve injury, and diabetic neuropathy, but this blockade is abrogated in Tlr5-deficient mice. In vivo electrophysiology demonstrated that co-application of flagellin/QX-314 selectively suppressed Aβ-fiber conduction in naive and chemotherapy-treated mice. TLR5-mediated Aβ-fiber blockade, but not capsaicin-mediated C-fiber blockade, also reduced chemotherapy-induced ongoing pain without impairing motor function. Finally, flagellin/QX-314 co-application suppressed sodium currents in large-diameter human DRG neurons. Thus, our findings provide a new tool for targeted silencing of Aβ-fibers and neuropathic pain treatment.

Figure 1. TLR5 is co-localized with A-fiber marker NF200 in DRG neurons, skin nerve fibers, and spinal cord axonal terminals in mice

(a) Co-localization of Tlr5 mRNA and NF200-IR in DRG neurons. Yellow, white, and blue arrows show double-labeled neurons, Tlr5 single-labeled neurons, and NF200 single-labeled neurons, respectively. Control (sense probe) shows no signal. Scale, 100 μm. (b) Size distribution frequency of Tlr5 mRNA-positive (Tlr5⁺), NF200-IR (NF200⁺) neurons, and total neurons in DRG. A total of 2238 neurons from 3 mice were counted. (c) Percentage of total DRG neurons expressing Tlr5, NF200, and both Tlr5 and NF200, and percentage of Tlr5⁺ neurons expressing NF200, IB4, CGRP, and TH. A total of 1320 Tlr5⁺ neurons from 3 mice were counted. (d) Co-localization of TLR5-IR and NF200-IR in DRG neurons. Note the TLR5 staining is absent in Tlr5^−/− mice. Scale, 100 μm. (e) Co-localization (shown by arrows) of TLR5 and NF200 in glabrous skin of hindpaw. Scale, 100 μm. (f) Co-localization of TLR5 and NF200 in deep laminae of spinal cord dorsal horn. Boxed region of the image on the left is enlarged in the images to the right. Scale bars, 100 μm.

Figure 2. Co-application of flagellin and QX-314 blocks sodium currents in large-diameter A-fiber neurons of mouse and human DRGs

(a) Traces of transient sodium currents in A-fiber and C-fiber neurons of WT and Tlr5^−/− mice. Transient sodium currents were evoked by a 200 ms voltage step from −70 mV to 0 mV (inset). (b) Time-course of sodium current amplitudes after perfusion with flagellin (0.3 nM) and QX-314 (5 mM) in mouse A-fiber neurons (30–55 μm, n = 20 from 7 WT mice, n = 5 from 5 Tlr5^−/− mice) and C-fiber neurons (10–25 μm, n = 22 from 7 WT mice, n = 5 from 3 Tlr5^−/− mice). *P<0.05, vs. WT C-fiber and Tlr5^−/− mice, two-Way ANOVA. (c) Dose-dependent inhibition of sodium currents by A-fiber blockade in mouse A-fiber neurons (n = 5, 20, and 8 for 0.03, 0.3, and 3 nM, respectively). Note flagellin (n = 6 neurons) or QX-314 (n = 5 neurons) alone has no effects. *P<0.05, vs. QX-314 alone, Two-Way ANOVA. (d) Left panel, traces of action potentials in WT and Tlr5^−/− DRG neurons before, during, and after flagellin (0.3 nM) + QX-314 (5 mM) application. Inset indicates the current injection protocol used for action potential generation. Dashed-lines indicate resting membrane potential. Right panel, action potential amplitudes of WT neurons (n = 17) and Tlr5^−/− neurons (n = 18). *P<0.05, vs. control. (e) Flagellin/QX-314 treatment causes entry of phallodin-rhodamine (10 μM) into large (red arrow) but not small (yellow arrow) neuron. Scale, 30 μm. Upper left, DIC image of a recorded mouse DRG neuron. Subsequent panels show fluorescence images (red filter, 540 nm) of the same neuron upon flagellin/QX-314-mediated entry of phallodin-rhodamine. Insets show action potential blockade by flagellin/QX-314 application. (f, g) Flagellin/QX-314 blocks sodium currents (50 ms step, from −70 mV to 0 mV) in large-diameter A-fiber neurons of human DRGs. (f) Left, DIC image of a recorded human DRG neuron. Scale, 30 μm. Right, traces of transient sodium currents from vehicle control and QX-314 (12 mM)/flagellin (30 ng/ml = 0.9 nM) treated neurons. (g) Time course of flagellin/QX-314-induced inhibition of sodium currents in human DRG neurons with large diameters (55–80 μm, A-fiber) but not small-diameters (< 50 μm, C-fiber). *P<0.05, Two-Way ANOVA. For control (no treatment) groups, n = 6 C-fiber, 7 A-fiber neurons. For QX-314 + flagellin groups, n = 8 C-fiber, 8 A-fiber neurons. Human DRG cultures were prepared from 4 donors. All data were expressed as mean ± s.e.m.

Figure 3. Co-application of flagellin and QX-314 selectively inhibits Aβ-fiber conduction in sciatic nerves of naive and chemotherapy-treated mice

(a) In vivo sciatic nerve recordings in naïve mice show traces of compound potentials evoked by hindpaw stimulation and the effects of 0.3 μg (0.3 μM in 30 μl) flagellin/60 mM QX-314 or 2% lidocaine applied via intraplantar injection of the hindpaw. Schematic beneath the traces indicates the relative timing of Aα, Aβ, and Aδ potentials. Control animals received no treatment, and PBS was used as vehicle. (b) Relative amplitude of Aα, Aβ, and Aδ potentials, expressed as area under curve (AUC), being normalized to control. *P<0.05, vs. vehicle control, n = 5 (blockade) and 6 (vehicle) mice/group. (c) In vivo sciatic nerve recordings of paclitaxel (PAX)-treated mice at 1 week show traces of compound potentials and the effects of 0.3 μg flagellin/60 mM QX-314 or 2% lidocaine. (d) Relative amplitude of Aα, Aβ, and Aδ potentials. *P<0.05, vs. control, n = 8 mice/group. Note a specific inhibition on Aβ-conduction by flagellin/QX-314. All data were expressed as mean ± s.e.m.

Figure 4. A-fiber blockade by co-application of flagellin/QX-314 inhibits mechanical allodynia and ongoing pain in different neuropathic pain conditions

(a) Reversal of paclitaxel (PAX)-induced mechanical allodynia by intraplantar A-fiber blockade (0.1–0.9 μg flagellin (Fla)/6 mM QX-314) but not C-fiber blockade (10 μg capsaicin (Cap)/6 mM QX-314). *P<0.05, vs. vehicle, Two-Way ANOVA. n = 5 mice/group. (b) Intraplantar A-fiber blockade inhibits PAX-induced mechanical allodynia in WT but not Tlr5^−/− mice. *P<0.05, Two-Way ANOVA. n = 5 mice/group. (c) Repeated applications of 0.3 μg flagellin/6 mM QX-314, once a day for 5 days, persistently inhibit PAX-elicited mechanical allodynia, assessed 1 h after each injection. QX-314 (6 mM) was used a vehicle. *P<0.05, Two-Way ANOVA. n = 5 mice/group. (d) Inhibition of ongoing pain by A-fiber (n = 7 mice) but not C-fiber (n = 5 mice) blockade, as revealed by CPP test showing difference score of time a mouse spent in nerve block-paired chamber (test time minus preconditioning time). *P<0.05, two-tailed student’s t-test. (e) Rota-rod test shows normal motor function following intraplantar A-fiber and C-fiber blockade. n = 5 mice/group. (f) Distinct effects of intraplantar A-fiber blockade vs. C-fiber blockade on CCI-induced mechanical allodynia. *P<0.05, Two-Way ANOVA. n = 5 mice/group. In b-d and f, A-fiber blockade was induced by 0.3 μg flagellin/6 mM QX-314 and C-fiber blockade was induced by 10 μg capsaicin/6 mM QX-314. All data were expressed as mean ± s.e.m.