Oxidative stress mediates thalidomide-induced pain by targeting peripheral TRPA1 and central TRPV4

Abstract

Background: The mechanism underlying the pain symptoms associated with chemotherapeutic-induced peripheral neuropathy (CIPN) is poorly understood. Transient receptor potential ankyrin 1 (TRPA1), TRP vanilloid 4 (TRPV4), TRPV1, and oxidative stress have been implicated in several rodent models of CIPN-evoked allodynia. Thalidomide causes a painful CIPN in patients via an unknown mechanism. Surprisingly, the pathway responsible for such proalgesic response has not yet been investigated in animal models.

Results: Here, we reveal that a single systemic administration of thalidomide and its derivatives, lenalidomide and pomalidomide, elicits prolonged (~ 35 days) mechanical and cold hypersensitivity in C57BL/6J mouse hind paw. Pharmacological antagonism or genetic deletion studies indicated that both TRPA1 and TRPV4, but not TRPV1, contribute to mechanical allodynia, whereas cold hypersensitivity was entirely due to TRPA1. Thalidomide per se did not stimulate recombinant and constitutive TRPA1 and TRPV4 channels in vitro, which, however, were activated by the oxidative stress byproduct, hydrogen peroxide. Systemic treatment with an antioxidant attenuated mechanical and cold hypersensitivity, and the increase in oxidative stress in hind paw, sciatic nerve, and lumbar spinal cord produced by thalidomide. Notably, central (intrathecal) or peripheral (intraplantar) treatments with channel antagonists or an antioxidant revealed that oxidative stress-dependent activation of peripheral TRPA1 mediates cold allodynia and part of mechanical allodynia. However, oxidative stress-induced activation of central TRPV4 mediated the residual TRPA1-resistant component of mechanical allodynia.

Conclusions: Targeting of peripheral TRPA1 and central TRPV4 may be required to attenuate pain associated with CIPN elicited by thalidomide and related drugs.

Keywords: Chemotherapeutic-induced peripheral neuropathy; Oxidative stress; TRPA1; TRPV4; Thalidomide.

Fig. 1

Thalidomide elicits mechanical and cold, but not heat, hypersensitivity that is dependent on TRPA1 and TRPV4. a–c Dose- and time-dependent mechanical and cold allodynia and heat hypersensitivity following intraperitoneal (i.p.) injection of thalidomide (Thal, 1, 10, 50, and 100 mg/kg) or Veh. d–f Mechanical allodynia at day 7 following Thal (50 mg/kg, i.p.) and Veh and after the administration of HC-030031 (HC03, 100 mg/kg, i.p.), HC-067047 (HC06, 10 mg/kg, i.p.), a combination of HC03 (100 mg/kg, i.p.) and HC06 (10 mg/kg, i.p.), or Veh. g, h Cold allodynia at day 7 following Thal (50 mg/kg, i.p.) or Veh and after the administration of HC03 (100 mg/kg, i.p.), HC-067047 (HC06, 10 mg/kg, i.p.), or Veh. BL, baseline. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Veh Thal or Veh Thal/Veh HC03/Veh HC06; ^§P < 0.05 vs. Thal/Veh HC03/Veh HC06. Two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 2

Genetic deletion of TRPA1 and TRPV4 attenuates mechanical and cold hypersensitivity evoked by thalidomide. a, b Time-dependent mechanical allodynia following intraperitoneal (i.p.) injection of thalidomide (Thal, 50 mg/kg) or Veh in Trpa1^+/+, Trpa1^−/−, Trpv4^+/+, and Trpv4^−/− mice. c, d Mechanical allodynia in Trpa1^+/+ and Trpa1^−/−, Trpv4^+/+ and Trpv4^−/− mice at day 7 following Thal (50 mg/kg, i.p.) or Veh and after the administration of HC-067047 (HC06, 10 mg/kg, i.p.) or Veh in Trpa1^−/− mice and HC-030031 (HC03, 100 mg/kg, i.p.) or Veh in Trpv4^−/− mice. e, f Time-dependent cold allodynia following Thal (50 mg/kg, i.p.) or Veh in Trpa1^+/+, Trpa1^−/−, Trpv4^+/+, or Trpv4^−/− mice. BL, baseline. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Trpa1^+/+/Veh Thal or Trpv4^+/+/Veh Thal or Trpa1^+/+/Veh Thal/Veh HC06 or Trpv4^+/+/Veh Thal/Veh HC03; ^§P < 0.05 vs. Trpa1^+/+/Thal or Trpv4^+/+/Thal or Trpa1^+/+/Thal/Veh HC06 or Trpv4^+/+/Thal/Veh HC03; ^#P < 0.05 vs. Trpa1^−/−/Thal/Veh HC06 or Trpv4^−/−/Thal/Veh HC03. Two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 3

Oxidative stress targets TRPA1 and TRPV4. a Typical traces and pooled data of patch-clamp inward currents elicited by thalidomide (Thal), pomalidomide (Poma) and lenalidomide (Lena) (all, 100 μM), allyl isothiocyanate (AITC, 100 μM), 4α-phorbol 12,13-didecanoate (4α-PDD, 100 μM), capsaicin (CPS, 1 μM), or Veh in mouse dorsal root ganglion (DRG) neurons. Data are mean ± SEM, n = 4–5 cells. *P < 0.05 vs. Veh. One-way ANOVA followed by Bonferroni’s post hoc test. b, c Mechanical and cold allodynia at day 7 following the intraperitoneal (i.p.) injection of thalidomide (Thal, 50 mg/kg) or Veh, and after the administration of phenyl-α-tert-butyl nitrone (PBN, 100 mg/kg, i.p.) or Veh. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Veh Thal/Veh PBN; ^§P < 0.05 vs. Thal/Veh PBN. Two-way ANOVA followed by Bonferroni’s post hoc test. d Pooled data of Ca²⁺ response to AITC (10 μM), 4α-PDD (1 μM), or Veh hTRPA1-HEK293 and hTRPV4-HEK293 cells in the presence of PBN (30 μM). e Concentration response (Ca²⁺ mobilization)-curve to H₂O₂ in cultured hTRPA1- and mTRPA1-HEK293 and hTRPV4- and mTRPV4-HEK293 transfected cells. f Pooled data of Ca²⁺ response to H₂O₂ (500 μM and 5 mM) or Veh in untransfected HEK293 or hTRPA1-HEK293, hTRPV4-HEK293 cells in the presence of HC-030031 (HC03, 30 μM) or HC-067047 (HC06, 10 μM). g Pooled data of the Ca²⁺ response to H₂O₂ (5 mM) in hTRPV4-HEK293 cells in the presence of dithiothreitol (DTT, 10 mM) or 2-mercaptoethanol (β-ME, 50 mM). h Pooled data of the Ca²⁺ response to H₂O₂ (500 μM) in DRG neurons from Trpa1^+/+ and Trpa1^−/− mice and H₂O₂ (5 and 10 mM) in DRG neurons from Trpa1^−/− mice in the presence of HC06 (10 μM). Data are mean ± SEM, n = 20–25 neurons or 80–100 cells. *P < 0.05 vs. Veh; ^§P < 0.05 vs. H₂O₂ (500 μM, 5 mM, or 10 mM). One-way ANOVA followed by Bonferroni’s post hoc test

Fig. 4

Thalidomide increases oxidative stress in the hind paw, sciatic nerve, and spinal cord. a H₂O₂ levels in the hind paw, sciatic nerve, and lumbar (L4–L6) spinal cord at day 7 following intraperitoneal (i.p.) injection of thalidomide (Thal, 50 mg/kg) or Veh and 60 min after the administration of phenyl-α-tert-butyl nitrone (PBN, 100 mg/kg, i.p.) or Veh. b, c Representative images and mean fluorescence intensity of 4-hydroxynonenal (4-HNE) staining in the hind paw, sciatic nerve, and lumbar (L4–L6) spinal cord and TRPA1 and TRPV4 staining in the spinal cord, at day 7 following Thal (50 mg/kg, i.p.) and 60 min after the administration of PBN (100 mg/kg, i.p.) or Veh. Data are mean ± SEM (n = 6 mice). *P < 0.05 vs. Veh Thal/Veh PBN; ^§P < 0.05 vs. Thal/Veh PBN. One-way ANOVA followed by Bonferroni’s post hoc test

Fig. 5

Peripheral TRPA1 and central TRPV4 contribute to thalidomide-induced mechanical allodynia. a, b Mechanical allodynia at day 7 following intraperitoneal (i.p.) thalidomide (Thal, 50 mg/kg) or Veh and after the administration of intraplantar (i.pl., 20 μl) or intrathecal (i.th., 5 μl) HC-030031 (HC03, 100 μg), HC-067047 (HC06, 100 μg), or Veh. c, d Mechanical allodynia at day 7 following Thal (50 mg/kg, i.p.) or Veh and after the administration of a combination of HC03 (100 μg, i.th.) and HC06 (100 μg, i.pl.) or HC06 (100 μg, i.th.) and HC03 (100 μg, i.pl.) or Veh. e Representative images of co-expression of PGP9.5 or S100 and TRPA1 in the peripheral nerve of hind paw. f Representative images of co-expression of CGRP or GFAP and TRPV4 in the lumbar (L4–L6) spinal cord. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Veh Thal/Veh HC03/Veh HC06; ^§P < 0.05 vs. Thal/Veh HC03/Veh HC06. Two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 6

Oxidative stress targeting peripheral TRPA1 and central TRPV4 mediates thalidomide-induced mechanical allodynia. a–c Mechanical allodynia at day 7 following thalidomide (Thal, 50 mg/kg) or Veh and after the administration of i.pl. or a combination of i.pl. and i.th. phenyl-α-tert-butyl nitrone (PBN, 100 μg) or Veh. d Cold allodynia at day 7 following Thal (50 mg/kg, i.p.) or Veh and after the administration of i.pl. PBN (100 μg) or Veh. e, f Mechanical allodynia in Trpv4^+/+, Trpv4^−/−, Trpa1^+/+, and Trpa1^−/− mice at day 7 following Thal (50 mg/kg, i.p.) or Veh and after the administration of i.th. PBN (100 μg) or Veh. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Veh Thal/Veh PBN; ^§P < 0.05 vs. Thal/Veh PBN; ^#P < 0.05 vs. Trpa1^−/−/Thal/Veh PBN. Two-way ANOVA followed by Bonferroni’s post hoc test