HDAC6 Inhibition Reverses Cisplatin-Induced Mechanical Hypersensitivity via Tonic Delta Opioid Receptor Signaling

Abstract

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6β-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin⁺ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity.SIGNIFICANCE STATEMENT Over one-fourth of cancer survivors suffer from intractable painful chemotherapy-induced peripheral neuropathy (CIPN), which can last for months to years after treatment ends. HDAC6 inhibition is a novel strategy to reverse CIPN without negatively interfering with tumor growth, but the mechanisms responsible for persistent reversal are not well understood. We built on evidence that the endogenous opioid system contributes to the spontaneous, apparent resolution of pain caused by nerve damage or inflammation, referred to as latent sensitization. We show that blocking the δ opioid receptor or its ligand enkephalin unmasks CIPN in mice treated with an HDAC6 inhibitor (latent sensitization). Our work provides insight into the mechanisms by which treatment with an HDAC6 inhibitor apparently reverses CIPN.

Keywords: CIPN; allodynia; latent sensitization; opioid receptors.

Figure 1.

Naltrexone and 6β-naltrexol reinstated cisplatin-induced mechanical hypersensitivity after reversal by HDAC6 inhibitor treatment. A, Timeline of the experimental design and von Frey test. Three days after completion of cisplatin treatment (2.3 mg/kg/d, i.p., 5 d on, 5 d rest, 5 d on), mice were treated with ACY-1083 for 14 consecutive days (10 mg/kg/d, i.p.). Naltrexone (3 mg/kg, s.c.) and 6β-naltrexol (10 mg/kg, s.c.) were injected after completion of ACY-1083. Mechanical hypersensitivity (von Frey testing) was measured at given time points. B, C, Paw withdrawal thresholds from von Frey test in (B) male (interaction: F_(45,179) = 4.915, p < 0.0001) and (C) female mice (interaction: F_(45,180) = 4.576, p < 0.0001). *p < 0.05; **p < 0.01; ***p < 0.001; cisplatin+ACY-1083 versus PBS (two-way ANOVA with Dunnett post hoc test). n = 4 mice per group for both males and females.

Figure 2.

Naloxone methiodide reinstated cisplatin-induced mechanical hypersensitivity after reversal by HDAC6 inhibitor treatment. (A) Timeline of the experimental design and von Frey test. Three days after completion of cisplatin treatment (2.3 mg/kg/day, i.p., 5 days on, 5 days rest, 5 days on), mice were treated with ACY-1083 for 14 consecutive days (10 mg/kg/day, i.p.). The peripherally-restricted opioid receptor antagonist naloxone methiodide (5 mg/kg, s.c.) was injected after completion of ACY-1083. Mechanical hypersensitivity (von Frey testing) was monitored at given time points. (B-C), Paw withdrawal threshold from von Frey test in (B) male (interaction: F_(21,84) = 8.658, p < 0.0001) and (C) female mice (interaction: F_(21,84) = 8.287, p < 0.0001). *p < 0.05; **p < 0.01; cisplatin+ACY-1083 versus PBS (two-way ANOVA with Dunnett post hoc test). n = 4 mice per groups for both males and females.

Figure 3.

Expression of DORs was decreased by cisplatin and reversed by ACY-1083 in DRG. Relative opioid receptor and opioid peptide precursor gene expression in (A) DRG and (B) spinal cord. Oprd1: interaction: F_(1,44) = 12.71, p = 0.0009. *p < 0.05; ***p < 0.001; two-way ANOVA with Tukey post hoc test. n = 12 mice (8 males and 4 females) per group.

Figure 4.

Expression of DORs was decreased by cisplatin and reversed by ACY-1083 in mouse DRG neurons. A, RNAScope was used to detect Oprd1 mRNA abundance in neurons positive for Nefh (encoding NF200 for mechanoreceptors) and P2rx3⁺ (encoding P2X3R for nonpeptidergic nociceptors). Scale bar, 100 μm. B, RNAScope was used to detect Oprd1 mRNA abundance in neurons positive for Calca2 (encoding CGRP for peptidergic nociceptors). Scale bar, 10 μm. C, Percentages of Nefh⁺ DRG neurons also positive for Oprd1. N = 8 sections from 4 male mice per group. D, Oprd1 mRNA abundance in Nefh⁺ DRG neurons. **p < 0.01; ***p < 0.001; Kruskal–Wallis test with Dunn's post hoc test. n = 99 neurons for PBS, 67 neurons for cisplatin, and 100 neurons for cisplatin+ACY-1083 from 4 male mice per group. E, Percentages of P2xr3⁺ DRG neurons also positive for Oprd1. N = 8 sections from 4 male mice per group. F, Oprd1 mRNA abundance in P2rx3⁺ DRG neurons. ***p < 0.001 (Kruskal–Wallis test with Dunn's post hoc test). n = 82 neurons for PBS, 89 neurons for cisplatin, and 96 neurons for cisplatin+ACY-1083 from 4 male mice per group. G, Percentages of Cgrp⁺ neurons also positive for Oprd1. N = 11-14 sections from 4 male mice per group. H, Oprd1 mRNA abundance in Cgrp⁺ DRG neurons. *p < 0.05 (Kruskal–Wallis test with Dunn's post hoc test). n = 74 neurons for PBS, 102 neurons for cisplatin, and 78 neurons for cisplatin+ACY-1083 from 4 male mice per group.

Figure 5.

DOR is required for persistent reversal of cisplatin-induced mechanical hypersensitivity by HDAC6 inhibition. A, Timeline of the experimental design and von Frey test. Three days after completion of cisplatin treatment (2.3 mg/kg/d, i.p., 5 d on, 5 d rest, 5 d on), mice were treated with ACY-1083 for 14 consecutive days (10 mg/kg/d, i.p.). The DOR antagonist naltrindole (3 mg/kg, s.c.); MOR antagonist CTOP (3 mg/kg, s.c.) or CTAP (1 mg/kg, s.c.); KOR antagonist norBNI (10 mg/kg, s.c.) was injected after completion of ACY-1083. Mechanical sensitivity (von Frey testing) was monitored at given time points. B, C, Paw withdrawal threshold from von Frey test in (B) male (interaction: F_(24,96) = 9.656, p < 0.0001) and (C) female mice treated with naltrindole (interaction: F_(24,96) = 11.99, p < 0.0001). *p < 0.05; **p < 0.01; ***p < 0.001; cisplatin+ACY-1083 versus PBS (two-way ANOVA with Dunnett post hoc test). n = 4 mice per group for both males and females. D, E, Paw withdrawal threshold from von Frey test in (D) male and (E) female mice treated with MOR and KOR antagonist. ***p < 0.001, cisplatin+ACY-1083 versus PBS (two-way ANOVA with Tukey post hoc test). n = 4 mice per group for both males and females.

Figure 6.

A, Validation of Oprd1 mRNA deletion from DRG neurons using RNAScope in Avil^Cre::Oprd1^fl/fl mice. Scale bar, 100 μm. B, Validation of Oprd1 mRNA deletion in DRG (t₍₆₎ = 9.788), (C) but not spinal cords of Avil^Cre::Oprd1^fl/fl mice using RT-PCR. ****p < 0.0001 (t test). n = 4 mice (2 males and 2 females) per group. D, Paw withdrawal threshold from von Frey test in Avil^Cre::Oprd1^fl/fl mice and littermate controls treated with cisplatin and ACY-1083 (interaction: F_(3,30) = 17.93, p < 0.0001). ***p < 0.001 (two-way ANOVA with Sidak post hoc test). n = 6 mice (3 males and 3 females) per group.

Figure 7.

Inhibition of enkephalin reinstated cisplatin-induced mechanical hypersensitivity after reversal by HDAC6 inhibitor treatment. A, Timeline of the experimental design and von Frey test. Three days after completion of cisplatin treatment (2.3 mg/kg/d, i.p., 5 d on, 5 d rest, 5 d on), mice were treated with ACY-1083 for 14 consecutive days (10 mg/kg/d, i.p.). Anti-enkephalin antibody (2 μg) or IgG control was intrathecally injected after completion of ACY-1083. Mechanical hypersensitivity (von Frey testing) was monitored at given time points. B, C, Paw withdrawal threshold from von Frey test in (B) male (interaction: F_(30,120) = 10.39, p < 0.0001) and (C) female mice (interaction: F_(30,120) = 13.84, p < 0.0001). **p < 0.01; ***p < 0.001; cisplatin+ACY-1083 versus PBS (two-way ANOVA with Dunnett post hoc test). n = 4 mice per groups for both males and females.

Figure 8.

Endogenous opioid signaling did not mediate prevention of cisplatin-induced mechanical hypersensitivity by HDAC6 inhibition. A, One day after completion of cisplatin treatment (2.3 mg/kg/d, i.p., 5 d on, 5 d rest, 5 d on) Hdac6^−/− and littermate WT mice were treated with naltrindole (3 mg/kg, s.c.) or naltrexone (3 mg/kg, s.c.). Mechanical sensitivity (von Frey testing) was monitored at given time points (interaction: F_(13,156) = 12.71, p < 0.0001). ***p < 0.001 (two-way ANOVA with Sidak post hoc test). N = 7 mice (4 males and 3 females) for each group. B, One day after coadministration of cisplatin (2.3 mg/kg/d, i.p., 5 d on, 5 d rest, 5 d on) and ACY-1083 (10 mg/kg, i.p., 1 h before each cisplatin injection) was complete, naltrexone (3 mg/kg, s.c.) was injected. Mechanical sensitivity (von Frey testing) was monitored at given time points (interaction: F_(6,60) = 5.307, p = 0.0002). ***p < 0.001 (two-way ANOVA with Sidak post hoc test). n = 6 male mice for each group.

Figure 9.

Summary of cellular mechanisms underpinning (A) cisplatin-induced hypersensitivity and (B) persistent reversal of cisplatin-induced hypersensitivity after treatment with an HDAC6 inhibitor. Created with www.BioRender.com.