Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain

Abstract

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.

Fig. 1.

Inhibition of α,β-methylene ATP evoked mechanical hyperalgesia in the hindpaw of naive rats after a 7 d treatment with P2X₃ receptor ASO, MSO, or saline. Paw withdrawal thresholds were measured before cannulation on day 0 and immediately before and 0.5 and 1 hr after administration of 0.1 μm α,β-methylene ATP. Each pointrepresents the mean ± SEM from three animals per treatment group. *p < 0.05 compared with saline treatment by ANOVA followed by Tukey's HSD test performed on gram threshold data.

Fig. 2.

Molecular analysis of P2X₃ receptor mRNA expression levels in naive rats after a 7 d treatment with P2X₃ receptor ASO, MSO, or saline. A, Representative DRG sections from saline-, ASO-, and MSO-treated animals after nonradioactive RNA in situ hybridization with a P2X₃ probe. Presence of P2X₃ receptor mRNA is indicated by dark-staining cells. P2X₃receptor mRNA levels are visibly reduced after ASO but not saline or MSO treatment. Scale bar, 200 μm. B, Quantification of P2X₃ receptor mRNA expression levels in the soma of DRG neurons visualized by in situ hybridization. P2X₃ receptor mRNA levels are expressed in arbitrary units. Each bar represents the mean ± SEM of 24 sections (4 sections per L4 and L5 DRG taken from 3 animals per treatment group). P2X₃ receptor ASO but not saline or MSO treatment decreases P2X₃ receptor mRNA expression levels. ***p < 0.001 compared with saline treatment by Kruskal–Wallis ANOVA with Dunn's multiple comparisons post-test.

Fig. 3.

P2X₃ protein expression levels in the dorsal horn of lumbar spinal cord in naive rats after a 7 d treatment with P2X₃ receptor ASO, MSO, or saline.A, Representative images of P2X₃ receptor immunoreactivity in dorsal horn sections of lumbar (L4/L5) spinal cord from animals treated with saline, ASO, or MSO. Red bandof P2X₃ immunoreactivity in lamina II is visibly reduced in ASO-treated animals (top panel). Coimmunostaining with CGRP (green) shows that equivalent regions of each spinal cord were analyzed. Scale bar, 200 μm.B, Quantification of P2X₃ protein expression levels in the dorsal horn of the spinal cord. Protein levels are expressed in arbitrary units. Each bar represents the mean ± SEM of 12 sections (4 sections per animal and 3 animals per treatment group). ***p < 0.001 compared with saline treatment by Kruskal–Wallis ANOVA with Dunn's multiple comparisons post-test.

Fig. 4.

Effect of P2X₃ receptor ASO, MSO, or saline treatment on development of mechanical hyperalgesia in a rat model of neuropathic pain. Four groups (n = 6) of animals were cannulated and infused with P2X₃ receptor ASO, MSO, or saline for 7 d. Twenty-four hours after cannulation, the sciatic nerve was partially ligated in three groups, whereas the fourth saline-treated group was maintained as a control for the effects of cannulation. Mechanical hyperalgesia was assessed on ipsilateral (A) and contralateral (B) hindpaws before cannulation and then daily until day 7. Eachpoint represents the mean ± SEM from six animals per treatment group. *p < 0.05 compared with neuropathic saline-treated group by ANOVA followed by Tukey's HSD test performed on gram threshold data.

Fig. 5.

Effect of P2X₃ receptor ASO, MSO, or saline treatment on established mechanical hyperalgesia in a rat model of neuropathic pain. The sciatic nerve was partially ligated in three groups of six animals. On day 13, all three groups were cannulated and treated with P2X₃ receptor ASO, MSO, or saline for 7 d. An addition saline group was set up on the same day as a control for cannulation. Mechanical hyperalgesia was assessed on ipsilateral (A) and contralateral (B) hindpaws before surgery and then daily from days 13–20. Eachpoint represents the mean ± SEM from six animals per treatment group. *p < 0.05 compared with neuropathic saline treatment group by ANOVA followed by Tukey's HSD test performed on gram threshold data.

Fig. 6.

Effect of P2X₃ receptor ASO, MSO, or saline treatment on the development and maintenance of mechanical allodynia in a rat model of neuropathic pain. A, Mechanical allodynia was assessed in the ipsilateral and contralateral (data not shown) hindpaws of animals described in Figure 4 before cannulation and then daily until day 6. Each pointrepresents the mean ± SEM from six animals per treatment group. No significant difference was detected on the development of mechanical allodynia. B, Mechanical allodynia was assessed in the ipsilateral and contralateral (data not shown) hindpaws of animals described in Figure 5 before surgery and then daily from days 13–20. Each point represents the mean ± SEM from six animals per treatment group. No significant difference was detected on the maintenance of mechanical allodynia.

Fig. 7.

Effect of P2X₃ receptor ASO, MSO, or saline treatment on the development of mechanical hyperalgesia in the complete Freund's adjuvant (CFA) model of inflammatory pain. Four groups (n = 6) of animals were cannulated and infused with P2X₃ receptor ASO, MSO, or saline for 7 d. Twenty-four hours after cannulation, three groups received an intraplantar stimulus of CFA into the hindpaw, and the fourth saline group was maintained as a control for cannulation. Mechanical hyperalgesia was assessed on ipsilateral (A) and contralateral (B) hindpaws before cannulation and then daily until day 7. Eachpoint represents the mean ± SEM from six animals per treatment group. *p < 0.05 compared with CFA saline treatment group by ANOVA followed by Tukey's HSD test performed on gram threshold data.

Fig. 8.

Inhibition of α,β-methylene ATP evoked mechanical hyperalgesia in the contralateral hindpaw of neuropathic rats after a 6 or 7 d treatment with P2X₃ receptor ASO, MSO, or saline. On the final experimental day, α,β-methylene ATP was administered by intraplantar injection to the contralateral hindpaws of the animals described in Figure 4. Paw withdrawal thresholds were determined before and 0.5 hr after α,β-methylene ATP administration. Doses of 0.1 or 1.0 μmol were used. In each case the mean ± SEM of the mean paw withdrawal thresholds ofn = 6 per treatment group are shown. In all cases ASO treatment results in a significant inhibition of the development of α,β-methylene ATP-evoked mechanical hyperalgesia. *p < 0.05 compared with saline treatment group by ANOVA followed by Tukey's HSD test performed on gram threshold data. However, the 100% inhibition seen in the ASO-treated group at the 0.1 μmol dose is reduced to 66% relative to predose values at a higher 1.0 μmol dose.

Fig. 9.

A, Inhibition of α,β-methylene ATP evoked mechanical hyperalgesia in the hindpaw of naive rats after a 2 d treatment with P2X₃ receptor ASO, MSO, or saline. Paw withdrawal thresholds were measured before cannulation on day 0 and immediately before and 1 hr after administration of 1 μmol α,β-methylene ATP. Each point represents the mean ± SEM from six animals per treatment group. *p < 0.05 compared with saline treatment by ANOVA followed by Tukey's HSD test performed on gram threshold data.B, P2X₃ receptor protein levels in DRG are reduced in ASO relative to MSO or saline groups after 2 d of treatment in naive rats. L4 and L5 DRG were isolated and pooled from naive rats and evaluated by Western blot analysis. Immunoblots were reprobed for transferrin to control for protein loading. Representative results are shown for three saline-, MSO-, and ASO-treated animals.