Sustained Morphine Administration Induces TRPM8-Dependent Cold Hyperalgesia

Abstract

It is not uncommon for patients chronically treated with opioids to exhibit opioid-induced hyperalgesia, and this has been widely reported clinically and experimentally. The molecular substrate for this hyperalgesia is multifaceted, and associated with a complex neural reorganization even in the periphery. For instance, we have recently shown that chronic morphine-induced heat hyperalgesia is associated with an increased expression of GluN2B containing N-methyl-D-aspartate receptors, as well as of the neuronal excitatory amino acid transporter 3/excitatory amino acid carrier 1, in small-diameter primary sensory neurons only. Cold allodynia is also a common complaint of patients chronically treated with opioids, yet its molecular mechanisms remain to be understood. Here we present evidence that the cold sensor TRPM8 channel is involved in opioid-induced hyperalgesia. After 7 days of morphine administration, we observed an upregulation of TRPM8 channels using patch clamp recording on sensory neurons and Western blot analysis on dorsal root ganglia. The selective TRPM8 antagonist RQ-00203078 blocked cold hyperalgesia in morphine-treated rats. Also, TRPM8 knockout mice failed to develop cold hyperalgesia after chronic administration of morphine. Our results show that chronic morphine upregulates TRPM8 channels, which is in contrast with the previous finding that acute morphine triggers TRPM8 internalization.

Perspective: Patients receiving chronic opioid are sensitive to cold. We show in mice and rats that sustained morphine administration induces cold hyperalgesia and an upregulation of TRPM8. Knockout or selectively blocking TRPM8 reduces morphine-induced cold hyperalgesia suggesting TRPM8 is regulated by opioids.

Keywords: 5°C; Cold receptor; cold plate; menthol; paw lift; whole cell recording.

Fig. 1

Sustained morphine administration induces cold hyperalgesia. Rats treated with morphine showed analgesia to the cold stimulus on day 1 (D1), but from day 4 (D4) onwards they started showing increased paw lifts in response to nociceptive cold (i.e. hyperalgesia). This increase lasted until the final testing day (D7). ** p < 0.01, *** p < 0.001. n = 25 for morphine group, and n = 8 for saline group.

Fig. 2

Sustained morphine administration induces an upregulation of TRPM8 receptors in DRGs after 7 days. A. Patch clamp recordings of small diameter DRG neurons (< 30 µm) from whole DRGs reveal that menthol application (100 µM, 1s) induces larger inward currents in the morphine treated group. Upper panel: representative current traces show menthol induced inward currents in small DRG neurons from the saline (Sal) and morphine (Mor) treated group. Lower panel: scatter plot of pooled inward currents. B. Bar graph showing no significant difference in the percentage of menthol responsive neurons between saline and morphine treated groups. C. Western blot analysis revealed an upregulation of TRMP8 channels in DRGs following sustained morphine administration. Upper panel: immunoblots from saline and morphine treated animals. Lower panel: statistical analysis shows that the relative expression of TRPM8 in the morphine group is significantly higher compared with saline group. β-actin serves as an internal control. Numbers in the columns stand for sample numbers. *, p < 0.05, and **, p < 0.01.

Fig. 3

In vivo administration of the TRPM8 selective antagonist RQ-00203078 (3 mg/kg) inhibits paw lifts during the cold plate test in both saline and morphine treated rats. * p < 0.05, ** p < 0.01. n = 8 for each group.

Fig. 4

TRPM8 knock out (KO) mice receiving sustained morphine administration did not develop cold hyperalgesia. In contrast, wild type (WT) mice receiving morphine developed obvious cold hyperalgesia. WT mice receiving saline serving as control did not develop cold hyperalgesia. ** p < 0.01, *** p < 0.001. n = 8 for each group.