Opioids enhance CXCL1 expression and function after incision in mice

Abstract

Chronic opioid consumption increases postoperative pain. Epigenetic changes related to chronic opioid use and surgical incision may be partially responsible for this enhancement. The CXCL1/CXCR2 signaling pathway, implicated in several pain models, is known to be epigenetically regulated via histone acetylation. The current study was designed to investigate the role of CXCL1/CXCR2 signaling in opioid-enhanced incisional sensitization and to elucidate the possible epigenetic mechanism underlying CXCL1/CXCR2 pathway-mediated regulation of nociceptive sensitization in mice. Chronic morphine treatment generated mechanical and thermal nociceptive sensitization and also significantly exacerbated incision-induced mechanical allodynia. Peripheral but not central messenger RNA levels of CXCL1 and CXCR2 were increased after incision. The source of peripheral CXCL1 appeared to be wound area neutrophils. Histone H3 subunit acetylated at the lysine 9 position (AcH3K9) was increased in infiltrating dermal neutrophils after incision and was further increased in mice with chronic morphine treatment. The association of AcH3K9 with the promoter region of CXCL1 was enhanced in mice after chronic morphine treatment. The increase in CXCL1 near wounds caused by chronic morphine pretreatment was mimicked by pharmacologic inhibition of histone deacetylation. Finally, local injection of CXCL1 induced mechanical sensitivity in naive mice, whereas blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision.

Perspective: Peripheral CXCL1/CXCR2 signaling helps to control nociceptive sensitization after incision, and epigenetic regulation of CXCL1 expression explains in part opioid-enhanced incisional allodynia in mice. These results suggest that targeting CXCL1/CXCR2 signaling may be useful in treating nociceptive sensitization, particularly for postoperative pain in chronic opioid-consuming patients.

Keywords: Chemokine; histone acetylation; opioid-induced hyperalgesia; postoperative pain; skin incision.

Figure 1

Assessment of mechanical and thermal sensitivity after chronic morphine treatment and/or hand paw incision. Chronic morphine treatment causes mechanical allodynia (A) and thermal sensitization (B). Chronic morphine treatment enhanced incision-induced mechanical allodynia (C), without alteration of thermal sensitivity (D). Animals received prior subcutaneous injection of escalating morphine or vehicle (saline) for 4 days. Incision and nociceptive testing procedures began approximately 18 hours after the final dose of morphine or saline. Incisions were made after measuring nociceptive thresholds at day 0. Values are displayed as the mean ± SEM. N=6. *p<0.05, **p<0.01 or *** p<0.001. Veh=vehicle; Mor=morphine; INC= incision.

Figure 2

Changes in CXCL1 and CXCR2 mRNA expression in skin and spinal cord tissue after incision and/or chronic morphine treatment. (A) The mRNA level of CXCL1 was significantly increased in skin after incision and further increased with chronic morphine treatment. (B) The mRNA level of CXCR2 was significantly increased after incision. Neither incision nor morphine treatment altered the mRNA expression of CXCLl (C) and CXCR2 (D) in spinal cord tissue. Values are displayed as the mean ± SEM. N=5. # p<0.05, ## p<0.01, ### p<0.001 vs. day 0 (before incision); * p<0.05, ** p<0.01 vs. vehicle treated group. Veh=vehicle; Mor=morphine; INC= incision.

Figure 3

Expression of CXCL1 in skin tissue at 1 day after incision. (A) Expression of CXCL1 was increased after incision and further increased in mice treated with morphine in the dermal layer. The epidermal layer was labeled by two dotted lines. Scale bar: 100 μm; (B) Double immunostaining showed that most CXCL1 (green) and neurtrophils (red) were co-localized (arrows) in the dermal layer at 1 day after incision in mice pretreated with morphine. Scale bar: 20 μm. Veh=vehicle; Mor=morphine; INC= incision.

Figure 4

Levels of acetylated histone H3 at lysine 9 (AcH3K9) in skin tissue at 1 day after incision. (A) Levels of acetylated H3K9 were unchanged in the epidermal layer after incision in both vehicle and morphine pretreated groups. Scale bar: 50 μm. (B) Expression of AcH3K9 was increased after incision and further increased in mice treated with morphine in the dermal layer. Scale bar: 50 μm. (C) Double immunostaining showed CXCL1 (green) and AcH3K9 (red) were colocolized (arrows) in the dermal layer at 1 day after incision in mice treated with morphine. Scale bar: 20 μm. (D) Quantification of AcH3K9 positive cells in the epidermis. (E) Quantification of AcH3K9 positive cells in the dermis. (D) Quantification of both CXCL1 and AcH3K9 positive cells in the dermis. Values are displayed as the mean ± SEM, n = 4, ### p<0.001 vs. day 0 (before incision); *** p<0.001 vs. vehicle treated group. Veh=vehicle; Mor=morphine; INC= incision.

Figure 5

Chromatin immunoprecipitation (ChIP) analysis of CXCL1 in skin tissue at day 1 after incision. Histone acetylation near the CXCL1 promoter in skin significantly increased at day 1 after incision in mice treated with morphine compared with mice treated with vehicle. Values are displayed as the mean ± SEM, n = 4, # p<0.05 vs. day 0 (before incision); ** p<0.01 vs. vehicle treated group. Veh=vehicle; Mor=morphine; INC= incision.

Figure 6

Effects of morphine or SAHA treatment on CXCL1 expression in skin tissues after incision. The protein level of CXCL1 was monitored by ELISA and increased after incision and further increased in mice with chronic morphine or SAHA treatment. Values are displayed as the mean ± SEM, n = 5, * p<0.05, ** p<0.01 vs. vehicle treated group. Veh=vehicle; Mor=morphine; INC= incision; SAHA = suberoylanilide hydroxamic acid.

Figure 7

Assessment of peripheral CXCL1/CXCR2 signaling on nociceptive sensitization after incision. Intraplantar injection of recombinant CXCL1 induced mechanical allodynia (A), without effects on thermal sensitivity (B). Intraplantar injection of the CXCR2 antagonist SB225002 at 1 day after incision significantly reversed incision-induced mechanical hypersensitivity (C), also without effects on thermal sensitivity (D). Values are displayed as the mean ± SEM, n = 6, *p<0.05, **p<0.01 or *** p<0.001 vs. vehicle treated group. Veh=vehicle; INC= incision.