CXCR4 signaling mediates morphine-induced tactile hyperalgesia

Abstract

Morphine and related compounds are the first line of therapy in the treatment of moderate to severe pain. Over time, individuals taking opioids can develop an increasing sensitivity to noxious stimuli, even evolving into a painful response to previously non-noxious stimuli (opioid-induced hyperalgesia; OIH). The mechanism underlying OIH is not well understood although complex intracellular neural mechanisms, including opioid receptor desensitization and down-regulation, are believed to be major mechanisms underlying OIH. However, OIH may also be associated with changes in gene expression. A growing body of evidence suggests that cellular exposure to mu agonists upregulate chemokines/receptors and recent work from our laboratory implicates chemokine upregulation in a variety of neuropathic pain behaviors. Here we characterized the degree to which chemokines/receptors signaling is increased in primary afferent neurons of the dorsal root ganglion (DRG) following chronic morphine sulfate treatment and correlated these changes with tactile hyperalgesic behavior in rodents. We demonstrate that mRNA expression of the chemokine, stromal-derived factor-1 (SDF1/CXCL12) is upregulated following morphine treatment in sensory neurons of the rat. The release of SDF1 was found to be constitutive when compared with the activity dependent release of the C-C chemokine, monocyte chemoattractant protein-1 (MCP1/CCL2) in a line of F11 neuroblastoma-sensory neuron hybrid cells. We further determined that there is pronounced CXCR4 expression in satellite glial cells and following morphine treatment, increased functional CXCR4 expression in sensory neurons of the DRG. Moreover, intraperitoneal administration of the specific CXCR4 antagonist, AMD3100, completely reversed OIH in the rat. Taken together; the data suggest that opioid-induced SDF1/CXCR4 signaling is central to the development of long lasting OIH and that receptor antagonists represent a promising novel approach to the management of the side effects associated with the use of opioids for chronic pain management.

Figure 1

Repeated morphine treatment paradigm. Animals underwent baseline testing for 2–3 days prior to the start of injections. Rats received once daily i.p. injections of morphine (10 mg/kg) for 5 days. Behavioral testing that occurred during the 5 days of injections (ID) were carried out 24 hours after the last morphine injection. Following the injection period, behavior was conducted on animals for up to 28 days following the last morphine injection (PID). Behavioral testing with the CXCR4 antagonist, AMD3100, occurred five days after the last morphine injection (PID5).

Figure 2

Repeated morphine treatment (10 mg/kg for 5 days) results in the development of tactile hyperalgesia as measured by von Frey filaments. Tactile hyperalgesic behavior persists for at least 28 days following the last morphine injection. ID, injection day, PID, post injection day (One-way ANOVA; *p<0.05, significant difference from baseline).

Figure 3

Repeated morphine injections reduces CXCR4-immunoreactivity (-IR) in satellite glial cells of rat lumbar DRG sections. Animals received repeated morphine injections (10 mg/kg for 5 days) and tissue was collected at 5 days (PID5) and 21 days (PID21) after the last morphine injection. A) CXCR4-IR (red) is largely restricted to satellite glial cells in the naïve rodent DRG. B) Following repeated morphine treatment, CXCR4-IR is reduced at PID5. C) By PID21 CXCR4-IR begins to return to naïve levels. Scale bar is 100 μm (n = 7 for day 5 and n = 7 for each day 21).

Figure 4

SDF1 mRNA expression is increased in the lumbar DRG following repeated morphine exposure. In situ hybridization was used to assess the expression pattern of SDF1 mRNA. A) High power photomicrograph of basal expression of SDF1 mRNA was observed in the lumbar DRG from saline injected rats in non-neuronal cells (black arrowhead indicates SDF1 mRNA transcripts in non-neuronal cell). Teal arrows indicate a lack of neuronal SDF1 mRNA transcripts. After a repeated morphine exposure, the level of SDF1 mRNA expression increased by post-injection day (PID) -5. SDF1 mRNA expression appears in both neuronal and non-neuronal cells. (Black arrows indicate neurons positive for SDF1 mRNA transcripts; arrowheads, presumptive glial cells positive for SDF1 mRNA transcripts). Scale bar A and B is 50 μm (n = 5 for each condition).

Figure 5

Chemokine specific release from transfected F11 cells is by regulated (MCP1) or constitutive release mechanisms (SDF1). F11 DRG neurons were differentiated and then depolarized by high K stimulation (50 mM; 50K), either with or without extracellular Ca (2 mM or 0 mM; 2Ca or 0CA). The amount of MCP1-RFP released into the culture medium was measured by sandwich ELISA. The release of SDF1-mRFP1 was examined in the same manner as MCP1-mRFP1. Baseline levels of MCP1-RFP (A) or SDF1-RFP (B) in F11 cells prior to addition of 50mM K were 6.3 ± 0.7 or 10 ± 1.1% of total media, respectively (A sandwich ELISA; *p<0.01 vs. any other group, Newman-Keuls Multiple Comparison Test).

Figure 6

The CXCR4 antagonist AMD3100 reverses loss of CXCR4 immunoreactivity in DRG derived from repeated morphine treated rats. Untreated rats (A), naive animal administered AMD3100 1 hour before sacrifice (B), repeated morphine treatment alone (i.p., 10 mg/kg, once daily for 5 days) (C) repeated morphine treatment in combination with different doses of AMD3100 1 hour before sacrifice (D, E) and 3 hours before sacrifice (F). Treatment with AMD3100 reverses repeated morphine treatment-induced loss of CXCR4 immunoreactivity at PID5 (C) in a dose-dependent manner (D, E). CXCR4-immunoreactivity (red label) in rat DRG sections following 10 mg/kg (E), but not 1 mg/kg (D) dose of AMD3100 returns CXCR4 immunoreactivity to levels observed in untreated control rats (A). White arrows indicate the presence of CXCR4-immunopositive neurons following AMD3100 treatment. Scale bar is 100 μm.

Figure 7

Morphine-induced tactile hyperalgesia in rodents is transiently reversed with CXCR4 antagonist, AMD3100 treatment. Five days following the last morphine injection (PID5) rats received an i.p. injection of AMD3100 (10 mg/kg) and tactile behavior was measured by von Frey filaments 1 and 3 hours post injection. AMD3100 treatment completely reversed morphine-induced tactile hyperalgesia by one hour post injection. Tactile hyperalgesia returned 3 hours following injection of AMD3100. *Significant difference from baseline; one-way ANOVA; p<0.01).