Article Figures & Data
Figures
- Figure 1.
EGFR expression in the spinal cord and DRG. A, EGFR expression (green) was concentrated in the SG of the spinal cord and colocalized with CGRP, IB4, OX42, and NF200. 20× objective. Scale bar = 100 μm. B, Higher magnification images of the boxed regions in panel A using a 60× objective lens demonstrated that EGFR co-localized with unmyelinated peptidergic (CGRP) and unmyelinated non-peptidergic (IB4) primary sensory afferent terminals in the SG. EGFR also co-localized with myelinated (NF200, arrowheads) primary sensory afferent terminals in in deeper layers of the dorsal horn of the spinal cord. The EGFR did not co-localize with neuronal cell bodies (NeuN) or astrocytes (GFAP) in the SG. Sparse co-localization within microglial cell bodies (OX42, arrowheads) was also observed in the SG. 60× objective. Scale bar = 50 μm. C, EGFR co-localized with unmyelinated non-peptidergic (IB4), unmyelinated peptidergic (CGRP) and myelinated (NF200) sensory primary afferent neuronal cell bodies in the DRG. The EGFR did not co-localize with GFAP-expressing satellite cells; 40× objective. Scale bar = 20 μm. Nikon A1 confocal microscope.
- Figure 2.
EGFR activation is both necessary and sufficient to cause morphine tolerance. A, Rats received daily intrathecal injections of either 455 ng morphine, 10 μg gefitinib, MS + gefitinib or vehicle for 4 d. On day 5, all animals received morphine alone; N = 6 rats per group, two-way ANOVA, interaction: F(15,100) = 20.92, p < 0.0001, days: F(5,100) = 47.51, p < 0.0001, treatment: F(3,20) = 68.17, p < 0.0001. B, Rats received daily subcutaneous injections of either 3.5 mg/kg morphine, 5 mg/kg gefitinib, MS + gefitinib or vehicle for 5 d. On day 6, all animals received MS alone; N = 5–6 rats per group, two-way ANOVA, interaction: F(15,95) = 14.75, p < 0.0001; days: F(5,95) = 21.87, p < 0.0001; treatment: F(3,19) = 166.4, p < 0.0001. C, Animals received daily intrathecal injections of either 455 ng MS, 63 ng EGF, or vehicle for 4 d. On day 5, all animals received MS alone; N = 6 rats per group, two-way ANOVA, interaction: F(10,75) = 19.45, p < 0.0001; days: F(5,75) = 20.43, p < 0.0001; treatment: F(2,15) = 12.01, p < 0.0001.
- Figure 3.
Inhibition of EGF signaling restores morphine analgesic effect against mechanical allodynia. A, Following a two-week recovery after SNL or sham surgery, rats received daily intrathecal injections of either 1.5 μg morphine, 10 μg gefitinib, or MS + gefitinib. Mechanical sensitivity was tested after daily injections using the von Frey method. Day 0: baseline after two-week recovery; N = 6 rats per group, two-way ANOVA, interaction: F(18,108) = 23.36, p < 0.0001; days: F(6,108) = 75.96, p < 0.0001; treatment: F(3,18) = 429.9, p < 0.001. B, Following a two-week recovery after SNL, animals received daily intrathecal injections of either 1.5 μg morphine, 200 ng EGFR-Fc (EGF scavenging molecule), MS + EGFR-Fc, or vehicle. Mechanical sensitivity was tested daily. Day 0: postsurgical baseline; N = 6 rats per group, two-way ANOVA, interaction: F(12,80) = 10.82, p < 0.0001; days: F(4,80) = 17.92, p < 0.0001; treatment: F(3,20) = 104.2, p < 0.0001.
- Figure 4.
EGFR and PDGFR-β signaling interact in the production of mechanical allodynia. A, Rats received daily intrathecal injections of either 63 ng EGF, 10 μg imatinib (PDGFR inhibitor), EGF + imatinib, or vehicle. Mechanical sensitivity was tested daily using the von Frey method; N = 6 rats per group, two-way ANOVA, interaction: F(12,80) = 4.455, p < 0.0001; days: F(4,80) = 13.21, p < 0.0001; treatment: F(3,20) = 43.01, p < 0.0001. B, Rats received daily intrathecal injections of either 63 ng EGF, 10 μg imatinib, EGF + imatinib, or vehicle. Thermal sensitivity was tested daily using the Hargreaves method; N = 6 rats per group, two-way ANOVA, interaction: F(12,80) = 1.112, p = 0.3625, days: F(4,80) = 2.745, p = 0.0340; treatments: F(3,20) = 0.141, p = 0.9342. C, Rats received daily intrathecal injections of either 250 ng PDGF-BB, 10 μg gefitinib (EGFR inhibitor), PDGF-BB + gefitinib, or vehicle. Mechanical sensitivity was tested daily; N = 6 rats per group, two-way ANOVA, interaction: F(12,80) = 6.72, p < 0.0001; days: F(4,80) = 11.12, p < 0.0001; treatment: F(3,20) = 33.73, p < 0.0001. D, Rats received daily intrathecal injections of either 250 ng PDGF-BB, 10 μg gefitinib, PDGF-BB+ gefitinib, or vehicle. Thermal sensitivity was tested daily; N = 4–5 rats per group, two-way ANOVA, interaction: F(12,52) = 1.324, p = 0.2338, days: F(4,52) = 4.353, p < 0.01; treatment: F(3,13) = 0.7291.
- Figure 5.
Graphic summary of findings. Pathway 1 shows that morphine administration causes tolerance by activation of the EGFR. We have shown that morphine administration causes tolerance by activating the EGFR. EGFR (or PDGFR) inhibition eliminates or reverses analgesic tolerance, restoring or maintaining the full analgesic effect of morphine. Pathway 2 details the effects of EGF administration on tolerance and mechanical sensitivity. Giving EGF (or PDGF) to animals chronically induces mechanical allodynia and reduces the analgesic effectiveness of a subsequent dose of morphine, in effect causing a pre-tolerant state in the absence of prior opioid exposure. Both of these phenomena can be blocked or reversed by EGFR or PDGFR inhibition. Pathway 3 shows that nerve injury induces release of EGF and PDGF, which subsequently activates the EGFR and PDGFR and induces mechanical allodynia. However, EGFR or PDGFR inhibition does not reverse the allodynia caused by nerve injury. This is likely due to the concomitant activation of a variety of other mediators of allodynia by nerve injury. Nerve injury also induces a pre-tolerant state that can be eliminated or reversed by EGFR or PDGFR inhibition, suggesting that pre-tolerance is selectively mediated by EGFR and/or PDGFR signaling. In sum, we can conclude that EGFR and PDGFR signaling mediate morphine analgesic tolerance and can induce a pre-tolerant state in opioid-naive animals. While EGFR and PDGFR signaling can induce mechanical allodynia, allodynia induced by nerve injury appears to involve a more complex set of mediators. However, EGFR or PDGFR inhibition still selectively reverse the decrease in morphine analgesia induced by nerve injury (pre-tolerance), suggesting that using these inhibitors clinically could permit the sustained treatment of nerve injury pain using far lower doses of opioids for extended periods of time.
Tables
Data structure Type of test Comparison 95% confidence interval Figure 2A Non-normally distributed Two-way ANOVA Dunett's multiple comparison test Morphine vs vehicle 0.864 to 1.719 Morphine vs gefitinib 0.7783 to 2.011 Morphine vs morphine + gefitinib –2.349 to –1.117 Figure 2B Non-normally distributed Two-way ANOVA Dunett's multiple comparison test Morphine vs vehicle 0.8614 to 1.635 Morphine vs gefitinib 0.7111 to 1.484 Morphine vs morphine + gefitinib –2.034 to –1.223 Figure 2C Non-normally distributed Two-way ANOVA Dunett's multiple comparison test Morphine vs vehicle 0.1181 to 1.828 Morphine vs EGF 0.8573 to 2.567 Figure 3A Non-normally distributed Two-way ANOVA Dunett's multiple comparison test Morphine vs sham –11.35 to –9.522 Morphine vs gefitinib –0.9836 to 0.9314 Morphine vs morphine + gefitinib –8.873 to –6.958 Figure 3B Normally distributed Two-way ANOVA Dunett's multiple comparison test Morphine vs vehicle –1.497 to 1.603 Morphine vs EGF-FC –1.904 to 1.196 Morphine vs morphine + EGF-FC –10.45 to –7.348 Figure 4A Normally distributed Two-way ANOVA Dunett's multiple comparison test Vehicle vs imatinib –1.891 to 2.498 Vehicle vs EGF 6.313 to 10.07 Vehicle vs EGF+ imatinib –6913 to 3.697 Figure 4B Normally distributed Two-way ANOVA Dunett's multiple comparison test Vehicle vs imatinib –1.515 to 1.977 Vehicle vs EGF –1.367 to 2.126 Vehicle vs EGF+ imatinib –1.732 to 1.761 Figure 4C Normally distributed Two-way ANOVA Dunett's multiple comparison test Vehicle vs gefitinib –1.408 to 2.104 Vehicle vs PDGF 4.401 to 7.913 Vehicle vs PDGF+ gefitinib –0.1661 to 3.346 Figure 4D Normally distributed Two-way ANOVA Dunett's multiple comparison test Vehicle vs gefitinib –0.921 to 1.743 Vehicle vs PDGF –0.9921 to 1.536 Vehicle vs PDGF+ gefitinib –0.606 to 2.058
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