Immunohistochemistry confirmed that Toll-like receptor 4 (TLR4) receptors were primarily expressed by microglial cells in the lumbar dorsal horn, and that morphine treatment amplified the post fracture/pin (FX) increase in TRL4-positive microglia at 2 weeks after injury. (A) Representative CD11b (green, microglia marker), TLR4 (red), and merged images for each of the treatment conditions are displayed. (B) Average number of TLR4-positive cells per high-power field (HPF) in the dorsal horn spinal cord. Data are expressed as mean values ± SD, N = 8 per cohort. Scale bar = 25 μm. ***P < 0.001 for FX + Vehicle or FX + Morphine versus Sham + Vehicle, ###P < 0.001 for FX + Morphine versus FX + Vehicle.