Immunolabeling of phosphorylated NR1 (pNR1), mGluR5, and GFAP in the spinal dorsal horn at day 7. Representative images are shown for (a-d) pNR1 (red) and (e-h) mGluR5 (red) and GFAP (green) after injury, sham, and injury with immediate or day 4 bupivacaine treatment. (i) The percentage of pNR1- and mGluR5-positive pixels normalized to sham levels increases in the dorsal horn after injury with immediate vehicle treatment (inj-VEH0h) (*p 0.046). Immediate bupivacaine treatment (inj-BP0h) prevents those increases in both mGluR5 and pNR1 (#p<0.0001), although GFAP remains increased (^p<0.0001). (j) The normalized percentages of pNR1-, mGluR5-, and GFAP-positive pixels increase after injury with day 4 vehicle treatment, relative to sham levels (*p≤0.003). However, bupivacaine administered 4 days after injury (inj-BPd4) does not prevent the increases in pNR1, mGluR5, and GFAP (^p≤0.024). Scale bar=100μm.