Figure 7.
The effect of cytokine inhibitors on NMDAR subunit NR1 phosphorylation in the RVM and behavioral hyperalgesia and allodynia at 14 d after CCI-ION. A, B, Double immunostaining shows colocalization of TNFR1 (Ab; red) or IL-1RI (Bb; red) with NR1 (Ac, Bc; green) in RVM neurons. Overlay of b and c reveals double labeling of RVM neurons with TNFR1/NR1 (Ad; yellow-orange) or IL-1RI/NR1 (Bd; yellow-orange). Note colocalization of these receptors with NR1 in the cell membrane. C, Compared with naive rats, the level of pNR1ser896 is increased at 14 d after CCI-ION (p < 0.001; n = 3 per group). D, The increased pNR1ser896 is totally blocked by intra-RVM microinjection of astrocytic inhibitor FC (100 fmol) compared with naive and sham-operated rats (n = 3 per group). E, F, Neutralizing endogenous TNF-α and IL-1β in the RVM using TNFR1/Fc (T/Fc) (50 fmol) (E) and IL-1ra (3 pmol) (F), respectively, blocks CCI-induced increase in pNR1 expression in the RVM at 14 d after CCI-ION compared with sham treatment (p < 0.05; n = 3 per group). No effect of these inhibitors on basal pNR1 expression is observed in sham-operated rats compared with naive rats (n = 3). G, Microinjection of NMDAR channel blocker MK801 (10 pmol) into the RVM abolishes CCI-induced mechanical hyperalgesia and allodynia compared with vehicle (Veh) injection at 14 d after CCI-ION. H, No effect of intra-RVM TNFR1/Fc (50 fmol) and IL-1ra (3 pmol) on basal mechanical threshold is observed in sham-operated rats compared with naive rats. However, these doses of TNFR1/Fc and IL-1ra attenuate CCI-ION-induced hyperalgesia/allodynia for 4–6 h after microinjection compared with vehicle treatment in CCI rats. D–F, **p < 0.01; *p < 0.05 versus naive; G, H, ***, ###p < 0.001; ** , ##p < 0.01; *, #p < 0.05 versus CCI plus vehicle. Scale bars: Aa, Ba, 0.015 mm; Ab–d, Bb–d, 0.005 mm. Error bars indicate SEM.
