Fig. 1.
Intraplantar complete Freund’s adjuvant (CFA) injection induces hyperalgesia and bromodomain-containing protein 4 (Brd4) expression in dorsal root ganglia (DRG) neurons. (A) Representative Western blot and statistical analyses (normalized to glyceraldehyde 3-phosphate dehydrogenase; GAPDH) demonstrating intraplantar CFA injection increased Brd4 expression in DRG. IB = Immunoblotting. **P < 0.01 versus saline; ##P < 0.01 versus naive; n = 6. (B) CFA decreased the paw withdrawal latency (PWL) measured after injection. BL = baseline. **P < 0.01 versus saline; ##P < 0.01 versus BL; n = 7. (C) In DRG, CFA (CFA 1d) increased the immunofluorescence of Brd4 (red, II), which colocalized with a neuronal marker (NeuN; green, III), a marker of A-fiber afferents and medium-large myelinated neurons (NF-200; green, VI), a marker of small peptidergic C-nociceptors called calcitonin gene-related peptide (CGRP; green, VII), and a marker of small nonpeptidergic C-nociceptors (IB4; green, VIII), but not a marker of satellite cells called glial fibrillary acidic protein (GFAP; green, IV) or a marker of macrophages called Iba-1 (green, V) at day 1 after injection. Scale bar = 50 μm. Thickness = 16 μm. **P < 0.01 versus saline 1d; n = 7. (D) Brd4-specific small interfering RNA (siRNA; Brd4 RNAi + CFA 1d, 10 μl, 5 μg) significantly increase the PWL of the CFA-treated group. MS RNAi = missense siRNA. **P < 0.01 versus CFA 1d; n = 7. (E) Representative Western blot and statistical analysis (normalized to GAPDH) demonstrating that Brd4-specific siRNA (Brd4 RNAi + CFA 1d, 10 μl, 5 μg) attenuated Brd4 expression in DRG of CFA-treated rats. **P < 0.01 versus CFA 1d; n = 7. (F) Injection with JQ1 (CFA 1d + JQ1; 10 μl; 10, 30, and 100 μM) dose-dependently attenuated the behavioral hyperalgesia in the CFA-treated rats. Veh = vehicle. JQ1 is an inhibitor of BET binding to acetylated histones. **P < 0.01 versus CFA 1d; n = 7. (G) Intrathecal administration with JQ1 (10 μl, 100 μM) failed to affect the Brd4 expression in DRG of CFA-treated rats; n = 6. (H) Representative tracks and statistical analyses of locomotion in an open field arena during the recording period (30 min). Bar charts are total moved distance, number of rears, and duration of rearing. **P < 0.01 versus saline 1d; #P < 0.05, ##P < 0.01 versus CFA 1d; n = 8.

Intraplantar complete Freund’s adjuvant (CFA) injection induces hyperalgesia and bromodomain-containing protein 4 (Brd4) expression in dorsal root ganglia (DRG) neurons. (A) Representative Western blot and statistical analyses (normalized to glyceraldehyde 3-phosphate dehydrogenase; GAPDH) demonstrating intraplantar CFA injection increased Brd4 expression in DRG. IB = Immunoblotting. **P < 0.01 versus saline; ##P < 0.01 versus naive; n = 6. (B) CFA decreased the paw withdrawal latency (PWL) measured after injection. BL = baseline. **P < 0.01 versus saline; ##P < 0.01 versus BL; n = 7. (C) In DRG, CFA (CFA 1d) increased the immunofluorescence of Brd4 (red, II), which colocalized with a neuronal marker (NeuN; green, III), a marker of A-fiber afferents and medium-large myelinated neurons (NF-200; green, VI), a marker of small peptidergic C-nociceptors called calcitonin gene-related peptide (CGRP; green, VII), and a marker of small nonpeptidergic C-nociceptors (IB4; green, VIII), but not a marker of satellite cells called glial fibrillary acidic protein (GFAP; green, IV) or a marker of macrophages called Iba-1 (green, V) at day 1 after injection. Scale bar = 50 μm. Thickness = 16 μm. **P < 0.01 versus saline 1d; n = 7. (D) Brd4-specific small interfering RNA (siRNA; Brd4 RNAi + CFA 1d, 10 μl, 5 μg) significantly increase the PWL of the CFA-treated group. MS RNAi = missense siRNA. **P < 0.01 versus CFA 1d; n = 7. (E) Representative Western blot and statistical analysis (normalized to GAPDH) demonstrating that Brd4-specific siRNA (Brd4 RNAi + CFA 1d, 10 μl, 5 μg) attenuated Brd4 expression in DRG of CFA-treated rats. **P < 0.01 versus CFA 1d; n = 7. (F) Injection with JQ1 (CFA 1d + JQ1; 10 μl; 10, 30, and 100 μM) dose-dependently attenuated the behavioral hyperalgesia in the CFA-treated rats. Veh = vehicle. JQ1 is an inhibitor of BET binding to acetylated histones. **P < 0.01 versus CFA 1d; n = 7. (G) Intrathecal administration with JQ1 (10 μl, 100 μM) failed to affect the Brd4 expression in DRG of CFA-treated rats; n = 6. (H) Representative tracks and statistical analyses of locomotion in an open field arena during the recording period (30 min). Bar charts are total moved distance, number of rears, and duration of rearing. **P < 0.01 versus saline 1d; #P < 0.05, ##P < 0.01 versus CFA 1d; n = 8.

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