Figure 4.
Inhibition of p38-MAPK ameliorates chronic hyperalgesia in HbSS mice. (A) Western immunoblotting revealed decreased p38-MAPK phosphorylation in the spinal cord of HbSS mice treated with PEA (IP, 14 days, 20 mg/kg per day). (B) PEA (30 μM) reduced phosphorylation of p38-MAPK and nuclear translocation of phosphorylated p38-MAPK in primary DRG neurons collected from female HbSS mice. Representative images of DRG neurons treated with Veh (complete media), TNF-α (1 ng/mL)/hemin (40 μM), and/or PEA (30 μM) immunolabeled with primary rabbit anti-mouse phospho-p38-MAPK (1:100; catalog no. 9211S, Cell Signaling Technology), secondary Cy3 AffiniPure donkey anti-rabbit immunoglobulin G (H+L), Absorption max: 550 nm and Excitation max: 570 nm (1:500; catalog no. 711-165-152, Jackson ImmunoResearch, West Grove, PA) antibodies, and DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (1:25 000; catalog no. D1306, Invitrogen, Thermo Fisher). (B-C) DRG neurons showing p38-MAPK phosphorylation and phospho-p38-MAPK nuclear colocalization were enumerated and averaged in 6 fields of view per subject. Incitement of a sickle microenvironment significantly increased phospho-p38-MAPK nuclear colocalization, which was completely attenuated with PEA cotreatment. Z-stacks of 10× 0.5-μm images were acquired on a laser scanning confocal microscope (Zeiss LSM 900, Carl Zeiss AG) using a plan-apochromat 63× oil M27 objective lens. (D-G) Targeting p38-MAPK with Nef dose-dependently (oral 6 or 12 mg/kg twice daily) reduced PWF in response to mechanical and cold stimuli and reduced cold aversion at days 7 and 14 of treatment, without affecting grip force in female HbSS mice, suggesting reduced hyperalgesia. Mean ± SD. In panels A-C, data were analyzed with unpaired Student 2-tailed t test. In panels D-G, data were analyzed with 2-way ANOVA and the Tukey post hoc multiple comparisons test. ∗Indicates difference compared with Veh; †indicates difference compared with corresponding BL. ∗,†P < .05; ∗∗,††P < .01; ∗∗∗,†††P < .001; ∗∗∗∗P < .0001. Age: 3.5 to 5.0 months. For panels A-C, n = 3 per condition; panels D-G; female HbSS Veh, n = 5; female HbSS Nef 6 mg/kg, n = 5; female HbSS Nef 12 mg/kg, n = 5; female HbAA Veh, n = 6; female HbAA Nef 12 mg/kg, n = 6. BW, body weight; IR, immunoreactivity; PWF, paw withdrawal frequency; Temp, temperature; VF, von Frey.

Inhibition of p38-MAPK ameliorates chronic hyperalgesia in HbSS mice. (A) Western immunoblotting revealed decreased p38-MAPK phosphorylation in the spinal cord of HbSS mice treated with PEA (IP, 14 days, 20 mg/kg per day). (B) PEA (30 μM) reduced phosphorylation of p38-MAPK and nuclear translocation of phosphorylated p38-MAPK in primary DRG neurons collected from female HbSS mice. Representative images of DRG neurons treated with Veh (complete media), TNF-α (1 ng/mL)/hemin (40 μM), and/or PEA (30 μM) immunolabeled with primary rabbit anti-mouse phospho-p38-MAPK (1:100; catalog no. 9211S, Cell Signaling Technology), secondary Cy3 AffiniPure donkey anti-rabbit immunoglobulin G (H+L), Absorption max: 550 nm and Excitation max: 570 nm (1:500; catalog no. 711-165-152, Jackson ImmunoResearch, West Grove, PA) antibodies, and DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (1:25 000; catalog no. D1306, Invitrogen, Thermo Fisher). (B-C) DRG neurons showing p38-MAPK phosphorylation and phospho-p38-MAPK nuclear colocalization were enumerated and averaged in 6 fields of view per subject. Incitement of a sickle microenvironment significantly increased phospho-p38-MAPK nuclear colocalization, which was completely attenuated with PEA cotreatment. Z-stacks of 10× 0.5-μm images were acquired on a laser scanning confocal microscope (Zeiss LSM 900, Carl Zeiss AG) using a plan-apochromat 63× oil M27 objective lens. (D-G) Targeting p38-MAPK with Nef dose-dependently (oral 6 or 12 mg/kg twice daily) reduced PWF in response to mechanical and cold stimuli and reduced cold aversion at days 7 and 14 of treatment, without affecting grip force in female HbSS mice, suggesting reduced hyperalgesia. Mean ± SD. In panels A-C, data were analyzed with unpaired Student 2-tailed t test. In panels D-G, data were analyzed with 2-way ANOVA and the Tukey post hoc multiple comparisons test. ∗Indicates difference compared with Veh; †indicates difference compared with corresponding BL. ∗,†P < .05; ∗∗,††P < .01; ∗∗∗,†††P < .001; ∗∗∗∗P < .0001. Age: 3.5 to 5.0 months. For panels A-C, n = 3 per condition; panels D-G; female HbSS Veh, n = 5; female HbSS Nef 6 mg/kg, n = 5; female HbSS Nef 12 mg/kg, n = 5; female HbAA Veh, n = 6; female HbAA Nef 12 mg/kg, n = 6. BW, body weight; IR, immunoreactivity; PWF, paw withdrawal frequency; Temp, temperature; VF, von Frey.

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