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. 2018 Aug 29;5(4):ENEURO.0101-18.2018. doi: 10.1523/ENEURO.0101-18.2018

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Copyright © 2018 McCreedy, Lee et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 8. — Diclofenac treatment reduces L-selectin on peripheral blood and infiltrated leukocytes but does not alter accumulation in the injured spinal cord. A, Flow cytometry gating for infiltrated myeloid cells and myeloid subsets. B, C, Representative flow cytometry histograms for CD62L staining in leukocyte populations from the blood (B) or spinal cord (C) of vehicle-treated (white) and diclofenac-treated (gray) mice at 24 h post-SCI. Representative isotype staining is shown in black. D, Flow cytometry analysis for CD62L expression on peripheral blood leukocytes from uninjured mice and injured mice treated with diclofenac or vehicle (PBS). Diclofenac induced a reduction of L-selectin on neutrophils and non-classical monocytes (p = 0.005 and 0.02, respectively). N = 4/uninjured and N = 6–7/SCI/treatment. One-way ANOVA with Tukey’s post hoc test (p = 0.004, <0.0001, 0.019, and 0.0004 and F_(2,14) = 8.47, 20.3, 5.30, and 14.8, respectively). *p < 0.05, **p < 0.01, ***p < 0.001. E, Flow cytometry analysis for CD11b levels on peripheral blood leukocytes from spinal cord-injured mice treated with diclofenac or vehicle (PBS). There were no differences in CD11b levels on myeloid cells or myeloid lineage subsets. N = 6–7/treatment. Unpaired two-tailed Student’s t tests. p = 0.15, 0.47, 0.99, and 0.16 and t₍₁₁₎ = 1.5, 0.75, 0.01, and 1.5, respectively. F, There were no differences in the accumulation of total myeloid cells (CD11b⁺) or any myeloid lineage subset in spinal cords of diclofenac-treated mice compared to vehicle-treated mice at 24 h post-SCI. N = 7/treatment. Unpaired two-tailed Student’s t tests. p = 0.37, 0.53, 0.92, and 0.23 and t₍₁₂₎ = 0.66, 0.64, 0.12, and 1.27, respectively. G, Flow cytometry analysis demonstrated loss of L-selectin on total leukocytes (CD45⁺, p = 0.020 and t₍₁₂₎ = 2.67), total myeloid cells (CD11b⁺, *p = 0.020 and t₍₁₂₎ = 2.69), non-classical monocytes (Ly6C^low/Ly6G^-, **p = 0.008 and t₍₁₂₎ = 3.16), and neutrophils (Ly6C^low/Ly6G⁺, *p = 0.049 and t₍₁₂₎ = 2.20) in the spinal cord of diclofenac-treated versus vehicle-treated mice at 24 h post-SCI. There was no effect on L-selectin on inflammatory monocytes (Ly6C^hi/Ly6G^-, p = 0.59 and t₍₁₂₎ = 0.56). N = 7/treatment. Unpaired two-tailed Student’s t tests. H, ELISA for sL-selectin in the peripheral blood at 8, 24, and 72 h post-SCI in mice receiving a vehicle (PBS) control injection or 1–60 mg/kg of diclofenac. Increased sL-selectin was observed at 8 and 24 h, but not 72 h, post-SCI in mice receiving 40 and 60 mg/kg diclofenac. N = 5/group for 8 and 72 h; N = 5/group at 24 h except for vehicle (N = 7) and 40 mg/kg diclofenac (N = 10). One-way ANOVA followed by Dunnett’s post hoc test (p = 0.005, 0.006, and 0.71 and F_(6,28) = 4.05, F_(6,35) = 3.72, and F_(6,28) = 0.63, respectively). *p < 0.05, **p < 0.01.