Figure 1.

Increased inflammatory monocyte infiltration in injured muscles of mice developing NHO. (A) C57BL/6 mice underwent either SCI or Sham surgery followed by an intra muscular injection of CDTX or PBS. Muscle leukocytes were extracted from hamstrings at day 4 post-surgery and isolated into four subsets based on forward scatter, side scatter as well as zombie aqua negativity (viable cells), F4/80, Ly6G, CD11b, and Ly6C expression. (B) Frequencies of each leukocyte population relative to total live muscle cells using the gating strategy outlined: (i) “Total F4/80⁺ cells” (CD11b⁺F4/80⁺, blue gates in A), (ii) “Ly6C^hi inflammatory monocytes” (F4/80⁺CD11b⁺Ly6G⁻ CD48⁺Ly6C^hi, blue gates in A), (iii) “Ly6C^mid/lo monocytes/macrophages” (F4/80⁺ CD11b⁺ Ly6G⁻CD48⁺ Ly6C^mid/lo, blue gates in A), and (iv) “granulocytes” (F4/80⁻CD11b⁺Ly6G⁺, red gates in A). The frequency of Ly6C^hi inflammatory monocytes (relative to total live muscle cells) in mice developing NHO (SCI+CDTX), compared to all other treatment groups was significantly higher (p = 0.0002 ANOVA n = 3–5/group). Each dot represents an individual mouse. Bars represent as mean ± SD. (C) Muscle leukocytes were extracted from hamstrings at day 4 post surgery, and 4 separate leukocyte populations were identified by flow cytometry using the following gating strategy: (i) “Ly6C^hi inflammatory monocytes” as CD45⁺ lineage (Ter119,CD3ε,B220)-negative F4/80⁺ CD11b⁺ Ly6G⁻ CD48⁺Ly6C^hi, red gates, (ii) “Ly6C^mid monocytes/macrophages” CD45⁺ Lin⁻ F4/80⁺ CD11b⁺ Ly6G⁻CD48⁺ Ly6C^mid, red gates, (iii) “Ly6C^neg monocytes” CD45⁺ Lin⁻ F4/80⁺ CD11b⁺ Ly6G⁻ CD48⁺ Ly6C^neg red gates, and (iv) “granulocytes” CD45⁺ Lin⁻ F4/80^neg CD11b⁺ Ly6G⁺, blue gates. (D) Frequencies of each myeloid subset relative to total live muscle cells and to total live CD45⁺ muscle leukocytes. There was a significant increase in frequency of Ly6C^hi monocytes relative to total live muscle cells (Di, p < 0.0001 Mann-Whitney test) and to CD45⁺ live muscle leukocytes (Di, p < 0.0001 Mann-Whitney test) after SCI+CDTX compared to Sham+CDTX. Each dot represents an individual mouse, n = 25–27/treatment group. Bars represent mean ± SD.