Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Inflammatory monocytes from the bloodstream infiltrated the cochlea after acoustic overstimulation. (A) Effects of acoustic overstimulation (120 dB, 1 h white band noise exposure) on the permanent threshold shift ( N = 8 ears). (B) Effects of noise exposure on outer hair cell degeneration ( N = 4 ears in each group). (C) Effects of acoustic stimulation on peripheral blood CD11b + Ly6G – F4/80 + CX3CR1 + populations and distinction between Ly6C ++ inflammatory monocytes and Ly6C – patrolling monocytes. (D) Effects of acoustic overstimulation on cell populations (CD11b + Ly6G – F4/80 + CX3CR1 + cells; Ly6C ++ possible inflammatory monocytes and Ly6C – possible resident macrophages) in cochlea at 2 days. (E) Clodronate liposome successfully reduced monocytes in peripheral blood (left) compared to neutrophils (right) in 1 dpn at which the monocytes begin to infiltrate to the cochlea. (F) Effects of clodronate liposome treatment after acoustic overstimulation on Ly6C ++ possible inflammatory monocyte populations in the cochlea after 2 days. (left) Effects of clodronate liposome treatment after acoustic overstimulation on Ly6C – possible resident macrophage populations. (right) Clod : clodronate liposome-treated group. ** : p < 0.01, *: p < 0.05, ns : not significant. (G) Immunofluorescence study comparing control and clodronate liposome at 2 dpn. White arrows indicate clusters of monocytes. The black box in the light microscopy image located on the left indicates the anatomical location of the immunofluorescence images. Scale bar = 50 μm.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Immunofluorescence, Light Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Inflammatory monocytes infiltrated within 2 days after acoustic stimulation, whereas neutrophil infiltration was not observed. (A) Percentages of cells sorted by Ly6C expression from the CD11b + Ly6G – F4/80 + CX3CR1 + population, demonstrating Ly6C ++ cell infiltration within 2 days after acoustic overstimulation. (B) Total cell counts showing the CD11b + Ly6G – F4/80 + CX3CR1 + population did not change after 2 dpn. (C) Distinction between Ly6G + CX3CR1 – neutrophils and CD11b + cells in the peripheral blood. (D) Lack of distinct neutrophils in the cochlea at 1 day after acoustic overstimulation compared with CD11b + cells. (E) Changes in the percentages and the cell count of neutrophils in cochlea after acoustic overstimulation. There was no statistically significant value. Ctr : control, N1d : 1 day after noise, N2d : 2 days after noise, N3d : 3 days after noise, N5d : 5 days after noise, Mo : monocytes, M φ: macrophages. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05, when compared with the control. (F) Immunofluorescence study of lower portion of spiral ligament at 1 dpn. Neutrophil (white) is mixed in the cluster of inflammatory monocytes (red and weak green). Scale bar = 20 μm.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Expressing, Cell Counting, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: The Time Course of Monocytes Infiltration After Acoustic Overstimulation

doi: 10.3389/fncel.2022.844480

Figure Lengend Snippet: Infiltrated monocytes transformed into macrophages within 5 days after acoustic stimulation. Concatenated contour plots (merging four biological replicates for each time point) showing CD11b + Ly6G – F4/80 + CX3CR1 + cells at different time points after acoustic overstimulation. Black contour : cochlea sample, red contour : control peripheral blood sample, dots : center of each population.

Article Snippet: 7-Aminoactinomycin D (7-AAD; cat. no. 420403; Biolegend) was used for live cell gating, phycoerythrin (PE)-Cy7-conjugated anti-CD11b (M1/70; cat. no. 25-0112-82; Invitrogen, Carlsbad, CA, United States) was used for myeloid cell gating, PE-conjugated anti-Ly6G (1A8; cat. no. 127608; Biolegend) was used for neutrophil gating, APC-conjugated anti-F4/80 (BM8; cat. no. 17-4801-82; Invitrogen) was used for monocyte/macrophage gating, and APC-Cy7-conjugated anti-Ly6C (HK1.4; cat. no. 128026; Biolegend) was used for inflammatory monocyte gating ( , ).

Techniques: Transformation Assay