Fig 2: SGN loss pattern and distribution of macrophages in the RC region from control and targeted-cell Cx26-null groups at P60. (A–C) Representative images of SGN in the apex (A), middle region (B), and base (C) from the control group at P60. (D–F) Parallel images in different regions from the targeted-cell Cx26-null group at P60. (G–I) Macrophages (CD45, green) and CX3CL1 (red) immunolabeling in the apex, middle region, and base of the control group, respectively. (J–L) Macrophages (CD45, green) and CX3CL1 (red) immunolabeling in corresponding regions of the targeted-cell Cx26-null group. White arrowheads indicate the SGN with high expression of CX3CL1. (M) Quantification of CX3CL1 fluorescence in SGNs in the different groups at P60 (n = 4 mice in each group). (N) Quantification of macrophage numbers per 10,000 µm2 of RCs in the different groups at P60 (n = 4 mice in each group). *Significant difference from the control group (p < 0.05).

Fig 3: Quantitative expression level of CX3CL1 in the OC and distribution of macrophages in the RC region from control and targeted-cell Cx26-null groups at P16. (A–C) Modiolar sections showed immunolabeling of CX3CL1 (red) of OCs in different regions from the control group. (D–F) Parallel images of CX3CL1 immunostaining in different regions from the targeted-cell Cx26-null group. The white boxes at the bottom right of the (C–F) show the DAPI staining in OCs, and * indicates missing hair cell. (G,H) Quantification of CX3CL1 immunostaining in OCs (G) or SGNs (H) in the different groups at P16 (n = 4 mice in each group). (I) Quantification of macrophage numbers per 10,000 µm2 of the RCs in different groups at P16 (n = 4 mice in each group). (J) Modiolar sections showed macrophages (CD45, green) and CX3CL1 (red) immunolabeling in RC regions from different groups. *Significant difference from the control group (p < 0.05). O1, OHC1; D1, DC1; IPC, inner pillar cell; OPC, outer pillar cell; ISC, inner sulcus cell; CC, Claudius cell.

Fig 4: CX3CR1 immunohistochemistry. (A-F) CX3CR1 immunohistochemistry in the CA1 field of the (A) sham group and ischemia operated groups at (B) 6 h, (C) 1 day, (D) 2 days, (E) 5 days and (F) 10 days after tgCI. Weak CX3CR1 immunoreactivity is detected in the SP (asterisk) of the sham operated gerbils and CX3CR1 immunoreactivity in the SP (asterisk) at 1 day after tgCI is strong. At 5 and 10 days after tgCI, strong CX3CR1 immunoreactivity is shown in cells (arrows) in the SO and SR. Scale bar, 50 µm. (G) ROD as % of CX3CR1-immunoreactive structures in the CA1 field after tgCI (n=7 at each point in time). *P<0.05 vs. the sham operated gerbils; †P<0.05 vs. the pre-time-point group. Bars indicate the mean ± standard error of the mean. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; tgCI, transient global cerebral ischemia; CX3CL1, C-X3-C motif ligand 1; CX3CR1, CX3C chemokine receptor 1; ROD, relative optical density.

Fig 5: Western blot analyses of CX3CL1 and CX3CR1 in the hippocampal CA1 field of sham operated and ischemia operated gerbils. ROD as % value of immunoblot band is represented (n=7 per group). *P<0.05 vs. the sham operated gerbils; †P<0.05 vs. the pre-time-point group. CX3CL1, C-X3-C motif ligand 1; CX3CR1, CX3C chemokine receptor 1; ROD, relative optical density.