Fig. 4

A microglial scar forms at the interface between the astrocytic and fibrotic scars. a–e Confocal immunofluorescence microscopy of representative spinal cord sections taken at the lesion epicenter at 7 (a), 14 (b, d), and 35 (c, e) days post-injury (dpi) showing formation of the microglial scar, characterized by the accumulation of TdT⁺ microglia (red) at the lesion borders, over time. The microglial scar is shown in relation to the infiltration of blood-derived myeloid cells (LysM-eGFP⁺, green) and formation of the astroglial scar (GFAP-immunoreactive astrocytes, blue). Panels (d) and (e) are insets of panels (b) and (c), respectively, showing close-ups of the microglial scar in Cx3cr1^creER::R26-TdT::LysM-eGFP mice at 14 and 35 dpi. f and g Immunoelectron microscopy images showing a gold-labeled microglia (Mi) (dense black dots, highlighted in red) located at the lesion border making direct contacts with immunolabeled astrocytic endfeet (As) (diffuse black, highlighted in blue and pointed by arrowheads) and a monocyte-derived macrophage (MDM, highlighted in green). The intimate relationship between the microglia and distal astrocytic processes is shown at high magnification in the inset (g). nu = nucleus, my = myelin debris. h Percentage of microglia (TdT⁺) that are actively proliferating (Ki67⁺ TdT⁺) at the lesion epicenter at 7, 14, and 35 dpi. i Counts of microglia (TdT⁺) at the lesion epicenter at 7, 14, and 35 dpi. j and k Confocal images showing the presence of TdT⁺ microglia (red), PDGFRβ⁺ pericytes/fibroblasts (green) and GFAP⁺ astrocytes (blue) at the lesion epicenter at 14 dpi. The distance from astrocyte endfeet is depicted by the yellow lines and indicated (k). l Percentage area occupied by microglia (TdT⁺, red bars in the histogram), pericytes/fibroblasts (PDGFRβ, green), and blood-derived myeloid cells (LysM-eGFP⁺, blue) as a function of distance from astrocyte endfeet (n = 4–9 mice). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the other time points. Statistical analysis was performed using a one-way ANOVA followed by a Bonferroni’s post hoc test. Scale bars: (a–c) 200 µm; (d and e) 20 µm; (f) 5 µm; (g) 2 µm; (j and k) 100 µm