Figure 2.
Identification of proliferative microglial aggregates in the fetal brain. (A and B) 3D stitching images of IBA-1, Ki67, and CD34 (A) or vimentin (B) antibody immunostaining in two fetal brains (13−15 gw) revealed large microglial aggregates with abundant Ki67+ microglia. Middle, the orientation of vimentin+ fibers in the center of the microglial aggregates with a D insert (B). Right, magnified orthogonal views (A and B). Scale bars (A), 400 µm (left), 50 µm (middle), and 20 µm (right); scale bars (B), 400 µm (left), 100 µm (middle), and 20 µm (right). (C) 3D stitching images of IBA-1, Ki67, and CD34 antibody immunostaining in the cortical layers of 13-gw fetal brains. White arrowheads, IBA-1+/Ki67+ cells. Scale bars, 200 µm (top) and 100 µm (bottom). (D) Percentage of Ki67+ microglia in the microglial aggregate and scattered microglia in other regions (fetuses, n = 8; data, mean ± SD; Mann–Whitney U test; dot, one brain; **P < 0.01). (E) Percentage of Ki67+ microglia in human fetal brain samples at 13−14 gw and 15−16 gw (fetuses, n = 6; mean ± SD; Mann–Whitney U test; ns, nonsignificant; dot, a section). (F) Illustration on the left shows sectional positions. The lower left shows a whole view of the fetal brain section. MGE, medial ganglionic eminence; V, ventricle; MZ, mantle zone. The middle shows the entire view of the microglial aggregate across four consecutive sections. The right two graphs show the number of IBA-1+ microglia and size of microglial aggregate in nine consecutive sections of a 15-gw fetal brain. Scale bars, 2 mm (lower left) and 100 µm (lower right). (G) High-resolution stitched images of Ki67 and IBA-1 antibody immunostaining. Bottom image shows a sectional view of the rectangle in the top panel. Scale bars, 100 µm (up) and 20 µm (below). (H) Shapes of microglial aggregate. Arrows, microglial orientation. The dashed line shows the IBA-1+ cell-enriched site. Scale bar, 200 µm. (I and J) Microglia in the border regions of proliferative microglial aggregates were imaged using SIM. The microglia on the border facing the cortical layers (superficial) lack long-oriented processes; the microglia on the border facing the caudate (deeper) show orientation and multiple extended processes (I). Microvascular scaffolding microglia are at the border of the caudate (J). Scale bars, 10 µm. (K and L) Illustration of the angle between the vertical line representing a straight line from the meninges to the ventricle and the cellular long-body axis (K). The average angle between the caudate surface facing the body and the vertical axes was 17−20°; the cortical-facing angle ranged from 35° to 41°. The vertical line is the line from the meninges to the ventricles (fetuses, n = 5; all data, mean ± SD; Mann–Whitney U test, **P < 0.01). Refer to the image caption for details. Panel A shows 3D stitched immunostaining of IBA-1, Ki67, and CD34 in fetal brain sections, revealing large microglia aggregates enriched with proliferating Ki67 positive cells, along with magnified orthogonal views. Panel B shows 3D stitched immunostaining images of IBA-1, Ki67, and vimentin staining, highlighting vimentin-positive fiber organization within aggregate cores and detailed inset views. Panel C shows stitched cortical immunostaining images demonstrating IBA-1 positive Ki67 positive microglia distributed across cortical layers, with highlighted proliferative cells. Panel D shows quantitative analysis comparing the percentage of Ki67 positive microglia within aggregates versus scattered regions, indicating significant enrichment in aggregates. Panel E shows comparison of Ki67 positive microglia percentages between different gestational stages, showing no significant difference. Panel F shows anatomical illustration of fetal brain sectional positions alongside whole-section imaging and serial section analysis quantifying microglia number and aggregate size across consecutive sections. Panel G shows high-resolution stitched immunostaining images of IBA-1 and Ki67 staining, confirming dense clustering and spatial organization of proliferative microglia. Panel H shows a large-scale view of fetal brain regions including cortex, caudate, and internal capsule, illustrating aggregate morphology, enrichment zones, and directional orientation of microglia. Panel I shows super-resolution imaging of microglia at aggregate borders, with cortical-facing cells lacking elongated processes and deeper caudate-facing cells exhibiting oriented extensions. Panel J shows juxtavascular microglia aligned along vascular structures at aggregate borders. Panel K shows a schematic illustrating measurement of the angle between the vertical axis (meninges to ventricle) and the long axis of microglia. Panel L shows quantitative analysis of microglial orientation relative to the vertical axis, comparing cortical-facing and caudate-facing populations.

Identification of proliferative microglial aggregates in the fetal brain. (A and B) 3D stitching images of IBA-1, Ki67, and CD34 (A) or vimentin (B) antibody immunostaining in two fetal brains (13−15 gw) revealed large microglial aggregates with abundant Ki67+ microglia. Middle, the orientation of vimentin+ fibers in the center of the microglial aggregates with a D insert (B). Right, magnified orthogonal views (A and B). Scale bars (A), 400 µm (left), 50 µm (middle), and 20 µm (right); scale bars (B), 400 µm (left), 100 µm (middle), and 20 µm (right). (C) 3D stitching images of IBA-1, Ki67, and CD34 antibody immunostaining in the cortical layers of 13-gw fetal brains. White arrowheads, IBA-1+/Ki67+ cells. Scale bars, 200 µm (top) and 100 µm (bottom). (D) Percentage of Ki67+ microglia in the microglial aggregate and scattered microglia in other regions (fetuses, n = 8; data, mean ± SD; Mann–Whitney U test; dot, one brain; **P < 0.01). (E) Percentage of Ki67+ microglia in human fetal brain samples at 13−14 gw and 15−16 gw (fetuses, n = 6; mean ± SD; Mann–Whitney U test; ns, nonsignificant; dot, a section). (F) Illustration on the left shows sectional positions. The lower left shows a whole view of the fetal brain section. MGE, medial ganglionic eminence; V, ventricle; MZ, mantle zone. The middle shows the entire view of the microglial aggregate across four consecutive sections. The right two graphs show the number of IBA-1+ microglia and size of microglial aggregate in nine consecutive sections of a 15-gw fetal brain. Scale bars, 2 mm (lower left) and 100 µm (lower right). (G) High-resolution stitched images of Ki67 and IBA-1 antibody immunostaining. Bottom image shows a sectional view of the rectangle in the top panel. Scale bars, 100 µm (up) and 20 µm (below). (H) Shapes of microglial aggregate. Arrows, microglial orientation. The dashed line shows the IBA-1+ cell-enriched site. Scale bar, 200 µm. (I and J) Microglia in the border regions of proliferative microglial aggregates were imaged using SIM. The microglia on the border facing the cortical layers (superficial) lack long-oriented processes; the microglia on the border facing the caudate (deeper) show orientation and multiple extended processes (I). Microvascular scaffolding microglia are at the border of the caudate (J). Scale bars, 10 µm. (K and L) Illustration of the angle between the vertical line representing a straight line from the meninges to the ventricle and the cellular long-body axis (K). The average angle between the caudate surface facing the body and the vertical axes was 17−20°; the cortical-facing angle ranged from 35° to 41°. The vertical line is the line from the meninges to the ventricles (fetuses, n = 5; all data, mean ± SD; Mann–Whitney U test, **P < 0.01).

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