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Effects of Maternal Exposure to Ultrafine Carbon Black on Brain Perivascular Macrophages and Surrounding Astrocytes in Offspring Mice

Onoda, Atsuto, Umezawa, Masakazu, Takeda, Ken, Ihara, Tomomi, Sugamata, Masao·2014·PLoS ONE
DOI 10.1371/journal.pone.0094336PMC PMC3983141
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Figure 1 Summarised scheme of animal treatments and sample collection.
Figure 1 Summarised scheme of animal treatments and sample collection.CC BY · PubMed Central
Figure 2 Characterisation of ultrafine carbon black (UfCB). A: Transmission electron microscopy images of UfCB particles in the instillation medium. The yellow numerical value means minor axis - major axis of the secondary UfCB particles. B: Dynamic light scattering data of UfCB in the instillation medium.
Figure 2 Characterisation of ultrafine carbon black (UfCB). A: Transmission electron microscopy images of UfCB particles in the instillation medium. The yellow numerical value means minor axis - major axis of the secondary UfCB particles. B: Dynamic light scattering data of UfCB in the instillation medium.CC BY · PubMed Central
Figure 3 Light micrographs of perivascular macrophages stained with PAS-haematoxylin. All scale bars represent 10 μm. Perivascular macrophages (PVMs) surrounding cerebral blood vessels of (A, B) 6-week-old control mouse, (C, D) 12-week-old control mouse, (E, F) 6-week-old UfCB-exposed offspring mouse, and (G, H) 12-week-old UfCB-exposed offspring mouse were shown. B, D, F, H: Enlarged view of A, C, E and G: PVMs of control mice contained many PAS-positive granules, sized 0.9 μm (B, arrow) and 1.3 μm (D, arrow), in the cytoplasm. Many PAS-positive granules were enlarged in UfCB-exposed offspring (F, arrow: 2.6 μm; H, arrow: 3.0 μm).
Figure 3 Light micrographs of perivascular macrophages stained with PAS-haematoxylin. All scale bars represent 10 μm. Perivascular macrophages (PVMs) surrounding cerebral blood vessels of (A, B) 6-week-old control mouse, (C, D) 12-week-old control mouse, (E, F) 6-week-old UfCB-exposed offspring mouse, and (G, H) 12-week-old UfCB-exposed offspring mouse were shown. B, D, F, H: Enlarged view of A, C, E and G: PVMs of control mice contained many PAS-positive granules, sized 0.9 μm (B, arrow) and 1.3 μm (D, arrow), in the cytoplasm. Many PAS-positive granules were enlarged in UfCB-exposed offspring (F, arrow: 2.6 μm; H, arrow: 3.0 μm).CC BY · PubMed Central
Figure 4 Quantitative observation of PAS-positive PVMs. A: The number of PAS-positive PVMs in each brain region (n = 5/group). Numbers in the following regions were significantly decreased in UfCB-exposed offspring; cerebral cortex (Cx), hippocampus (HIP), hypothalamus (Hy), midbrain (MBr), cerebellum (Cb), and medulla oblongata (MO). Asterisks indicate statistical significance (* P<0.05, **P<0.01, ***P<0.001). Data are shown as mean ±SD. B: The regions of the brain. Abbreviations: Olf, olfactory bulb; Cx, cerebral cortex; cc, corpus callosum; Str, striatum; HIP, hippocampus; Th, thalamus; Hy, hypothalamus; MBr, midbrain; Po, pons; Cb, cerebellum; MO, medulla oblongata.
Figure 4 Quantitative observation of PAS-positive PVMs. A: The number of PAS-positive PVMs in each brain region (n = 5/group). Numbers in the following regions were significantly decreased in UfCB-exposed offspring; cerebral cortex (Cx), hippocampus (HIP), hypothalamus (Hy), midbrain (MBr), cerebellum (Cb), and medulla oblongata (MO). Asterisks indicate statistical significance (* P<0.05, **P<0.01, ***P<0.001). Data are shown as mean ±SD. B: The regions of the brain. Abbreviations: Olf, olfactory bulb; Cx, cerebral cortex; cc, corpus callosum; Str, striatum; HIP, hippocampus; Th, thalamus; Hy, hypothalamus; MBr, midbrain; Po, pons; Cb, cerebellum; MO, medulla oblongata.CC BY · PubMed Central
Figure 5 Transmission electron micrographs of perivascular macrophages (PVMs) and astrocytic end-foot. All scale bars represent 2 μm. A–C: Electron micrograph of perivascular regions of the cerebral cortex (grey matter) of (A, B) 6- and (C) 12-week-old mouse in the control group. (A, C) Perivascular macrophages [PVM] were found between endothelial cells and glia limitans with many round lysosomal granules [LG] of moderate intensity and some pale vacuoles. (B, C) The astrocytic end-foot [AE] was attached to a endothelial cells [EC] and a perivascular macrophage. D, E: Electron micrographs of perivascular regions of the cerebral cortex (grey matter) of (D) 6- and (E) 12-week-old UfCB-exposed offspring mouse. (D) Arrow head: Crescent-shaped spaces, an ultrastructural feature of apoptotic bodies [57], of EC were shown. (E) Honeycomb-like structured lysosomal granules (*) were shown in the perivascular macrophages [PVM]. (E) Severe swelling of astrocytic end-feet (**) was found at sites attached to perivascular macrophages with denatured granules.
Figure 5 Transmission electron micrographs of perivascular macrophages (PVMs) and astrocytic end-foot. All scale bars represent 2 μm. A–C: Electron micrograph of perivascular regions of the cerebral cortex (grey matter) of (A, B) 6- and (C) 12-week-old mouse in the control group. (A, C) Perivascular macrophages [PVM] were found between endothelial cells and glia limitans with many round lysosomal granules [LG] of moderate intensity and some pale vacuoles. (B, C) The astrocytic end-foot [AE] was attached to a endothelial cells [EC] and a perivascular macrophage. D, E: Electron micrographs of perivascular regions of the cerebral cortex (grey matter) of (D) 6- and (E) 12-week-old UfCB-exposed offspring mouse. (D) Arrow head: Crescent-shaped spaces, an ultrastructural feature of apoptotic bodies [57], of EC were shown. (E) Honeycomb-like structured lysosomal granules (*) were shown in the perivascular macrophages [PVM]. (E) Severe swelling of astrocytic end-feet (**) was found at sites attached to perivascular macrophages with denatured granules.CC BY · PubMed Central
Figure 6 Light micrographs of GFAP-positive astrocytes of the wide-field. Scale bars represent (A, C) 200 m or (B, D) 100 m. A, C: The frontal cortex of 6-week-old male mice of (A) the control group (B) UfCB-exposed offspring. B, D: Enlarged views of A and C. (B) Few GFAP-positive astrocytes were observed in the grey matter in the control group, while (D) many GFAP-positive astrocytes were detected in the grey matter in UfCB-exposed offspring. GFAP-positive astrocytes were not observed at any sites attached to blood vessels with PVMs with small (approximately 1 m) PAS-positive granules (Figure 7A), but were found at blood vessels with PVMs that had enlarged (approximately 2-3 m) PAS-positive granules in the UfCB-exposed offspring (Figure 7B, C). Moreover, GFAP-positive astrocytic end-feet were detected at sites attached to PVMs with enlarged PAS-positive granules (Figure 7E, G, H), and not surrounding PVMs with small PAS-positive granules (Figure 7F, I) around one blood vessel in the UfCB-exposed offspring (Figure 7D-I). These results suggested that the increase in the expression level of GFAP in astrocytic end-feet around blood vessels in the grey matter was correlated with enlargement of PAS-positive granules of PVM .
Figure 6 Light micrographs of GFAP-positive astrocytes of the wide-field. Scale bars represent (A, C) 200 m or (B, D) 100 m. A, C: The frontal cortex of 6-week-old male mice of (A) the control group (B) UfCB-exposed offspring. B, D: Enlarged views of A and C. (B) Few GFAP-positive astrocytes were observed in the grey matter in the control group, while (D) many GFAP-positive astrocytes were detected in the grey matter in UfCB-exposed offspring. GFAP-positive astrocytes were not observed at any sites attached to blood vessels with PVMs with small (approximately 1 m) PAS-positive granules (Figure 7A), but were found at blood vessels with PVMs that had enlarged (approximately 2-3 m) PAS-positive granules in the UfCB-exposed offspring (Figure 7B, C). Moreover, GFAP-positive astrocytic end-feet were detected at sites attached to PVMs with enlarged PAS-positive granules (Figure 7E, G, H), and not surrounding PVMs with small PAS-positive granules (Figure 7F, I) around one blood vessel in the UfCB-exposed offspring (Figure 7D-I). These results suggested that the increase in the expression level of GFAP in astrocytic end-feet around blood vessels in the grey matter was correlated with enlargement of PAS-positive granules of PVM .CC BY · PubMed Central
Figure 7 Light micrographs of GFAP-positive astrocytes and PAS-positive PVMs of 6-week-old male mice. All scale bars represent 10 μm. (A–C) 6-μm paraffin sections and (D–I) 10-μm frozen sections. A: PVM surrounding the cerebral blood vessels of a 6-week-old control mouse. GFAP-positive astrocytes were very few in number at sites attached to blood vessels with PVMs with small (approximately 1 μm) PAS-positive granules (arrow). B, C: PVM surrounding cerebral blood vessels of a mouse in the UfCB group. The PVM possessed enlarged granules (arrows). Many GFAP-positive astrocytes were observed at sites attached to PVMs with enlarged granules. D: GFAP-positive astrocytes and PAS-positive PVM (arrows) surrounding cerebral blood vessels of mouse in the UfCB group. E–I: Enlarged views of (D). Around one cerebral vessel, GFAP-positive astrocytic end-feet were detected (E, G, H) at a site attached to PVM with enlarged PAS-positive granules (G, 2.2 μm; H, 1.6 μm), but (F, I) not surrounding PVMs with small PAS-positive granules (smaller than 1.1 μm).
Figure 7 Light micrographs of GFAP-positive astrocytes and PAS-positive PVMs of 6-week-old male mice. All scale bars represent 10 μm. (A–C) 6-μm paraffin sections and (D–I) 10-μm frozen sections. A: PVM surrounding the cerebral blood vessels of a 6-week-old control mouse. GFAP-positive astrocytes were very few in number at sites attached to blood vessels with PVMs with small (approximately 1 μm) PAS-positive granules (arrow). B, C: PVM surrounding cerebral blood vessels of a mouse in the UfCB group. The PVM possessed enlarged granules (arrows). Many GFAP-positive astrocytes were observed at sites attached to PVMs with enlarged granules. D: GFAP-positive astrocytes and PAS-positive PVM (arrows) surrounding cerebral blood vessels of mouse in the UfCB group. E–I: Enlarged views of (D). Around one cerebral vessel, GFAP-positive astrocytic end-feet were detected (E, G, H) at a site attached to PVM with enlarged PAS-positive granules (G, 2.2 μm; H, 1.6 μm), but (F, I) not surrounding PVMs with small PAS-positive granules (smaller than 1.1 μm).CC BY · PubMed Central

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Metadata

Authors
Onoda, A., Umezawa, M., Takeda, K., Ihara, T., & Sugamata, M.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-04-10
Volume
9
Issue
4
Pages
e94336
ISSN
1932-6203
DOI
10.1371/journal.pone.0094336
PMC
PMC3983141
Cited by
56
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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Onoda, A., Umezawa, M., Takeda, K., Ihara, T., & Sugamata, M. (2014). Effects of Maternal Exposure to Ultrafine Carbon Black on Brain Perivascular Macrophages and Surrounding Astrocytes in Offspring Mice. PLoS ONE. https://doi.org/10.1371/journal.pone.0094336