Hyperglycemia increases the secretion of SCO-spondin- and CD63-positive vesicles, which is present at the apex of ependymal cells.

(A-C) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in sections of the SCO under normoglycemic conditions (A) and hyperglycemic conditions (CSF glucose concentration of 5 mM (B) or 10 mM (C)). Nuclear staining with Hoechst (blue). Scale bar: 50 μm. SCO-spondin–positive blebs, ER, BV, and cilia are depicted. The pattern of intracellular SCO-spondin immunoreactivity was altered under different glycemic conditions. (D and Dˋ) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in frontal brain sections from hyperglycemic rats (CSF glucose concentration of 10 mM). Nuclear staining with Hoechst (blue). Scale bar: 30 μm. Most secreted SCO-spondin was detected at the apex of ependymal cells (yellow arrows). Immunofluorescence and DIC (D`) microscopy confirmed that cilia and SCO-spondin interacted. (E) TEM revealed the presence of aggregated extracellular proteins at the apex of ependymal cells (arrows) in hyperglycemia. Scale bar: 5 μm. (Eˋ) Gold immunolabeling and TEM using anti-SCO-spondin in ependymal cells, caudal area; 10 mM glucose CSF. SCO-spondin–positive reaction (black gold particles) were detected in the cilia. Scale bar: 5 μm. (F and Fˋ) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in frontal brain sections (caudal area) from hyperglycemic rats (CSF glucose concentration of 10 mM). Nuclear staining with Hoechst (blue). Scale bar: 30 μm. Most secreted SCO-spondin was detected at the apex of ependymal cells (yellow arrows). Immunofluorescence and DIC (Fˋ) microscopy confirmed that cilia and SCO-spondin interacted. (Fˋˋ) Gold immunolabeling and TEM using anti-SCO-spondin in ependymal cells; medial area. SCO-spondin–positive reaction (black gold particles) was detected in the cilia. Scale bar: 5 μm. (G-I) Immunohistochemical staining of SCO-spondin (green) and CD63 (red) in frontal brain sections containing SCO cells under different glycemic conditions. Intensity reactivity was detected for the exosome marker CD63, mainly in ER cisternae. Scale bar: 80 μm. (J) Immunohistochemical staining of SCO-spondin (green) and CD63 (red) in sections containing ependymal cells under hyperglycemic conditions. CD63 (large arrows) was detected at the apex of ependymal cells, which its colocalization with SCO-spondin was lower (arrowheads). Scale bar: 30 μm. (K) Gold immunostaining with anti-OSC-spondin and TEM analysis in ependymal cells. Scale bar: 2 μm. (L) Quantification of Mander’s overlap coefficient for SCO-spondin and CD63 in SCO and ependymal cells under different glycemic conditions. In normoglycemia, CD63 and SCO-spondin are not detected in ependymal cells. The graph shows data from 4 biologically independent samples. The error bars represent the SD. Data used to generate graph can be found in S1 Data. (D) SCO-spondin immunoreactivity was also observed in SCO cells and pre-RF (Fig 4D, inset and arrowheads), and positive immunoreaction was detected close to the apex of ependyma cilia (Fig 4D, yellow arrows). Using DIC analysis in ependymal cells (Dˋ), we also observed positive immunoreaction for SCO-spondin and acetylated α-tubulin on the ependymal cilia (Fig 4Dˋ, white arrows). TEM analysis showed that aggregated proteins were present in the apex of ependymal cells cilia at 10 mM glucose in CSF (Fig 4E, arrows). These secreted materials were not observed in the control ependymal cells (Fig 4E, normoglycemia). Gold immunostaining with anti-SCO-spondin and TEM analysis confirmed SCO-spondin associated with the apex of ependymal cells and MVBs (Fig 4E’and inset; S3F and S3G Fig). BV, blood vessels; DIC, differential interference contrast; d3v, dorsal third ventricle; ER, endoplasmic reticulum; MVB, multivesicular body; SCO, subcommissural organ.