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Fig. 4 | Fluids and Barriers of the CNS

Fig. 4

From: Posthemorrhagic hydrocephalus associates with elevated inflammation and CSF hypersecretion via activation of choroidal transporters

Fig. 4

Isolated choroid plexus is viable and retains it transport activity upon culturing. Isolated rat choroid plexus demonstrated calcein fluorescence, indicative of viable cells, upon tissue culturing for 0, 16 and 24 h (a–c), whereas fluorescence was absent in control choroid plexus kept in H2O for 24 h (d). Inserts are magnification of the white boxes. Scale bars 500 μm. Choroid plexus tissue fixed directly after isolation (e) or following 16 h (f) or 24 h (g) of tissue culture and stained with toluidine blue. The boxed areas have been enlarged (insets) to reveal nuclear detail and presence of an organelle-free apical zone that includes the brush border (bounded by arrowheads). Note that after 24 h of culture, the brush border is greatly diminished (open arrowheads), and epithelial cells display small, pyknotic nuclei. Art, arteriole; cap, capillary; N, nucleus. Scale bars 5 mm. Electron micrographs of the choroid plexus acutely isolated (h) or after 16 h of tissue culture (i). At both time points tight junctions (TJ; small, white arrows) and basolateral membrane invaginations (MI; pointed arrows) are well developed, and the extracellular basal membrane remains juxtaposed to underlying loose connective tissue (CT). In tissue cultured for 16 h (i), there is a diminution or disappearance of microvilli in many cell profiles (large, open arrows), whereas other cells retain microvilli (large, closed arrow). Most nuclei are euchromatic, but chromatin condensation has commenced in a minority of cells (nucleus on the right), and in the cytoplasm there are large, empty vacuoles (small, open arrows), but organelles remain intact. Inset in i shows the epithelium after 24 h of culture. Although tight junctions are intact, there are no microvilli left, nuclei are pyknotic, cytolysis has commenced, basal membrane invaginations have disappeared, and the contact with underlying extracellular matrix has been lost (asterisk). Scale bars 500 nm; 2 μm; 2 μm. Quantification of Western blots of acutely isolated rat choroid plexus (0 h) and following tissue culturing (16 h) with anti-NKCC1 antibody (j) or the α1 subunit of the Na+/K+-ATPase (k). Data illustrated as normalized to GAPDH. l 86Rb+ efflux rates obtained in choroid plexus acutely isolated (0 h, n = 4) or after tissue culturing (16 h, n = 6; 24 h, n = 4) with or without the NKCC1 inhibitor bumetanide (BUM, 20 µM). m 86Rb+ influx obtained in choroid plexus acutely isolated (0 h, n = 5) or after 16 h tissue culturing with or without the Na+/K+-ATPase inhibitor ouabain (OUA, 2 mM). Error bars represent standard deviation and statistical significance was tested with an unpaired two-tailed t-test or a one-way ANOVA followed by Sidak’s multiple comparisons test (l and m). ***P < 0.001, NS not significant.

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