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. 2010 Nov 5:7:20.
doi: 10.1186/1743-8454-7-20.

Hydrocephalus induces dynamic spatiotemporal regulation of aquaporin-4 expression in the rat brain

Anders D Skjolding  1 , Ian J Rowland, Lise V Søgaard, Jeppe Praetorius, Milena Penkowa, Marianne Juhler
Affiliations

Hydrocephalus induces dynamic spatiotemporal regulation of aquaporin-4 expression in the rat brain

Anders D Skjolding et al. Cerebrospinal Fluid Res. .

Abstract

Background: The water channel protein aquaporin-4 (AQP4) is reported to be of possible major importance for accessory cerebrospinal fluid (CSF) circulation pathways. We hypothesized that changes in AQP4 expression in specific brain regions correspond to the severity and duration of hydrocephalus.

Methods: Hydrocephalus was induced in adult rats (~8 weeks) by intracisternal kaolin injection and evaluated after two days, one week and two weeks. Using magnetic resonance imaging (MRI) we quantified lateral ventricular volume, water diffusion and blood-brain barrier properties in hydrocephalic and control animals. The brains were analysed for AQP4 density by western blotting and localisation by immunohistochemistry. Double fluorescence labelling was used to study cell specific origin of AQP4.

Results: Lateral ventricular volume was significantly increased over control at all time points after induction and the periventricular apparent diffusion coefficient (ADC) value significantly increased after one and two weeks of hydrocephalus. Relative AQP4 density was significantly decreased in both cortex and periventricular region after two days and normalized after one week. After two weeks, periventricular AQP4 expression was significantly increased. Relative periventricular AQP4 density was significantly correlated to lateral ventricular volume. AQP4 immunohistochemical analysis demonstrated the morphological expression pattern of AQP4 in hydrocephalus in astrocytes and ventricular ependyma. AQP4 co-localized with astrocytic glial fibrillary acidic protein (GFAP) in glia limitans. In vascular structures, AQP4 co-localized to astroglia but not to microglia or endothelial cells.

Conclusions: AQP4 levels are significantly altered in a time and region dependent manner in kaolin-induced hydrocephalus. The presented data suggest that AQP4 could play an important neurodefensive role, and may be a promising future pharmaceutical target in hydrocephalus and CSF disorders.

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Figures

Figure 1
Figure 1
MRI imaging. A T2-weighted fast spin-echo sequence was used for quantification of lateral ventricular volume (A: control, B: 2W kaolin). Twenty-two contiguous T2-weighted axial slices were acquired in interleaved order, as shown in C. For quantification of water diffusion, four different regions of interest (cortex (yellow), periventricular (red), periaqueductal (purple) and CSF (green)) were drawn, using the b = 0 images, and apparent diffusion coefficient values were calculated for each ROI (D). In addition, T1-weighted pre (E) and post (F) contrast images were acquired. Blood brain barrier integrity was determined by subtraction of pre-contrast images from post-contrast images (G).
Figure 2
Figure 2
Lateral ventricular volume. Quantitative MRI data, presented as box plot (median, interquartiles and range, n = 10 for all groups except 2W when n = 7). In each group the median and interquartile range were as follows (Kruskal-Wallis), control: 5.0 mm3 (4.0-7.5); two days (2D): 34.5 mm3 (19.5-37.5); one week (1W): 39.5 mm3 (25.0-76.0); two weeks (2W): 91.0 mm3 (58.0-123.0). The hydrocephalic groups had significantly larger ventricles, p < 0.01, 0.001 and 0.001 at 2 D, 1W and 2W, respectively.
Figure 3
Figure 3
Apparent diffusion coefficient (ADC) values. Box plot showing each region of interest in each group (control and 2-day, 1-week and 2-weeks hydrocephalus) quantified by MRI (median, interquartiles and range, n = 10 for all groups except 2W when n = 7). Periventricular ADC values showed a significant difference (Kruskal-Wallis) between the control group and both the one week and two week group, p < 0.05 for both. Control: 75.5*10-5mm2/s (70.4-83.2; two days: 84.3*10-5mm2/s (81.6-90.4); one week: 88.1*10-5mm2/s (83.0-99.1); two weeks 89.1*10-5mm2/s (85.9-90.3). No significant differences were found between groups in any other regions of interest.
Figure 4
Figure 4
Aquaporin-4 western blotting. Immunoreactivity signal at ~42 kD corresponds to β-actin, and ~30-32 kD corresponds to aquaporin-4. Western blots from the periventricular region of interest are shown as example. Two of the periventricular samples (one control and one two day) did not meet the minimum total protein level for western blotting, and therefore data from these two rats could not be obtained. Densitometry of ECL films was performed using QuantityOne software (Bio-Rad Laboratories). Aquaporin-4 signal was normalized against the β-actin signal. Relative aquaporin-4 expression values for each ROI are presented as box plots (median, interquartile range and total range). At day two a significant (Kruskal-Wallis) decrease compared to control in aquaporin-4 expression was found in both periventricular region (control: 1.00, 0.95-1.04, two day: 0.78, 0.63-0.084, p < 0.05) and cortex (control: 1.00, 0.93-1.14, two day: 0.81, 0.67-0.97, p < 0.05). Expression was normalized after one week, but after two weeks we found significantly increased periventricular expression (control: 1.00, 0.88-1.21; two weeks: 1.40, 1.27-1.64, p < 0.05). No significant changes were found in the periaqueductal region.
Figure 5
Figure 5
Immunohistochemistry of aquaporin-4 in the periventricular region. A: low magnification picture representing a control rat. The boxed area shows the studied periventricular region. B and C: different patterns of aquaporin-4 immunoreactivity. Aquaporin-4 positive glial processes were identified as reticular pattern and perivascular "vessel-like" structures. "Vessel-like" immunoreactivity was identified either as strongly labeled round/eliptical structures where we expect a vessel is cut across (B, arrow) or as parallel lines with branches where a vessel is cut in its length direction (C, arrow). "Vessel-like" immunoractivity was identified in all cases. The reticular pattern could further be categorized as either continuous (B) or discontinuous (C). In cases of discontinuous reticular pattern, larger areas were found devoid of the reticular pattern. In controls the continuous pattern prevailed, in the acute phase (two day and one week) a trend pointed towards the discontinuous pattern, and finally at 2 weeks the reticular pattern was again prominent. The inserted table summarizes the proportion of rats in each group presenting either continuous or discontinuous reticular pattern and the proportion of rats showing perivascular "vessel-like" immunoreactivity. This suggests a dynamic response in the expression pattern of aquaporin-4. No evidence of immunoreactivity in cell soma of astrocytes was found. Negative immunohistochemistry controls did not show any immunoreactivity. * = lateral ventricle. A scale bar 100 μm; B and C scale bar 50 μm.
Figure 6
Figure 6
Aquaporin-4 immunohistochemistry of the ependyma. The aquaporin-4 positive ependymal cell lining of each rat was categorized as belonging to one of four different aquaporin-4 immunoreactivity morphologies (single-layered cuboidal (A), multilayered cuboidal (B), no staining (C) and flattened ependyma (D)). The table shows the proportion of animals in each group belonging to each morphological category. Cuboidal ependyma in single- or multilayered patterns were found in all groups. In the two day group, two cases were found to have lost ependymal staining. At two weeks a diversity of morphologies were reported ranging from single layered cuboidal to flattened in the case of most extreme hydrocephalus. No clear evidence of apical expression of aquaporin-4 in ependymal cells was found at any time point. Scale bar 50 μm.
Figure 7
Figure 7
Double immunofluorescence for aquaporin-4 and glial fibrillary acidic protein in control and two week hydrocephalic rat. Aquaporin-4 is visualized with TxRED (red) and glial fibrillary acidic protein with FITC (green). Nuclear counterstaining with DAPI (blue). Pictures represent glia limitans (A-F), periventricular region (including ependyma) (G-L) and vessels (M-R). Yellow colours in merged pictures confirm co-localization of aquaporins-4 and astrocytic GFAP in glia limitans and in perivascular endfeet. Both in control and in all hydrocephalic groups aquaporin-4 and glial fibrillary acidic protein showed co-localisation in the astroglial endfeet surrounding the vessels (M-R) and in glia limitans (A-F). Astrocytic cell soma were mainly without evidence of aquaporin-4 expression. * = lateral ventricle. Scale bar 100 μm.
Figure 8
Figure 8
Combined aquaporin-4 immunofluorescence and flouorescein linked lectin staining in control and two week hydrocephalic rat. Aquaporin-4 visualized with TxRED (red) and lectin with FITC (green). Nuclear counterstaining with DAPI (blue). Pictures represent glia limitans (A-F), ependyma (G-L) and vessels (M-R). In both contol and hydrocephalic rats aquaporin-4 and lectin staining showed complementary immunoreactivity, clearly not co-localized in glia limitans (A-F). In the ependyma lectin intensively stained the apical membrane of the cells bordering the ventricular wall (H, I, K, L). Immunoreactivity of aquaporin-4 in controls clearly localised to the basolateral membrane (G). In hydrocephalic animals presenting changed aquaporin-4 morphology of the ependyma (J-L), the aquaporin-4 immunoreactivity of the cells lining the ventricle was found not to co-localize with the continuous apical staining of the ependymal cells by lectin (K), except in specific areas corresponding to the lateral membrane of the ependymal cells (arrows, K). This confirmed basolateral expression of aquaporin-4 in the ependymal cells of hydrocephalic rats. In vessels (M-R) aquaporin-4 was clearly located to perivascular areas without signs of endothelial expression. No evidence of aquaporin-4 expression in microglia was found; neither in controls nor in hydrocephalic rats. * = lateral ventricle. Scale bar 50 μm.
Figure 9
Figure 9
Correlation between quantitative MRI and relative AQP4 expression. Plots of lateral ventricular volume vs. relative aquaporin-4 expression (A) and periventricular apparent diffusion coefficient value vs. relative aquaporin-4 expression (B) in both controls, hydrocephalic (two days, 2 D, one week,1W and two weeks, 2W) and non-hydrocephalic kaolin-injected animals. In hydrocephalic animals we found a significant linear positive correlation between lateral ventricular volume and periventricular aquaporin-4 expression (r2 = 0.33, p = 0.002). No correlation between periventricular apparent diffusion coefficient value and relative aquaporin-4 expression was found.
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