Fig 2: Schematic representation of the expression of AQP1 and AQP4 in 6 months and 12 months WKY and SHR rats. In 6 months and 12 months WKY (a,c), rat expression of AQP 1 is detected in the apical pole of the ChP epithelium associated with active production of CSF. It is also found in the CSF, most likely associated with EVs. Per contra, AQP4 is expressed in the astrocyte end-feet and the basolateral membrane of the ependymal cells associated with brain CSF homeostasis. In addition, it is found in the CSF. In 6 months SHR rats (b), AQP1 expression is significantly increased in the apical pole of the ChP epithelium, and AQP4 expression increases in the astrocytes end-feet but not in the ependymal cells. This AQP disbalance may contribute to the mechanisms that trigger ventriculomegaly. In the CSF, the expression of AQPs remains the same as in 6 months WKY. In the 12 months SHR rats (d), AQP1 expression significantly decreased in the ChP epithelium while proportionally increasing in the CSF compared to 12 months WKY (c). Thus, it could reduce CSF production as a possible compensatory mechanism for ventriculomegaly. In turn, AQP4 decreased in the ependymal layer while it increased in the CSF and in the astrocytes’ end-feet, which could also contribute to reducing the production of CSF (ependymal layer) and the increase of the CSF absorption (astrocyte end-feet).

Fig 3: Confocal microscopy images of AQP4 in the ependymal cells of the lateral ventricle of WKY and SHR rats. A slight decrease of AQP4 was detected in the ependyma of SHR in the 6 months group when compared to WKY (a–d), while in the 12 months group, a significant decrease of AQP4 was observed in the SHR (e–h) rats. White arrows point to AQP4 immunoreactive material (b). At the bottom, stain intensities in relative units (gray units) for AQP4 (i,j) are represented as the means ± SD (n = five animals per group). The differences between WKY and SHR were significant when applied to a One-way ANOVA test with post hoc analysis using the Tukey post hoc test (* p < 0.05). LW: lateral wall; SVZ: subventricular zone; EP: ependyma; LV: lateral ventricle; GU: grey units. Scale bars 20 μm.

Fig 4: Confocal and optical microscopy images of AQP4 in the blood vessels of brain parenchyma of WKY and SHR rats. A significant increase of AQP4 was observed in SHR in the 6 months group when compared to WKY (a,b,e,f), in the 12 months group was found a more significant presence of AQP4 in the SHR (c,d,g,h) rats. Stain intensities in relative units for AQP4 (i,j) are represented as the means ± SD (n = five animals per group). The differences between WKY and SHR were significant when applied to a One-way ANOVA test with post hoc analysis using the Tukey post hoc test (* p < 0.05). BV: blood vessel; GU: grey units. Scale bars 10 μm.