Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference

Abstract

Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels.

Figure 1

Hypothesis for apparent diffusion coefficient (ADC) value changes in brain tissue. Schema depicting the standard (A, B) and our working hypothesis (C, D) to explain decreased brain ADC values following silencing of AQP4 expression. Classically, decreased ADC is associated with decreased extracellular space due to cellular swelling (B). The water channel, AQP4, is expressed in astrocyte membranes and can facilitate water movement (C). After silencing APQ4, we hypothesize that lower ADC values are caused by decreased water permeability due to a decreased number of AQP4 channels (D).

Figure 2

In vitro application of small interference RNA against AQP4 (siAQP4) decreases AQP4 expression. (A) AQP4 expression was quantified from dot blot experiments (inset) in primary astrocyte cultures treated with siGLO (control) and siAQP4. AQP4 expression was decreased 3 days after siAQP4 application (76%±4%, ^**t-test, P<0.001, n=6). (B) AQP4 (red) and GFAP (green) immunolabeling in primary astrocyte cultures treated with siGLO (control, n=6) and siAQP4 (n=6) showed no morphological differences between groups. Nuclei were stained with DAPI (blue). (C) Confocal images of AQP4 immunoreactivity (AQP4-ir) in CA1 of control (siGLO, C1) and siAQP4-treated organotypic hippocampal slices (C2) demonstrated a large decrease in AQP4 staining. (D) Optical densities of immunohistochemistry from organotypic slices showed a significant decrease in AQP4-ir from siAQP4-treated slices (67%) compared with siGLO-treated slices (analysis of variance and Tukey–Kramer multiple comparisons tests, ^**P<0.001, n=8). These results provided the basis for our small interference RNA (siRNA) transfection protocol for in vivo experiments. (E) AQP4 expression quantified from Western blot experiments (inset) in organotypic hippocampal slice cultures treated with siGLO (control), or siAQP4. Treatment with siAQP4 decreased AQP4 levels (inset: red) (54%±7%) compared with siGLO-treated (i.e., control) hippocampal slices (Kruskal–Wallis test, nonparametric analysis of variance, ^*P<0.05, n=6). GFAP, glial fibrillary acid protein.

Figure 3

Small interference RNA (siRNA) for AQP4 diffuses within brain tissues. (A) Diffusion of nontargeted siRNA (siGLO) tagged with CY3 in a rat coronal section at the site of injection. CY3-siGLO (large arrow) was observed in the cortex at the site of injection and siRNA diffused within the brain parenchyma toward the contralateral striatum via the corpus callosum (CC, small arrows and inset box, higher magnification B2). The presence of siRNA was also detected at the surface (B1) of the ipsilateral and contralateral cortex (arrow heads) and within the ipsilateral striatum (not shown). (B) Confocal images (B1, B2, B3) of GFAP (green) and siGLO-CY3 (red) immunostaining at 3 days after the initial injection. (B1) Double staining revealed positive CY3 in astrocytes (arrows) around blood vessels and also in the glia limitans (arrowheads) in close proximity to the injection site. Several astrocytes were transfected by the tagged siRNA. (B2) In the CC, siRNA was detected in astrocytes (arrows), showing that the siRNA is able to diffuse via the CC to the contralateral hemisphere. (B3) Far from the injection site, siGLO-CY3 was observed in astrocytes in contact with blood vessels (arrows) within the contralateral striatum. GFAP, glial fibrillary acid protein.

Figure 4

Efficiency of AQP4 inhibition with small interference RNA against AQP4 (siAQP4). (A) AQP4 expression in siGLO- and siAQP4-treated rats was analyzed by Western blot where a specific band at 30 kDa was observed (red) in siGLO-treated rats. The intensity of the signal was decreased in siAQP4-treated rats, with no change in the intensity of the actin band (green). (B) Expression of AQP4 (n=4) was decreased (0.9±0.09 arbitrary units, A.U.) in siAQP4 compared with siGLO-treated animals (1.22±0.11 A.U., ^*P<0.05, unpaired t-test). (C) AQP4 immunolabeling was also performed to examine variations in AQP4 expression in situ. (C1, C2) Confocal images of AQP4 staining in siGLO- (C1) and siAQP4 (C2)-treated rats showed a significant decrease in the intensity of AQP4 staining in the ipsilateral cortex of siAQP4-treated rats (P<0.05, unpaired t-test). (D) AQP4 labeling in the contralateral striatum of the siGLO- (D1) and siAQP4-treated rats (D2) revealed decreased AQP4 expression. These immunohistochemical results are in accordance with the Western blot decreases in AQP4 expression (Figures 2A and 2B). (E) AQP4 immunolabeling (n=6) was quantified using optical densitometry demonstrating a decrease in all brain areas (^*P<0.05 and ^**P<0.001, unpaired t-test). The color reproduction of this figure is available on the html full text version of the manuscript.

Figure 5

Effects of small interference RNA against AQP4 (siAQP4) injection on neuronal survival and blood–brain barrier (BBB) integrity. (A) Neuronal changes were evaluated using NeuN (red) immunolabeling and counterstained with DAPI staining for cell nuclei (blue) in the ipsilateral cortex of siGLO- (A1) and siAQP4 (A2)-treated rats. (B) The number of NeuN-positive cells in four different regions of interest (ROIs) was not significantly altered in the siAQP4-treated compared with the siGLO-treated rats (analysis of variance, n=5 rats). In ipsi-cortex, the number of neurons in siAQP4-treated rats (1,139±30 neurons per mm²) was higher than in the siGLO rats (998±62 neurons per mm², analysis of variance, P=0.4). Similarly, no significant results were seen in the contralateral striatum (1,145±38 neurons per mm²) of siAQP4-treated versus siGLO-treated rats (1,229±53 neurons per mm², analysis of variance, P=0.2). The data are consistent with the idea that siAQP4 is not toxic in brain regions where small interference RNA (siRNA) is detected. (C) IgG staining was used to evaluate BBB integrity 3 days after siGLO and siAQP4 injection in four ROIs (ipsilateral and contralateral cortex and striatum). In siGLO-treated rats, IgG staining was increased within the ipsilateral cortex (162%±16%, analysis of variance and Tukey–Kramer multiple comparisons tests, ^*P<0.05, n=5) compared with controls and siAQP4-treated rats (128%±16%). In the other ROIs, there were no significant modifications of IgG staining compared with control values suggesting that the BBB was not affected.

Figure 6

Small interference RNA against AQP4 (siAQP4) has no effect on astrocyte morphology. (A, B) Astrocyte morphology using GFAP staining following AQP4 silencing was evaluated in animals undergoing neuroimaging. GFAP staining was examined in siGLO-treated rats (A1, B1) and in siAQP4-treated rats (A2, B2) in the ipsilateral cortex adjacent to the injection site (A1, A2) and in contralateral striatum (B1, B2). There was increased GFAP staining intensity in siGLO-treated (A1) compared with siAQP4-treated rats (A2) in the ipsilateral cortex, that was confirmed by of fluorescence quantification (C). In the contralateral striatum (contra-striatum), GFAP staining in siGLO-treated rats (B1) and in siAQP4-treated rats (B2) showed no differences in staining intensity. The presence of siAQP4 did not significantly affect the morphology of the astrocytes in the ipsilateral cortex (A1, A2) or contralateral cortex (B1, B2). (C) Quantification of GFAP-IR showed a significant increase in the ispilateral cortex (ipsi-cortex) of the siGLO-treated rats (7.95±0.40 A.U.) compared with the siAQP4-treated rats (4.63±0.43 A.U., analysis of variance and Tukey–Kramer multiple comparisons tests, ^*P<0.05, n=7) demonstrating that siAQP4 prevents an increase in GFAP expression. (D) Astrocyte morphology was quantified using convexity factor and demonstrated that the presence of siAQP4 did not affect astrocyte morphology identical to our in vitro results. GFAP, glial fibrillary acid protein; IR, immunoreactivity.

Figure 7

Apparent diffusion coefficient (ADC) and T2 values after small interference RNA against AQP4 (siAQP4) injection. (A) The in vivo ADC was calculated in siGLO- and siAQP4-treated rats from anterior to posterior brain regions. A global decrease in ADC values was observed at several levels (+1, injection, −1, −2, −3). Enlarged ADC images focusing on the contra-cortex showed a decrease in ADC in siAQP4 compared with siGLO-treated rats (right panel). Arrows illustrate decreased ADC location in the cortex (dotted line indicates corpus callosum). The quantification of the ADC was performed at the site of injection at the same level used for the histology. (B) The in vivo T2 was recorded in siGLO- and siAQP4-treated rats from anterior to posterior brain regions. No changes were observed within the brains of both groups. (C) In the ipsilateral cortex (ipsi-cortex), ADC values in siAQP4-treated rats were significantly decreased to 60.8±2.5 compared with 91.1±2.5 × 10⁻⁵ mm²/s in siGlo rats representing a 33% reduction (^*P<0.05, analysis of variance and Tukey–Kramer multiple comparison test, n=4). Similarly, a 50% decrease was observed in ipsilateral striatum and in contralateral cortex and striatum. In the contralateral striatum, ADC values were 45.4±1.7 in siAQP4-treated rats compared with 92.1±1.0 × 10⁻⁵ mm²/s in siGLO representing 51% decrease (^**P<0.01, analysis of variance and Tukey–Kramer multiple comparison test). (D) T2 values, measured in siGLO- and siAQP4-treated rats were not significantly different between brain regions suggesting that water content was not changed.