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. 2009 Jun;40(6):2182-90.
doi: 10.1161/STROKEAHA.108.523720. Epub 2009 Apr 16.

Acute vascular disruption and aquaporin 4 loss after stroke

Beth Friedman  1 , Christian Schachtrup, Philbert S Tsai, Andy Y Shih, Katerina Akassoglou, David Kleinfeld, Patrick D Lyden
Affiliations

Acute vascular disruption and aquaporin 4 loss after stroke

Beth Friedman et al. Stroke. 2009 Jun.

Abstract

Background and purpose: Ischemic protection has been demonstrated by a decrease in stroke-infarct size in transgenic mice with deficient Aquaporin 4 (AQP4) expression. However, it is not known whether AQP4 is rapidly reduced during acute stroke in animals with normal AQP4 phenotype, which may provide a potential self-protective mechanism.

Methods: Adult male rats underwent transient occlusion of the middle cerebral artery (tMCAo) for 1 to 8 hours followed by reperfusion for 30 minutes. Protein and mRNA expression of AQP4 and glial fibrillary acidic protein (GFAP) were determined by Western blot and rtPCR. Fluorescence quantitation was obtained with laser scanning cytometry (LSC) for Cy5-tagged immunoreactivity along with fluorescein signals from pathological uptake of plasma-borne high-molecular-weight fluorescein-dextran. Cell death was assessed with in vivo Propidium Iodide (PI) nucleus labeling.

Results: In the ischemic hemisphere in tissue sections, patches of fluorescein-dextran uptake were overlapped with sites of focal loss of AQP4 immunoreactivity after tMCAo of 1 to 8 hours duration. However, the average levels of AQP4 protein and mRNA, determined in homogenates of whole striatum, were not significantly reduced after 8 hours of tMCAo. Tissue section cytometry (LSC) of immunoreactivity in scan areas with high densities of fluorescein-dextran uptake demonstrated reductions in AQP4, but not in IgG or GFAP, after tMCAo of 2 hours or longer. Scan areas with low densities of fluorescein-dextran did not lose AQP4. There was sparse astrocyte cell death as only 1.7+/-0.85% (mean, SD) of DAPI labeled cells were PI- and GFAP-labeled after 8 hours of tMCAo.

Conclusions: During acute tMCAo, a rapid loss of AQP4 immunoreactivity from viable astrocytes can occur. However, AQP4 loss is spatially selective and occurs primarily in regions of vascular damage.

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Figures

Figure 1
Figure 1
Immunostaining of AQP4 after 1 hour of tMCAo. Panels on left are scans of a single section respectively imaged for Cy5-AQP4 immunoreactivity (top left), for pathological fluorescein-dextran uptake (middle left); the bottom left panel shows the merged image. Middle panels are views of the section that are converted to grey scale and inverted for clarity. Right panels show areas at arrows at higher magnification. The pale area that lacks Aquaporin 4 staining overlies a dense patch of high-molecular weight fluorescein dextran uptake.
Figure 2
Figure 2
Whole brain sections from cases with tMCAo of 1, 2, 4 or 8 hours duration imaged for AQP4 immunoreactivity (left panels) and for fluorescein-dextran uptake (right panels). The striatum in the ischemic hemisphere shows pale areas with reduced AQP4. Fluorescein images demonstrate overlapping areas with high molecular weight fluorescein-dextran uptake. In the ischemic neocortex, after 8 hours of tMCAo, periodic discontinuities emerge in the AQP4 immunoreactivity along with patches of fluorescein-dextran leakage. The contralateral cortex retains a normal, continuous AQP4 staining pattern. Scale bar = 1mm.
Figure 3
Figure 3
Average AQP4 mRNA and protein expression levels in homogenates of whole ischemic and contralateral striata after 8 hours of tMCAo as determined from Western blot and rtPCR analyses. (A) Quantitation of AQP4 and GFAP protein by Western blot from whole ischemic striata (I) and contralateral striata (C) after 8 hours tMCAo (n=4). Each lane was loaded with 30 μg of protein. Loading variations were controlled for by normalization to actin levels in each sample. Bars show mean +/− SEM. There were no significant changes in AQP4 or GFAP protein levels in ischemic striata relative to the contralateral side. (B) Quantitation of AQP4 and GFAP mRNA by rtPCR of whole ischemic and contralateral striata and cortices after 8 hours tMCAo (n=7). Gene expression data was normalized to GAPDH and presented as fold change (mean ± SE) versus the contralateral striata or cortex. The differences in bar height were not significant for AQP4 (ns). GFAP mRNA in ischemic striata and cortices was significantly greater than levels on the contralateral side (ANOVA, p<0.005). (C) High resolution images of fluorescein-dextran labeling (green) and perivascular AQP4 staining (red) in a microvessel after tMCAo of 8 hours. Rotated view (labeled X–Z) shows vessel in cross-section; note intensely fluorescent vessel walls labeled with fluorescein-dextran uptake, unlabelled vessel lumen and perivascular AQP4. Images illustrate heterogeneity of perivascular AQP4 expression in single sections of striata.
Figure 4
Figure 4
The use of laser scanning cytometry (LSC) to compare of the effects of increased occlusion duration on the frequency of scan areas that are co-labelled by fluorescein-dextran and immunoreactivity to AQP4, IgG or GFAP. (A1 and A2) illustrate a brain section that shows fluorescein-dextran labeling in ischemic hemisphere after 8 hours of tMCAo in a photomicrograph (A1) and in a companion LSC-generated digitization of this section (A2). (A3) Example of 40 μm-diameter scan areas used to quantify fluorescence. This panel illustrates positive scan areas which result from vascular uptake of high molecular weight fluorescein-dextran from a case after 2 hours of tMCAo. (A4) LSC generated scattergram with data from scan areas rastered across an entire section. Each quadrant provides the per cent area for 1 of 4 different types of counts: 1. The upper left quadrant demonstrates single-labeled Cy5 immunoreactivity; 2. The upper right quadrant demonstrates counting regions that are double positive for fluorescein-dextran and Cy5; 3. The lower right quadrant demonstrates counting regions single-labeled with fluorescein-dextran; 4. The lower left quadrant demonstrates counting regions with only background levels of signal. The double labeling indices for whole sections shown in graphs in panel (F) were determined from the per cent of total scan areas per section that are double positive (in the upper right quadrant) and this value is then normalized to the summed percent of scan areas with fluorescein-dextran label (obtained from the upper and lower right quadrants). (A5) Changes in the density of scan areas with fluorescein-dextran label as a function of occlusion duration of tMCAo. The number of cases that were assayed at individual occlusion durations was: 1 hour tMCAo, n=5; 2 hour tMCAo, n=6; 4 hour tMCAo n=5; 8 hour tMCAo, n=5. Different sections from each brain, at the level of the anterior commissure, were immunostained with AQP4, IgG, and GFAP. From 1 to 2 sections per stained series were LSC assayed for fluorescein-dextran uptake so that the dextran density index shown in panel (A5) represents an average value across 16 to 19 sections per occlusion duration. The dextran density index is the total number of fluorescein-dextran labeled scan areas divided by the total number of scan areas for that brain section. The areal density of fluorescein-dextran uptake increases significantly with occlusion duration (1 vs 4 hours p< 0.05 and 1 vs 8 hours; p<0.001: ANOVA followed by Tukey’s test). (B) Images of immunostaining merged with fluorescein-dextran label in a 50 μm section from one brain after 2 hours of tMCAo. Sections are within a 1 mm thick block. (B1) Fluorescein-dextran uptake concentrates among segments of microvessels. (B2) Higher magnification of inset (white box in B1) shows heterogeneity in label intensity in affected vessels of varying caliber. (C1) Fluorescent merged image of fluorescein-dextran labeled vasculature (green with yellow-green borders) and AQP4 immunoreactivity (red). (C2) The inset panel shows overlap of vessels marked by fluorescein-dextran uptake within the vessel (green), and by perivascular AQP4 which is yellow near vessels with fluorescein-dextran leakage or red near vessels that are unlabelled by fluorescein-dextran. (D1) Merged images of fluorescein-dextran labeled vasculature and IgG leakage (red). (D2) High magnification of merged image demonstrates nearly complete overlap of fluorescein-dextran fluorescence and Cy5-IgG immunoreactivity (yellow). This section is the same section shown in panels B1 and B2 where the fluorescein-dextran fluorescence appears green. Fluorescein-dextran labeled vessels are also surrounded by Cy5-IgG immunoreactivity, consistent with widespread extravasation of IgG after stroke. (E1) Fluorescent merged image of fluorescein-dextran labeled vasculature (green) and GFAP immunoreactivity (red). (E2) Fluorescein- dextran-labeled vasculature intersperses with densely stained GFAP stained astrocytes. (F) Comparison of the effect of increased occlusion duration on co-labeling with fluorescein-dextran and Cy5 labelled immunoreactivity for AQP4, IgG and GFAP in scan areas rastered across whole brain sections. Relative to the AQP4 double label index after 1 hour of tMCAo, the AQP4 double-label index was significantly reduced after 2, 4, and 8 hours of tMCAo. In contrast, the IgG and GFAP double-label index did not decline after 1 hour of tMCAo. Double-label index data was obtained from 5 to 6 sections (1–2 sections per rat brain) for each occlusion-duration.
Figure 5
Figure 5
Immunoreactivity of AQP4 as quantified with LSC, in brain areas with minimal uptake of high molecular weight fluorescein–dextran. (A) Regions with low levels of fluorescein-dextran signal on the ischemic and contralateral sides of the brain show similar levels of AQP4 immunoreactivity after 1 and 4 hours of tMCAo. Larger vessels in these images appear more densely stained than the network of microvessels. At 8 hours of tMCAo regions of low fluorescein-dextran are more heterogeneously stained and exhibit areas of reduced and elevated AQP4 immunoreactivity relative to the contralateral side. The ovoid structures in 8 hour images are clusters of myelinated fibers which typically show little AQP4 immunoreactivity (e.g. also illustrated by sparsely stained white matter tracts in Figure 2). (B) Laser scanning cytometry (LSC) quantitation of tissue fluorescence from rasterized scans of operator-defined subregions that encompass about 300 scan areas in regions with low or high levels of fluorescein-dextran signal (see methods). The y axis plots the number of scan areas with fluorescein signal divided by the total number of scan areas for that subregion. Subregions of low and high levels of fluorescein dextran were operator-segmented within brain sections. Data for total fluorescence measures was taken from 5 to 6 sections per occlusion duration (1–2 sections per rat brain). (C) Quantitation of AQP4 Cy5 signal in operator-defined subregions. The y axis plots the number of scan areas with Cy5 label divided by the total number of scan areas of that subregion. There was a progressive loss of AQP4 staining in the region of high leakage, but not in the regions of low leakage within the ischemic or contralateral hemispheres (repeated measures ANOVA, p<0.02).
Figure 6
Figure 6
Cell death induced by acute stroke was identified by in vivo uptake of circulating Propidium Iodide. A series of images of one field are shown. Panels A1 and A2 show DAPI stained nuclei in ischemic striatum (in A1) and the corresponding distribution of PI nuclear uptake consequent to loss of plasmalemma permeability barriers in dying cells (in A2). Merged image (white arrowhead) shows white halo at site of overlap of DAPI and PI. Panels B1 and B2 show the GFAP immunoreactivity of a cell co-labeled by PI (arrowhead) and in an adjacent PI negative cell (double arrow). (C) Pie chart of proportion of cells (DAPI positive) which are co-labeled with propidium iodide or GFAP. The blow-up chart on the right delineates the proportion of propidium iodide positive cells which are co-labeled by GFAP (in tan sector). Data are based on 2 sections per rat brain (n=3) in fields imaged at 60× in the ischemic striatum. The number of DAPI cells counted per rat brain was 594, 566 and 678.
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