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Comparative Study
. 2006 Sep 5;103(36):13532-6.
doi: 10.1073/pnas.0605796103. Epub 2006 Aug 28.

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

Affiliations
Comparative Study

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

Didrik S Frydenlund et al. Proc Natl Acad Sci U S A. .

Abstract

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Immunofluorescence analysis of brains subjected to MCAO (neocortex; 24 hr of reperfusion). (A and C) Contralateral neocortex, area opposite to ischemic core. (B and D) Central part of ischemic cortex (Fig. 5, region 4). Yellow labeling surrounding vessels in contralateral cortex indicates colocalization (arrows) of AQP4 (green) and dystrophin (red in A) and α-syntrophin (red in C). In contrast, vessels in the ischemic cortex are associated with a red signal (open arrowheads), indicating the absence of AQP4 and retention of dystrophin (B) and α-syntrophin (D). Filled arrowheads indicate the subpial endfeet. Dys, dystrophin; α-syn, α-syntrophin. (Scale bar: 20 μm.)
Fig. 2.
Fig. 2.
Immunogold analysis of AQP4 expression immediately (0 hr in AC) and 24 hr (DF) after the onset of reperfusion (0 hr). At 24 hr, there is a pronounced reduction in the number of gold particles (arrows) over perivascular membranes in central part of the ischemic cortex (F) compared with the border zone (E) and contralateral side (D). In the ischemic cortex, AQP4 labeling remains over the abluminal membrane of the perivascular endfeet (open arrows in F). E, endothelial cells; L, vessel lumen; P, pericyte. (Scale bar: 0.5 μm.)
Fig. 3.
Fig. 3.
Time course of AQP4 expression after MCAO. Values along the ordinate represent linear density of gold particles over perivascular membranes (same samples as in Fig. 2). Density values were obtained from the central part of the ischemic cortex (blue), striatal part of the core (green), and cortical border zone (red). The horizontal black line indicates the reference level (calculated from the neocortex and striatum contralateral to the lesion). The values for the ischemic cortex (blue) and striatal core (green) are significantly different from the reference values for reperfusion times of 24, 48, and 72 hr. The corresponding P values are 0.0058, 0.0122, and 0.0477 (cortex) and 0.0043, 0.0076, and 0.0052 (striatum), respectively.
Fig. 4.
Fig. 4.
Conventional freeze fracture images from contralateral cortex (A and B), cortical border zone (C and D), and central part of ischemic lesion (E and F), all from 24 hr of reperfusion after MCAO. (A) Low-magnification image from contact region between astrocyte endfoot (white asterisk) and the edge of capillary (black asterisk in cytoplasm). Boxed area encloses P-face of astrocyte, shown at higher magnification in B. Nu, endothelial nucleus. (B) High-magnification view of P-face of astrocyte endfoot plasma membrane, showing >100 AQP4 arrays in ≈0.3 μm2. (C) P-face of astrocyte endfoot in border zone has AQP4 arrays at approximately the same density as in contralateral control. (D) E-face view of AQP4 imprints, also from border zone. (E) Low-magnification view of capillary in central part of ischemic lesion. Membrane debris is present in the lumen of the capillary. Boxed area including presumptive astrocyte endfoot shown at higher magnification in F. The black asterisk indicates endothelial cytoplasm. (F) High-magnification image of E-face of presumptive astrocyte endfoot in central part of ischemic lesion. AQP4 arrays were not detected in this or any other membrane adjacent to capillaries. The black asterisk indicates endothelial cytoplasm. BD and F are at the same magnification. (Calibration bars: A and E, 1 μm; B, C, and F, 0.1 μm.)
Fig. 5.
Fig. 5.
Diagram of coronal slice through the forebrain. The shaded area on the right side is the area affected by ischemia. Specimens were dissected from the following: 1, contralateral neocortex (control); 2, contralateral striatum (control); 3, cortical border zone; 4, central part of ischemic cortex; and 5, striatal part of the ischemic core.

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