Deletion of astroglial connexins weakens the blood-brain barrier

Abstract

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood-brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and β-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.

Figure 1

Developmental expression of Cx43 in astrocyte endfeet. Confocal microscopy projections in the mouse cortex performed at the indicated ages. Cx43 immunostaining is shown in green. Endothelial cells are labeled with Pecam1 (red), and astrocyte endfeet enwrapping blood vessels are labeled with aquaporin-4 (Aqp4) (blue). Images in (a) and (b) are enlarged views of the boxed areas, which correspond to vessels with a diameter of 10 to 20 μm and capillaries (5 μm), respectively. Scale bars: 10 μm.

Figure 2

Developmental expression of Cx30 in astrocyte endfeet. Confocal microscopy projections in the mouse cortex performed at the indicated ages. Cx30 immunostaining is shown in green. Endothelial cells are labeled with Pecam (red), and astrocyte endfeet enwrapping blood vessels are labeled with aquaporin-4 (Aqp4) (blue). Images in (a) and (b) are enlarged views of the boxed areas, which correspond to vessels with a diameter of 10 to 20 μm and capillaries (5 μm), respectively. Scale bars: 10 μm.

Figure 3

Ultrastructural characterization of the gliovascular interface in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30. In the cortex of WT mice, thin perivascular endfeet with a dense intracellular content were in close contact with the basal lamina surrounding the microvessels. In DKo mice cortex, perivascular astrocytes appeared systematically swollen and edematous (asterisks). Images on the right are enlarged views of the endothelial tight junction (TJ) areas (rectangles) in WT and DKo. Note that the morphology of TJs was comparable in WT and DKo. a, denotes astrocyte endfeet; b, denotes basal lamina; e, denotes endothelial cell. Scale bars: 1 μm (left images) and 100 nm (right images).

Figure 4

Comparison of aquaporin-4 (Aqp4) and β-dystroglycan levels in hippocampus of wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30. Protein extracts from hippocampus were analyzed on Western blots using anti-β-dystroglycan anti-Aqp4, and anti-GAPDH (loading control) antibodies. The averaged ratio of β-dystroglycan (β-DG) or Aqp4 with GAPDH was calculated from three independent experiments. In DKo mice, β-DG and Aqp4 quantity was reduced by about half compared with WT. Data are mean values±s.d., ^*P<0.05 comparing WT and DKo mice. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Comparison of brain vascular volume in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30 by in situ brain perfusion assays. [¹⁴C]sucrose distribution volume in the brain of WT and DKo mice were measured with albumin-free-bicarbonate buffer (open bars) and with albumin addition (40 g/L; filled bars) delivered by in situ brain perfusion at 2.5 mL/min for 120 seconds. Data are mean values±s.d. of five animals. ^*P<0.05 comparing WT and DKo mice; ^†P<0.05 comparing DKo with and without albumin perfusion fluid.

Figure 6

Ultrastructural characterization of the gliovascular interface interface in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30 under high vascular pressure conditions. (A) The brain sections of WT and DKo mice subjected to in situ brain perfusion with albumin and horseradish peroxidase (HRP) revealed with DAB. A massive extravasation of HRP was observed in many areas of DKo brain compared with WT brain. (B) Transmission electron microscopy images of cortex ultrathin sections of WT and DKo mouse brains subjected to in situ brain perfusion with albumin and HRP. In WT mice, almost no sign of HRP extravasation was detected. In DKo mice, perivascular astrocytes appeared systematically swollen and edematous (asterisks), and a strong HRP staining was present at the level of the basal lamina around vessels (arrows) and in intercellular spaces in the parenchyma. a, astrocyte endfeet; e, endothelial cell. Scale bars: 5 μm. DAB, 3,3′-diaminobenzidine.