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. 2017 Jul 24;127(8):3136–3151. doi: 10.1172/JCI91301

Figure 2. CLDN4 and JAM-A are expressed within TJ strands of reactive astrocytes in vivo.

Figure 2

(A) Immunostaining within an AdIL-1 lesion at 7 dpi for GFAP (green), the basement membrane marker pan-laminin (blue), and the astrocytic TJ protein CLDN4 (red) demonstrates CLDN4 expression at reactive astrocytic endfeet surrounding the vasculature (arrows). Scale bar: 10 μm. See also Supplemental Figure 1C. (B) Transmission electron microscopy of astrocytic endfeet within cortical AdIL-1 and AdCtrl injection sites demonstrates TJs in AdIL-1 lesions but not in controls. Immunogold staining shows colocalization of CLDN4 and JAM-A to the TJ structures (arrows point to gold particles). A, astrocyte; EC, endothelial cell. Original magnification, ×10,000. (C) Immunostaining within an EAE lesion at 21 days for GFAP (blue), pan-laminin (red), and CLDN4 (green) demonstrates the structural organization of the reactive GL. This cross section shows basement membranes of the endothelial BBB (EBM) and astrocytic GL (ABM), demarcated by pan-laminin staining and differentiated by astrocytic endfeet, stained by GFAP and CLDN4. Leukocytes, identified in gray based on morphologic features and DAPI nuclear staining, are seen within the endothelial lumen (LUM) and PVS. White arrows highlight colocalization of CLDN4 and GFAP; pink arrows indicate areas of irregular CLDN4 staining, possibly reflecting irregularities of expression in the plane of staining or degradation in proximity to leukocytes. Scale bars: 10 μm. See also Supplemental Figure 1, D and E. (D) Schematic of the endothelial BBB and astrocytic GL in health and inflammatory disease. Under healthy conditions, endothelial cells express TJ proteins CLDN5 and occludin (OCLN), which reinforce a closed BBB. In response to inflammation, CLDN5 and OCLN are downregulated, opening the BBB. In turn, astrocytes of the GL upregulate TJ proteins CLDN1, CLDN4, and JAM-A, closing the GL and restricting incoming leukocytes to the PVS (blue). Data are representative of findings from at least 3 (A–C) biological replicates.