Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice

Glia. 2003 May;42(3):235-51. doi: 10.1002/glia.10216.


In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Animals
  • Antigens, Surface / metabolism
  • Aquaporin 4
  • Aquaporins / metabolism
  • Astrocytes / metabolism*
  • Astrocytes / pathology
  • Astrocytes / ultrastructure
  • Biomarkers
  • Blood-Brain Barrier / genetics*
  • Brain / blood supply*
  • Brain / metabolism*
  • Brain / physiopathology
  • Claudin-1
  • Disease Models, Animal
  • Down-Regulation / genetics
  • Endothelium, Vascular / metabolism*
  • Endothelium, Vascular / pathology
  • Endothelium, Vascular / physiopathology
  • Female
  • Horseradish Peroxidase
  • Immunohistochemistry
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred mdx
  • Microscopy, Electron
  • Muscular Dystrophy, Duchenne / metabolism*
  • Muscular Dystrophy, Duchenne / pathology
  • Muscular Dystrophy, Duchenne / physiopathology
  • Phosphoproteins / metabolism
  • Tight Junctions / metabolism*
  • Tight Junctions / pathology
  • Tight Junctions / ultrastructure
  • Zonula Occludens-1 Protein


  • Actins
  • Antigens, Surface
  • Aqp4 protein, mouse
  • Aquaporin 4
  • Aquaporins
  • Biomarkers
  • CLDN1 protein, human
  • Claudin-1
  • Cldn1 protein, mouse
  • Membrane Proteins
  • Phosphoproteins
  • TJP1 protein, human
  • Tjp1 protein, mouse
  • Zonula Occludens-1 Protein
  • Horseradish Peroxidase