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Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We investigated cerebral cortex microvessels during immune-mediated demyelination in the MS model chronic murine experimental autoimmune encephalomyelitis (EAE) by immunolocalization of the endothelial cell tight junction (TJ) integral proteins claudin-5 and occludin, a structural protein of caveolae, caveolin-1, and the blood-brain barrier-specific endothelial transporter, Glut 1. In EAE-affected mice, there were areas of extensive subpial demyelination and well-demarcated lesions that extended to deeper cortical layers. Activation of microglia and absence of perivascular inflammatory infiltrates were common in these areas. Microvascular endothelial cells showed increased expression of caveolin-1 and a coincident loss of both claudin-5 and occludin normal junctional staining patterns. At a very early disease stage, claudin-5 molecules tended to cluster and form vacuoles that were also Glut 1 positive; the initially preserved occludin pattern became diffusely cytoplasmic at more advanced stages. Possible internalization of claudin-5 on TJ dismantling was suggested by its coexpression with the autophagosomal marker MAP1LC3A. Loss of TJ integrity was confirmed by fluorescein isothiocyanate-dextran experiments that showed leakage of the tracer into the perivascular neuropil. These observations indicate that, in the cerebral cortex of EAE-affected mice, there is a microvascular disease that differentially targets claudin-5 and occludin during ongoing demyelination despite only minimal inflammation.

Cerebral cavernous malformations (CCMs) often cause hemorrhages that can result in severe clinical manifestations, including hemiparesis and seizures. The underlying mechanisms of the aggressive behavior of CCMs are undetermined to date, but alterations of vascular matrix components may be involved. We compared the localization of the tight junction proteins (TJPs) in 12 CCM specimens and the expression of glucose transporter 1 (GLUT-1), which is sensitive to alterations in TJP levels, in 5 CCM specimens with those in 5 control temporal lobectomy specimens without CCM by immunofluorescence microscopy. The TJPs occludin, claudin-5, and zonula occludens ZO-1 were downregulated at intercellular contact sites and partly redistributed within the surrounding tissue in the CCM samples; there was also a marked reduction of GLUT-1 immunoreactivity compared with that in control specimens. Corresponding analysis using quantitative real-time reverse transcription polymerase chain reaction on 8 CCM and 8 control specimens revealed significant downregulation of mRNA expression of occludin, claudin-5, ZO-1, and GLUT-1. The altered expression and localization of the TJPs at interendothelial contact sites accompanied by a reduction of GLUT-1 expression in dilated CCM microvessels likely affect vascular matrix stability and may contribute to hemorrhages of CCMs.

Gliomas, particularly glioblastoma multiforme, perturb the blood-brain barrier and cause brain edema that contributes to morbidity and mortality. The mechanisms underlying this vasogenic edema are poorly understood. We examined the effects of cocultured primary cultured human glioblastoma cells and glioma-derived growth factors on the endothelial cell tight junction proteins claudin 1, claudin 5, occludin, and zonula occludens 1 of brain-derived microvascular endothelial cells and a human umbilical vein endothelial cell line. Cocultured glioblastoma cells and glioma-derived factors (e.g. transforming growth factor beta2) enhanced the paracellular flux of endothelial cell monolayers in conjunction with downregulation of the tight junction proteins. Neutralizing anti-transforming growth factor beta2 antibodies partially restored the barrier properties in this in vitro blood-brain barrier model. The involvement of endothelial cell-derived matrix metalloproteinases (MMPs) was demonstrated by quantitative reverse-transcriptase-polymerase chain reaction analysis and by the determination of MMP activities via zymography and fluorometry in the presence or absence of the MMP inhibitor GM6001. Occludin, claudin 1, and claudin 5 were expressed in microvascular endothelial cells in nonneoplastic brain samples but were significantly reduced in anaplastic astrocytoma and glioblastoma samples. Taken together, these in vitro and in vivo results indicate that glioma-derived factors may induce MMPs and downregulate endothelial tight junction protein and, thus, play a key role in glioma-induced impairment of the blood-brain barrier.

During brain development and blood-brain barrier (BBB) differentiation the expression of P-glycoprotein (P-gp) may complement the protective function of the placental barrier against xenobiotic substances. To establish an immunohistochemical procedure for P-gp detection, different anti-P-gp monoclonal antibodies were first tested on a fibrosarcoma cell line and colonic carcinoma tissue. The protocol was then tested on adult human brains as a BBB-P-gp tissue-specific control and for double labeling with anti-P-gp and the astroglia marker glial fibrillary acidic protein (GFAP). The protocol was then used to analyze the expression and localization of P-gp in human fetuses during cerebral cortex formation. At the earliest examined stage, 12 weeks of gestation (wg), P-gp was detectable as diffuse cytoplasmic labeling of the endothelial cells lining the primary cortex microvessels. At 18 wg, a punctate P-gp staining pattern was detected on cortex and subcortical vessels and on their side branches. At 22 wg, P-gp staining was linear and concentrated on endothelial cell membranes. In all examined ages, GFAP-positive radial glial cells and astrocytes did not stain for P-gp, even at their perivascular processes, whereas faint P-gp labeling was seen on vimentin-reactive radial glia at the earliest examined fetal age. At midgestation, P-gp colocalized with caveolin-pY14 on the abluminal endothelial cell membrane. These results demonstrate that P-gp is expressed early during human cerebral cortical microvessel development, and suggest that at midgestation there may be efflux activity that is regulated by interactions with the caveolar endothelial cell compartment.