Outer brain barriers in rat and human development

Abstract

Complex barriers at the brain's surface, particularly in development, are poorly defined. In the adult, arachnoid blood-cerebrospinal fluid (CSF) barrier separates the fenestrated dural vessels from the CSF by means of a cell layer joined by tight junctions. Outer CSF-brain barrier provides diffusion restriction between brain and subarachnoid CSF through an initial radial glial end feet layer covered with a pial surface layer. To further characterize these interfaces we examined embryonic rat brains from E10 to P0 and forebrains from human embryos and fetuses (6-21st weeks post-conception) and adults using immunohistochemistry and confocal microscopy. Antibodies against claudin-11, BLBP, collagen 1, SSEA-4, MAP2, YKL-40, and its receptor IL-13Rα2 and EAAT1 were used to describe morphological characteristics and functional aspects of the outer brain barriers. Claudin-11 was a reliable marker of the arachnoid blood-CSF barrier. Collagen 1 delineated the subarachnoid space and stained pial surface layer. BLBP defined radial glial end feet layer and SSEA-4 and YKL-40 were present in both leptomeningeal cells and end feet layer, which transformed into glial limitans. IL-13Rα2 and EAAT1 were present in the end feet layer illustrating transporter/receptor presence in the outer CSF-brain barrier. MAP2 immunostaining in adult brain outlined the lower border of glia limitans; remnants of end feet were YKL-40 positive in some areas. We propose that outer brain barriers are composed of at least 3 interfaces: blood-CSF barrier across arachnoid barrier cell layer, blood-CSF barrier across pial microvessels, and outer CSF-brain barrier comprising glial end feet layer/pial surface layer.

Keywords: SSEA-4; YKL-40; arachnoid blood-CSF barrier; development; outer CSF-brain barrier; pial microvessel blood-CSF barrier; subarachnoid space.

Figure 1

Distribution of claudin-11 immunoreactivity in sagittal sections of E18 rat (A,B) and 21st wpc human (C) brain. (A) Immunostaining for claudin-11 in a developing rat brain at E18 demonstrates a strong reactivity of the entire arachnoid barrier cell layer (= arachnoid blood-CSF barrier (aB-CSFB)) (arrowheads) in marked contrast to unstained leptomeningeal cells in the subarachnoid space (SAS) and in the entire brain. Cisterns at the base of the brain and cisterna magna (CM) are well-developed. Note the very strongly stained arachnoid covering of the tentorium cerebelli (TC) and the interrupted barrier cell layer where the trigeminal nerve perforates the dura-arachnoid (arrow). The area outlined with a rectangle is shown at higher magnification in (B) and demonstrates that the barrier cell layer (aB-CSFB) covering the forebrain consists of a strongly stained single cell layer. (C) At mid-gestation the human fetal arachnoid barrier cell layer (aB-CSFB) is also formed by a single claudin-11 positive cell layer. In (B,C) note that neither the E18 rat radial glial end feet layer (EFL) and the pial surface layer forming the outer CSF brain barrier (oCSF-BB) facing the subarachnoid space (SAS) nor the human fetal radial glial end feet layer (EFL) and pial surface layer show claudin-11 immunoreactivity. Scale bars: (A) 1000 μm, (B) 50 μm, (C) 100 μm.

Figure 2

Arachnoid blood-CSF barrier, subarachnoid space, pial surface, and glial end feet layers immunostained for collagen 1 (A) and double immunolabeled with antibodies against BLBP and SSEA-4 (B,C) from occipital cortex of a 21st wpc human fetus. (A) Immunostaining for collagen 1 depicts the limitation of the subarachnoid space (SAS) of a 21st wpc human fetus. The arachnoid barrier cell layer (aB-CSFB) is unstained (arrows) and the strongly immunostained pial surface layer (PSL) faces an unstained glial end feet layer. The basement membranes of the pial microvessels (BV) loose their stainability abruptly when the vessels enter the end feet layer and fuse with the inner-most part of the PSL (arrowheads). (B) In a section adjacent to that shown in (A), the entire end feet layer is immunolabeled with anti-BLBP corresponding to the radial glial end feet. Note the spaces in the end feet layer where blood vessels penetrate into the parenchyma. (C) Double-labeling with antibodies against BLBP and SSEA-4, nuclear stained with DAPI, reveals an evenly distributed co-localization in the end feet layer. Where pial microvessels penetrate into the marginal zone, the perivascular canals are lined with BLBP, revealing Virchow-Robins space (illustrated with rectangles in B,C). The subarachnoid space contains SSEA-4 positive leptomeningeal cells but shows no BLBP reactivity. (B,C) same magnification. Scale bars: 100 μm.

Figure 3

Arachnoid blood-CSF barrier, subarachnoid space, pial surface, and glial end feet layers double immunolabeled with antibodies against YKL-40 and SSEA-4 (A–C) in parietal cortex of a 15th wpc human fetus. Parietal cortex of a 15th wpc fetus, immunolabeled with anti-YKL-40 (A) and anti-SSEA-4 (B), counterstained with DAPI and merged (A1,B) in (C). The images are representations of a stacked series of 10 steps, with 1 μm intervals, processed as maximum projection intensity. The staining of the end feet layer (EFL) shows multiple delicate and curled end feet membranes. YKL-40 immunoreactivity is also present in the arachnoid blood-CSF barrier (aB-CSFB in (A) and in leptomeningeal cells in the subarachnoid space (arrows in A1). In (B) the pattern of SSEA-4 distribution is seemingly equal to that of YKL-40, and the merged image of the stacked images confirms the co-localization of YKL-40 and SSEA-4 in leptomeningeal cells within the subarachnoid space (SAS) and in the end feet layer of the radial glial cells (C). Scale bar: 10 μm.

Figure 4

Marginal zone in parietal cortex of a 15th wpc human fetus double immunolabeled with antibodies against YKL-40 and IL-13Rα2 (A) and in occipital cortex of a 21st wpc human fetus (B,C) labeled with antibodies against EAAT1 (B) and against EAAT1 and YKL-40 (C). Parietal cortex of a 15th wpc fetus, immunolabeled with antibodies against IL-13Rα2 and YKL-40, merged and nuclear counterstained with DAPI (A). The YKL-40 immunoreactive end feet layer contains evenly distributed interleukin-13Rα2 receptors (white arrowheads). (B) The excitatory amino acid transporter (EAAT1) is visualized in occipital cortex of a 21st wpc fetus, below the subarachnoid space (SAS) within the end feet layer (EFL) and in surrounding parenchymal blood vessels (BV). The merged image with YKL-40 (C) shows a similar distribution within the end feet layer and around the blood vessels. (A) Scale bar: 20 μm. (B,C) same magnification. Scale bar: 50 μm.

Figure 5

Glia limitans in adult human brain stained for GFAP, MAP2, and YKL-40 (A–C). Adult human cerebral cortex immunostained for GFAP in (A) depicts the glia limitans (GL) as a dense multilayered network of GFAP positive astrocytic processes and few small fibrous astrocytes (arrowheads). Note the larger protoplasmic astrocytes (arrows) deeper in the molecular layer. Protrusions of astrocytic processes, which look like remnants of end feet (RE) are seen as patches facing the subarachnoid space (SAS). Immunostaining for MAP2 (B) defines the inner border of glia limitans (GL) toward the rest of the molecular layer and the unstained outer border with the empty-looking protrusions/remnants of end feet (RE) toward the subarachnoid space (SAS) is clearly depicted. Immunoreactivity for YKL-40 of glia limitans (GL) is seen in (C). Many protrusions exhibit distinct apical membrane reactivity for YKL-40, and YKL-40 positive spheroid bodies corresponding to corpora amylacea are seen within the glia limitans but also in the border zone toward the MAP2 positive part of the molecular layer (arrowheads). (A–C) same magnification. (A) Scale bar: (A) 20 μm.