Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain

Abstract

During development and in various diseases of the CNS, new blood vessel formation starts with endothelial tip cell selection and vascular sprout migration, followed by the establishment of functional, perfused blood vessels. Here we describe a method that allows the assessment of these distinct angiogenic steps together with antibody-based protein detection in the postnatal mouse brain. Intravascular and perivascular markers such as Evans blue (EB), isolectin B4 (IB4) or laminin (LN) are used alongside simultaneous immunofluorescence on the same sections. By using confocal laser-scanning microscopy and stereological methods for analysis, detailed quantification of the 3D postnatal brain vasculature for perfused and nonperfused vessels (e.g., vascular volume fraction, vessel length and number, number of branch points and perfusion status of the newly formed vessels) and characterization of sprouting activity (e.g., endothelial tip cell density, filopodia number) can be obtained. The entire protocol, from mouse perfusion to vessel analysis, takes ∼10 d.

Nogo-A regulates vascular network architecture in the postnatal brain

“…To quantitatively assess these differences, we next determined the vascular volume fraction (percent of the tissue volume occupied by vessels) in the computationally reconstructed 3D datasets. The vascular volume fraction in WT animals in the different regions investigated was between 2% and 3.5% and thus very similar to previous studies of our 3,17 and other groups. 7,30 Moreover, the vascular volume fraction in Nogo-A (e-h) Quantification of the 3D vascular volume fraction in P10 cortex (e), hippocampus (f), superior colliculus (g), and brainstem (h) by computational analysis using global morphometry.…”

“…3,4,7,31,32 In particular, the commonly used immunofluorescent methods are limited with regard to their capacity to analyze the architecture of the brain vasculature in three dimensions or at the level of vascular networks. 3,6,7 To circumvent these limitations and in order to study the cerebral vasculature in three dimensions, we employed vascular corrosion casting in combination with highresolution, synchrotron radiation-based mCT and computational network analysis, techniques that provide quantitative, characteristic features of 3D blood vessel networks. 19,21,33,34 Previously, these or similar methods were only used in adult mice, for example to investigate the effects of VEGF 165 overexpression on angiogenesis in the adult mouse brain, 17 the vascular alterations in a mouse model of Alzheimer, 29 and to study microvascular networks in rat brain tumors.…”

“…Moreover, this also suggests that Nogo-A specifically represses the number of newly formed endothelial tip cells and capillary sprouts. How Nogo-A precisely acts in this regard remains unclear, but several underlying mechanisms are possible: Nogo-A may either directly inhibit the generation of endothelial tip cells [1][2][3][4] or may, once an endothelial tip cell has been formed, negatively influence its function, e.g., by antiadhesive or collapse-inducing effects on the filopodia and lamellipodia, or may negatively regulate the stabilization of the transient endothelial tip cell phenotype. We previously showed that the Nogo-A specific fragment Nogo-A Delta 20 suppressed brain endothelial cell sprouting and migration by inducing lamellipodia and filopodia retraction in vitro.…”

“…Nogo-A deficiency does not disrupt the morphologic integrity of the newly formed vessel segments Angiogenesis involves various cellular and molecular sub-mechanisms such as vessel sprouting, branching, endothelial cell proliferation and migration, and finally lumen formation [1][2][3][4][5] that could be affected in a different proportion in Nogo-A-deficient animals. For example, increased vessel sprouting and branching would result in more vessel segments, enhanced endothelial cell proliferation and migration would lead to increased vessel length, and increased vessel proliferation or lumen formation would be associated with a bigger vessel diameter.…”

Size-selective opening of the blood–brain barrier by targeting endothelial sphingosine 1–phosphate receptor 1

“…CNS vasculature undergoes angiogenesis and remodeling at early postnatal stages (19,20), alterations of which can affect subsequent BBB properties (2,21). To determine whether S1P 1 regulates the BBB directly or indirectly via vascular developmental defects (2, 21), we deleted endothelial S1pr1 in adult mice (>8 wk) and analyzed them for BBB function 4 wk after gene deletion.…”