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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.

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Figure 1: Flowchart, summary and time frame of the protocol.
Figure 2: Intracardial EB injection and brain dissection.
Figure 3: Visualization of blood vessel endothelium and endothelial tip cells in the postnatal mouse brain at P8.
Figure 4: Discrimination between perfused blood vessels and newly forming sprouting blood vessels and endothelial tip cells in the P8 mouse cortex.
Figure 5: Stereological analysis of the vascular network in the P8 mouse cortex.
Figure 6: Characterization of endothelial tip cells in the postnatal mouse brain at P8.

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Acknowledgements

We thank L. Slomianka for help with stereological analysis and A. Wacker for critical reading of the manuscript. T.W. was supported by an MD-PhD fellowship of the Swiss National Science Foundation, by the Olga Mayenfisch Foundation, the Hartmann Muller Foundation, the EMDO Foundation and by the MD-PhD student allowance of the Swiss Society for Microvascular Research (SSMVR). J.V. was supported by the Swiss National Science Foundation (no. 310000 120321/1). All the animal experiments were conducted in J.V.'s laboratory and were approved by the Veterinary office of the Canton of Zurich. The histological studies were performed in M.E.S.'s laboratory. Microscopy image acquisition and analysis was performed in M.E.S.'s laboratory and at the Center for Microscopy and Image Analysis, University of Zurich.

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Contributions

T.W. and J.V. designed the experiments and wrote the manuscript. T.W., O.W. and J.V. conducted the experiments. T.W. and J.M.M. analyzed the data. J.V. and M.E.S. supervised the experiments in their respective laboratories. D.B. helped with the endothelial tip cell-marker experiments and gave critical inputs to the manuscript. H.G., S.P.H. and L.R. gave critical inputs to the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Thomas Wälchli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimization of the tissue fixation protocol to combine EB perfusion and IB4 endothelial tip cell staining in the postnatal mouse brain.

a-l Labeling of EB-perfused P8 mice with biotinylated IB4 in combination with immunfluorescence (e.g. LN to label the basement membrane). Different fixation methods of EB-perfused P8 mice to optimize the combination of EB perfusion with IB4 endothelial tip cell labeling and antibody stainings were tested. Combinations of immersion-fixation with 4% FA and different amounts of GA were used. Subsequently, 40 µm coronal brain sections were labeled with biotinylated IB4 to visualize blood vessel endothelial cells (red) and with an antibody against LN (green) to test if antigenicity is still present despite GA treatment (a-l,). EB (perfused blood vessels, cyan). Whereas postfixation in 4% FA only (no GA) resulted in a considerable loss of the EB signal (a-d), the combination of 4% FA + 0.025% GA (e-h) or 4% FA + 0.05% GA (i-l) were optimal for combination of the different label procedures (IB4, LN) with EB-perfused vessels.

Scale bars: 50 µm (a-l).

Supplementary Figure 2 Classical endothelial tip cell markers in direct comparison to IB4 labeling in the P8 mouse brain cortex.

Immunofluorescent labeling of 40 µm P8 coronal mouse brain sections labeled with the classical endothelial tip cells markers VEGFR2 (a-d, green), VEGFR3 (e-h, green) and Dll4 (i-l, green) and with biotinylated IB4 to visualize blood vessel endothelial (tip) cells (red). Cell nuclei (DAPI, blue).

a-l IB4 as well as antibodies against VEGFR2 (a-d), VEGFR3 (e-h) or Dll4 (i-l) visualize CNS blood vessel structures and endothelial tip-, stalk-, and phalanx cells. Boxed areas (a,b,e,f,i,j) highlight endothelial tip cells that are enlarged on the right hand (c,d,g,h,k,l). White arrowheads (a,b,i,j) mark additional endothelial tip cells

c,d,g,h,k,l IB4+ endothelial tip cells with clearly identifiable, multiple filopodia forming a typical “hand-like” structure (c,g,k). Note that neither VEGFR2 (d), VEGFR3 (h) nor Dll4 (l) label endothelial tip cell filopodia as accurately as IB4. As filopodia are the key morphological criterion for identifying endothelial tip cells, the classical tip cell markers VEGFR2, VEGFR3 and Dll4 do not facilitate the identification of endothelial tip cells in the postnatal mouse brain. Moreover, none of these markers allows a clear delineation of endothelial stalk- or phalanx cells from endothelial tip cells (d,h,l).

Scale bars: 50 µm (a,b,e,f,i,j) ; 10 µm (c,d,g,h,k,l).

Supplementary Figure 3 Immunofluorescence of perivascular cells in the vicinity of endothelial tip cells in the P8 mouse brain cortex.

a-p All samples used for the immunofluorescence shown in this figure have been immersion fixed with 4% FA and 0.025% GA, which was the final fixation protocol of the present study. This ensures optimal retaining of EB inside the vessels but also good antigenicity for a variety of cellular markers (see also Supplementary Fig. S1). For example a-d shows GFAP+ astrocytes and GFAP+ radial glia (green), IB4+ blood vessel endothelial cells (red) including an endothelial tip cell and an established blood vessel in the P8 mouse brain cortex. Endothelial tip cell filopodia do not follow a template of GFAP+ astrocytes and radial glia (a). Boxed area with zoom on endothelial tip cell is enlarged in b-d. Cell nuclei (DAPI, blue). e-h PDGFRB+ pericytes (green) and IB4+ blood vessels (red) in the P8 mouse cortex. Boxed area is enlarged in f-h. Endothelial tip cell filopodia are PDGFRB-. Cell nuclei (DAPI, blue). i-l LN+ (green) common basement membrane of IB4+ blood vessels (red) and PDGFRB+ pericytes. Boxed area with zoom on endothelial tip cell is enlarged in j-l. Note the faint LN-staining of endothelial tip cell filopodia at the base of the endothelial tip cell body (l). Cell nuclei (DAPI, blue). Note that no pericytes are present at the tip cell (f,h) whereas LN is ensheathing the tip cell body (j,l). m-p Nf160+ axons (cyan) and IB4+ endothelial tip cell (red, filopodia) in the P8 mouse corpus callosum. Boxed area with zoom on endothelial tip cell is enlarged in n-p. Cell nuclei (DAPI, blue).

Scale bars: 50 µm (a,e,i,m); 10 µm (b-d, f-h, j-l, n-p).

Supplementary information

Supplementary Figures

Supplementary Figures 1–3 (PDF 4937 kb)

Intracardial Evans blue injection of a P8 mouse.

This video shows the different steps of intracardial Evans blue (EB) injection of a P8 mouse pub. All steps are precisely explained in the “PROCEDURE” part of this manuscript. (MOV 9204 kb)

Brain dissection of a P8 mouse.

This video shows the different steps of brain dissection of an Evans-blue (EB) injected P8 mouse pup. All steps are precisely explained in the “PROCEDURE” part of this manuscript. (MOV 12461 kb)

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Wälchli, T., Mateos, J., Weinman, O. et al. Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain. Nat Protoc 10, 53–74 (2015). https://doi.org/10.1038/nprot.2015.002

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