title: 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.
authors.affiliation: 1] Group of CNS Angiogenesis and Neurovascular Link, and Physician-Scientist Program, Swiss Center for Regenerative Medicine and Division of Surgical Research, and Neuroscience Center Zurich and Division of Neurosurgery, University and University Hospital Zurich, and Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland. [2] Division of Neurosurgery and Laboratory of Molecular Neurooncology, University Hospital Zurich, Zurich, Switzerland. [3] Brain Research Institute, University of Zurich and Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wälchli
authors.affiliation: Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland.
authors.forename: José María
authors.initials: JM
authors.lastname: Mateos
authors.affiliation: Brain Research Institute, University of Zurich and Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Weinman
authors.affiliation: Division of Cardiology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Daniela
authors.initials: D
authors.lastname: Babic
authors.affiliation: Division of Neurosurgery and Laboratory of Molecular Neurooncology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Regli
authors.affiliation: Swiss Center for Regenerative Medicine and Division of Surgical Research, University and University Hospital Zurich, Zurich, Switzerland.
authors.forename: Simon P
authors.initials: SP
authors.lastname: Hoerstrup
authors.affiliation: 1] Department of Oncology, Katholieke Universiteit (KU) Leuven, Vesalius Research Center, Vascular Patterning Lab, Leuven, Belgium. [2] Vlaams Instituut voor Biotechnologie (VIB), Vesalius Research Center, Vascular Patterning Lab, Leuven, Belgium. [3] Vascular Biology Laboratory, London Research Institute, Cancer Research UK, London, UK.
authors.forename: Holger
authors.initials: H
authors.lastname: Gerhardt
authors.affiliation: Brain Research Institute, University of Zurich and Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Schwab
authors.affiliation: Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Vogel
mesh_terms: D000818:Animals; D001921:Brain / Q000098:blood supply* / Q000166:cytology*; D042783:Endothelial Cells / Q000378:metabolism*; D005070:Evans Blue; D007797:Laminin; D037102:Lectins; D051379:Mice; D018613:Microscopy, Confocal; D018919:Neovascularization, Physiologic / Q000502:physiology*; D055420:Perfusion Imaging / Q000379:methods*
chemical_list: D007797:Laminin; D037102:Lectins; D005070:Evans Blue