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.

SUMMARYWiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching. Nogo-A KO mice were generated by homologous recombination of exons 2 and 3 in the Nogo-A gene, as described previously (Dimou et al., 2006;Simonen et al., 2003). Antibodies11C7 antibody was raised against an 18-amino acid Nogo-A peptide corresponding to the rat sequence of amino acids 623-640 (Oertle et al., 2003). 7B12 antibody has an epitope in the C-terminal part of the Nogo-A-specific region (aa 763-820) (Oertle et al., 2003). Both antibodies are function-blocking antibodies (Liebscher et al., 2005) and are monospecific for Nogo-A (Dodd et al., 2005;Oertle et al., 2003). Polyclonal rabbit antibody Rb173A (Laura) recognizes the Nogo-A-specific region (aa 174-979) and the antibody Rb1 (Bianca) is specific for the N terminus of Nogo-A and Nogo-B (aa 1-172) (Dodd et al., 2005;Oertle et al., 2003). Nogo-A receptor complex components were blocked by anti-NgR (R&D Systems) or anti-Lingo1 (Abcam) antibodies. Compound Y27632 was used to inhibit ROCK (Sigma). Rabbit anti-neurofilament 160 antibody (Chemicon) was used for whole-mount staining and mouse anti--tubulin III (Abcam) was used for staining of dissociated DRG cultures. DRG cultures DRG explant culturesDRGs of newborn rats, wild-type and Nogo-A KO mice were plated on 20 g/ml poly-L-lysine (PLL)-coated four-well tissue culture plates. Cultures were incubated for 5-7 days at 37°C and 5% CO 2 atmosphere in F12 medium (Invitrogen) supplemented with 10% foetal bovine serum (Sigma), 100 g/ml nerve growth factor (NGF) and 10 g/ml gentamycin (Sigma). Cytosine arabinoside was added to inhibit mitosis of non-neuronal cells. Control, monoclonal mouse IgG antibody directed against wheat auxin, anti-Nogo-A antibodies 11C7 or 7B12, antibodies against NgR-1 or Lingo1 or the ROCK blocker Y27632 were added to the culture medium at a concentration of 10 g/ml at the beginnin...