Histological assessment of angiogenesis in the hypoxic central nervous system

Angiogenesis, the sprouting of new capillaries from preexisting vessels, is an integral part of both normal development and numerous pathological conditions such as tumor growth, inflammation, and stroke. The development of angiogenesis assays has been critical in understanding this process in both the context of disease and normal physiology. With the growing availability of antibodies against angiogenic markers as well as advances in microscopy and imaging analysis software, a more comprehensive assessment of the angiogenesis process is beginning to take form (Milner et al., Stroke 39:191-197, 2008; Freitas-Andrade et al., J Cereb Blood Flow Metab 32:663-675, 2012; Li et al., Glia 58:1157-1167, 2010; Dore-Duffy and LaManna, Antioxid Redox Signal 9:1363-1371, 2007). This chapter describes an in vivo method of inducing brain angiogenesis in mice by chronic exposure to mild hypoxia. In addition, a detailed procedure of quantifying angiogenesis using multiple immunofluorescent labeling of mouse brain tissue sections is also presented.