title: Stereological and flow cytometry characterization of leukocyte subpopulations in models of transient or permanent cerebral ischemia.
abstract: Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid.
authors.forename: Iván
authors.initials: I
authors.lastname: Ballesteros
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid; Maribel_cd@hotmail.com.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Cuartero
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid.
authors.forename: Ana
authors.initials: A
authors.lastname: Moraga
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid.
authors.forename: Juan
authors.initials: J
authors.lastname: de la Parra
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Lizasoain
authors.affiliation: Unidad de Investigacón Neurovascular, Departmento de Farmacología, Falcultad de Medicina, Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid.
authors.forename: María Ángeles
authors.initials: MÁ
authors.lastname: Moro
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000097:blood* / Q000473:pathology*; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods*; D002546:Ischemic Attack, Transient / Q000097:blood / Q000473:pathology; D007962:Leukocytes / Q000473:pathology*; D051379:Mice; D017628:Microglia / Q000473:pathology; D009000:Monocytes / Q000473:pathology; D022423:Myeloid Cells / Q000473:pathology; D009504:Neutrophils / Q000473:pathology