Treatment strategies in
acute ischemic stroke are still limited. Considering numerous translation failures, research is tending to a preferred use of human-like animal models, and a more-complex perspective of tissue salvaging involving endothelial, glial and neuronal components according to the neurovascular unit (NVU) concept. During
ischemia, blood-brain barrier (BBB) alterations lead to
brain edema and hemorrhagic transformation affecting NVU components. The present study aims on a novel quantification method of BBB damage and affected tissue following experimental
cerebral ischemia, closely to the human condition. Wistar rats underwent embolic
middle cerebral artery occlusion, followed by an intravenous application of
fluorescein isothiocyanate (
FITC)-tagged
albumin (≈70kDa) and/or biotinylated rat
IgG (≈150kDa) as BBB permeability markers. Both
fluorescent agents revealed similar leakage and allow quantification of BBB permeability by fluorescence microscopy, and after immunohistochemical conversion into a permanent diaminobenzidine label at light-microscopical level. The following markers were identified for sufficient detection of NVU components: Rat endothelial cell antigen-1 (RECA) and
laminin for vessels, Lycopersicon esculentum and Griffonia simplicifolia
agglutinin for vessels and microglial subpopulations, ionized
calcium binding adaptor molecule 1 (Iba1), CD68 and CD11b for macrophages, activated microglia, monocytes and neutrophils, S100β for astroglia, as well as NeuN and HuC/D for neurons. This is the first report confirming the usefulness of simultaneously applied
FITC-albumin and biotinylated rat
IgG as BBB permeability markers in experimental
stroke, and, specifying
antibodies and
lectins for multiple fluorescence labeling of NVU components. Newly elaborated protocols might facilitate a more-complex outcome measurement in
drug development for
cerebral ischemia.