A novel quantification of blood–brain barrier damage and histochemical typing after embolic stroke in rats

• Published in: Brain research 2010-11, Vol.1359, p.186-200
• Main Authors: Michalski, Dominik, Grosche, Jens, Pelz, Johann, Schneider, Dietmar, Weise, Christopher, Bauer, Ute, Kacza, Johannes, Gärtner, Ulrich, Hobohm, Carsten, Härtig, Wolfgang
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood-Brain Barrier - injuries; Blood-Brain Barrier - pathology; Blood–brain barrier; Capillary Permeability; Cerebral circulation. Blood-brain barrier. Choroid plexus. Cerebrospinal fluid. Circumventricular organ. Meninges; Embolic cerebral ischemia; FITC-albumin; Fluorescein-5-isothiocyanate - analogs & derivatives; Fluorescein-anti-fluorescein; Fundamental and applied biological sciences. Psychology; Griffonia simplicifolia; Histological Techniques; Immunoglobulin G; Immunohistochemistry; Immunoperoxidase; Infarction, Middle Cerebral Artery - complications; Infarction, Middle Cerebral Artery - pathology; Lycopersicon esculentum; Medical sciences; Microscopy, Confocal; Microscopy, Fluorescence; Neurology; Rats; Rats, Wistar; Serum Albumin; Stroke; Stroke - etiology; Stroke - pathology; Vascular diseases and vascular malformations of the nervous system; Vertebrates: nervous system and sense organs
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/j.brainres.2010.08.045

Abstract 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 (≈ 70 kDa) and/or biotinylated rat IgG (≈ 150 kDa) 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.