A new method for determining blood-brain barrier integrity based on intracardiac perfusion of an Evans Blue-Hoechst cocktail

A new method for determining brain regions with blood-brain barrier (BBB) alterations is described. In this method, mice were perfused intracardially with Evans Blue (EB) and Hoechst tracers added in a standard formaldehyde fixative solution. This cocktail method was tested after a localized cryolesion induced in the brain had produced an edematous brain region with disrupted BBB in the animals. The results were then compared with the intravenous and intraperitoneal administration of the tracers prior to intracardiac perfusion. When using the cocktail method, red EB fluorescence locates the cryoinjured brain region while the Hoechst tracer stains the nuclei in that same region. EB and Hoechst fluorescence can also be observed in the choroid plexus and circumventricular organs, where there is no functional BBB. The cocktail gives more intense EB staining in zones of disrupted BBB than that given by traditional methods which use this tracer. The Hoechst tracer is also more useful when administered in the cocktail, since when administrated intravenously it stains all the brain nuclei. The cocktail method permits the immunostaining of brain sections, enabling researchers to characterize and analyze structural and cellular changes in regions where BBB disturbances are present. Thus, immunohistochemistry has been used here to determine the nature of intense EB fluorescent cells that appear in the perilesional rim, which were identified here as neuronal cells.