Horseradish peroxidase retention and washout in blood-brain barrier lesions

Horseradish peroxidase (HRP) is widely used to visualize blood-brain barrier (BBB) breakdown. In addition to its visual properties, HRP activity can be measured and can, therefore, be used to quantitate barrier leakage rapidly and simply. In addition, the same tissue can be used for electron microscopic studies in which the structural basis for the leakage can be resolved. Here we describe two methods for quantitation of barrier damage using HRP: one in small, multifocal barrier lesions caused by ethanol combined with barbiturates, and a second in large, single-focus, necrotic lesions. We determined the optimal circulation times and fixation methods for each. In the case of multifocal barrier damage, short-term HRP circulation for up to 1 min precisely marked individual leaking vascular segments, which could then be counted and their density calculated. Immersion fixation was necessary since perfusion fixation washed the extravasated HRP out of the tissue. In freeze-lesions, circulation times of at least 5 min were necessary to deliver acceptable amounts of HRP to the tissue. Tissue retention increased with longer circulation times to an optimum of 30 min, but then fell. Perfusion fixation for times as long as 1 h did not affect the tissue retention of HRP.