Charge-related alterations of the cerebral endothelium

In a short-term rat brain perfusion model, the luminal surface of cerebral endothelium was exposed to the following solutions: (a) the polycation protamine sulfate (PS) in a dose of 50, 100, and 500 micrograms/ml for 1 or 2 minutes; (b) PS in a dose of 100 micrograms/ml (or 500 micrograms/ml) for 1 or 2 minutes followed by the polyanion heparin in an equivalent dose of 12 units/ml (or 60 units/ml) for 1 or 2 minutes; (c) heparin alone for 1 or 2 minutes, and (d) Krebs-Ringer-bicarbonate solution as control for 1, 2, or 4 minutes. We studied in the cerebral endothelium: (a) structural alterations by electron microscopy, (b) permeability changes to horseradish peroxidase (HRP) by light and electron microscopy, and (c) charge alterations of luminal surface visualized with colloidal iron at pH 1.8 by electron microscopy. We found that: (a) PS resulted in extravasation of HRP throughout the perfused hemispheres in a time- and dose-dependent fashion. In this experimental group, colloidal iron binding decreased on the luminal surface of the cerebral blood vessels; (b) heparin perfusion following PS reversed the colloidal iron staining but failed to prevent the blood barrier opening to HRP; (c) heparin perfused alone also induced extravasation of HRP in the treated brain hemispheres; (d) in Krebs-Ringer-bicarbonate-perfused control brains extravasation of HRP was encountered only in occasional vascular segments. In all brain hemispheres showing tracer extravasation, electron microscopy revealed HRP reaction product in compartments of endothelial tight junctions suggesting opening of interendothelial routes as the structural basis of blood-brain barrier opening. Endothelial cell death reflected by swelling and influx of HRP into endothelial cytoplasm in PS- and/or heparin-perfused hemispheres was probably an additional mechanism explaining tracer extravasation into the neuropil. Our results indicate a correlation between the effect of polycation PS and a decrease in the anionic sites of cerebral endothelium. The relationship between charge alteration and barrier opening in the short-term rat brain perfusion model is not clear.