IMMUNOCYTOCHEMICAL STUDIES OF PROTAMINE-INDUCED BLOOD-BRAIN-BARRIER OPENING TO ENDOGENOUS ALBUMIN

The cellular mechanisms of blood-brain barrier (BBB) opening to endoge nous albumin in the mouse brain after intracarotid infusion of solutio ns of protamine free base (PB) or protamine sulfate (PS) were studied using quantitative immunocytochemistry. Ultrathin sections of brain sa mples embedded at low temperature in Lowicryl K4M were exposed to anti -mouse albumin antiserum followed by protein A-gold. Using morphometry , the density of immunosignals (gold particles per mu m(2)) was record ed over four compartments: vascular lumen, endothelial profiles, suben dothelial space (including the basement membrane), and brain parenchym a (neuropil). In addition, the adsorption of endogenous albumin eviden ced by the number of gold particles per mu m of the endothelial lumina l plasmalemma was quantitatively evaluated. In the applied experimenta l conditions, PB was found to be strongly cytotoxic as indicated by th e appearance of rapid degenerative changes and the disruption of the e ndothelial lining with concomitant clumping of the blood plasma. The a ction of PS was milder, offering a better opportunity for detailed ult rastructural and morphometric examination of brain samples during cons ecutive steps of PS action (2, 5, 10 and 30 min). As early as 10 min a fter infusion of PS solution, the adsorption of blood plasma albumin t o the endothelial luminal surface was increased 2.5 times. Simultaneou sly, the immunolabelling of the endothelial profiles and subendothelia l space was significantly increased. These results suggest that BBB di sruption occurs through enhanced adsorption of albumin or albumin-prot amine complexes to the luminal plasmalemma, followed by transendotheli al vesicular transport, rather than through modification of interendot helial junctional complexes. Th:is process appears to be focally disse minated throughout the cerebral vascular network and declines at 30 mi n following infusion of PS solution.