A QUANTITATIVE IMMUNOCYTOCHEMICAL STUDY OF THE OSMOTIC OPENING OF THE BLOOD-BRAIN-BARRIER TO ENDOGENOUS ALBUMIN

The time sequence of the blood-brain barrier opening to endogenous albumin in rat brain after intracarotid infusion of hyperosmolar L(+)arabinose was studied using quantitative immunocytochemistry. Brain samples obtained 1, 5, and 30 min after insult were immersion-fixed in formaldehyde-glutaraldehyde mixture and embedded at low temperature in Lowicryl K4M. Untreated rats or rats exposed only to Ringer's solution were used as a control. Ultrathin sections were exposed to anti-rat albumin antiserum followed by protein A-gold, The density of immunosignals (gold particles per square micrometre) was recorded over four compartments: vascular lumen, endothelium, subendothelial (perivascular) space including basement membrane, and brain parenchyma (neuropil). The labelling density of the vessel lumen, containing blood plasma, was considered to represent 100% of the circulating albumin. Morphometric and statistical analysis indicated that in control animals only 0.4-0.6% of circulating albumin appears in the subendothelial space and in the basement membrane. As soon as one minute after L(+)arabinose infusion, this value increased to 3%, followed by a further increase to 25% and 56% after 5 and 30 min, respectively. A slow increase of the labelling density in the adjacent neuropil suggests that the basement membrane represents an obstacle for escaping albumin, which apparently sticks to or is trapped by this membrane. The results indicate that the applied procedure, although based on morphometric analysis of static electron micrographs can also be used for studying dynamic processes such as transvascular passage of albumin after disruption of the brain-blood barrier.