Ultrastructure of Brain Endothelium

Abstract

In vertebrate brains, either fixed in aldehydes directly or rapidly frozen first and then substituted with organic solvent before fixation at low temperatures, tracer molecules that had been infused intravascularly are prevented from reaching the interstitial fluid (IF) of the central nervous system (CNS). This barrier to the passage of hydrophilic solutes is due to the inability of the molecules to pass through zonular or circumferential junctions between adjacent endothelial cells. The second basis for the barrier is the inability of the pits or caveolae of the endothelial cells to transfer the molecules across the endothelium (Reese and Karnovsky 1967; Brightman and Reese 1969). Unlike the mammal, some of the pial vessels in the anuran brain are capillaries. Consisting as they do of one cell layer, the endothelium, these pial vessels of the frog can be rapidly frozen. As the depth of rapid freezing that is free of ice-crystal artefact is only about 15–30 μm, the tunica media of arterioles or venules would be preserved but not the underlying endothelium. For this reason, the frog was selected to assess the possible artefacts that could be introduced by primary fixation in aldehydes. Another constraint of the rapid-freeze method is that tracers such as horseradish peroxidase (HRP), the demonstration of which depends on its enzymatic activity, cannot be used at such low temperatures. Instead, a molecule such as ferritin (M _r 950 000) is infused into the heart of normal frogs and those in which the blood-cerebrospinal fluid (CSF) barrier is opened by the topical application to the pial surface 3 M urea (Nagy et al. 1998).