The passage of macromolecules across the blood-CNS barrier is severely restricted by the paucity of transendothelial vesicular transport (VT). It is clear that in injury states and during development, VT does occur and in the case of the former is linked to edema formation and subsequent pathologic events. Although in the developing CNS VT may serve as an important mechanism for nutrient transport across immature endothelial cells, we believe that this relatively non-selective mechanism may render the developing CNS more vulnerable. This is particularly relevant to understanding the toxic effects of environmental agents or drugs which impair the developing CNS but do not effect the mature CNS. Therefore, in this proposal we have selected developmental and injury models in order to evaluate changes in the structural features of the endothelial cell which are associated with VT. The overall purpose of this proposal is to 1: examine the role of endothelial surface charge and glycoprotein structure in macromolecular transport across spinal cord microvessels and 2: evaluate the relationship between endothelial enzymes and transendothelial VT. We propose that the blood-spinal cord barrier to macromolecular transport is dependent upon a distinctive glycocalyx, resulting in an anionic surface charge, and that an immature or disrupted glycocalyx is associated with macromolecular transport. Furthermore, we hypothesize that vascular membrane alterations are not confined to VT but are also reflected in the loss or redistribution of endothelial enzymes. Outcome measures include quantitative light and electron microscopy of endothelial enzymes and surface charge and structure. The development of enzymes (alkaline phosphatase, gamma glutamyl transpeptidase, and Na, K, ATPase) will be studied cytochemically beginning at embryonic day II. Furthermore, development of constituents of the endothelial glycocalyx will be analyzed using biotinylated lectins (Concanavalin A, Ricinus communis, and soybean agglutinin) to localize oligosaccharide residues on the luminal plasma membrane. Finally, the relationship between VT and luminal surface charge during development will be examined using the macromolecular tracer horseradish peroxidase in conjunction with anionic and cationized ferritin. Similar outcome measures will be applied to both spinal cord contusive and freeze injury models. The former model provides a source of vessels which exhibit transient VT. The latter model was selected in order to study the relationship between and orderly pattern of vascular regeneration and mechanisms associated with reconstitution of vascular integrity. These studies will provide a necessary foundation for understanding regulatory control of macromolecules across the barrier with an ultimate goal of identifying factors which may contribute to barrier modulation and/or stabilization after CNS trauma or during early CNS development.
Showing the most recent 10 out of 15 publications
