The objective of this proposal is to examine the function of the endothelial glycocalyx in modulating protein permeability across the blood-spinal cord barrier. The glycocalyx is a negatively charged, amorphous layer that is situated between the endothelial cell and the humoral compartment. Plasma proteins must first confront this structure in order to access the endothelial cell. We hypothesize that the charged glycocalyx repels negatively charged plasma proteins but mediates transport of positively charged plasma proteins. To test this hypothesis we will study endothelial permeability to horseradish peroxidase (HRP) that has been modified from its neutral state (nHRP) to yield negatively (aHRP) and positively (cHRP) charged derivatives. Protein permeability will be examined in spinal cord vessels in vivo under normal conditions and after neutralization of the charged glycocalyx with protamine sulfate. We expect to find that normal spinal cord vessels will exhibit permeability to only cHRP and that the negative charged glycocalyx is necessary to maintain this restrictive permeability. Similar complementary studies will be conducted in cerebral endothelium in vitro to confirm the selective cellular uptake of cHRP and to define the kinetics of endocytosis. We propose that cellular uptake of cHRP occurs by adsorptive-mediated endocytosis and that neutralization of the negativelv charged glycocalyx facilitates nonselective, fluid-phase endocytosis. After contusive spinal cord injury abnormal protein permeability to nHRP develops along the axis of the cord. In this injury model the glycocalyx is disrupted in regions adjacent to the injury while remaining intact in regions distant from the injury. We hypothesize that the glycocalyx regulates protein permeability in this pathologic state. This hypothesis will be tested by comparing barrier permeability to nHRP, aHRP, and cHRP. We expect to find selective permeability to cHRP in regions with intact glycocalyx, while finding nonselective permeability in regions of disrupted glycocalyx. Outcome measures include isoelectric focusing and SDS/PAGE to characterize charged derivatives of HRP. Cellular uptake of HRP in vitro will be determined by colorimetric assay. The kinetics of endocytosis will be studied in vitro, including time and temperature dependency and saturation characteristics. For studies in vivo quantitative light and electron microscopy will be employed to assess vascular leakage to HRP.
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