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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS023324-08
Application #
2264780
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1985-08-01
Project End
1994-09-30
Budget Start
1993-04-01
Budget End
1994-09-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Neurology
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Mautes, A E; Weinzierl, M R; Donovan, F et al. (2000) Vascular events after spinal cord injury: contribution to secondary pathogenesis. Phys Ther 80:673-87
Mautes, A E; Bergeron, M; Sharp, F R et al. (2000) Sustained induction of heme oxygenase-1 in the traumatized spinal cord. Exp Neurol 166:254-65
Mautes, A E; Noble, L J (2000) Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat. Brain Res 883:233-7
Richmon, J D; Fukuda, K; Maida, N et al. (1998) Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier. Brain Res 780:108-18
Mautes, A E; Kim, D H; Sharp, F R et al. (1998) Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat. Brain Res 795:17-24
Noble, L J; Mautes, A E; Hall, J J (1996) Characterization of the microvascular glycocalyx in normal and injured spinal cord in the rat. J Comp Neurol 376:542-56
Westmark, R; Noble, L J; Fukuda, K et al. (1995) Intrathecal administration of endothelin-1 in the rat: impact on spinal cord blood flow and the blood-spinal cord barrier. Neurosci Lett 192:173-6
McKenzie, A L; Hall, J J; Aihara, N et al. (1995) Immunolocalization of endothelin in the traumatized spinal cord: relationship to blood-spinal cord barrier breakdown. J Neurotrauma 12:257-68
Aihara, N; Hall, J J; Pitts, L H et al. (1995) Altered immunoexpression of microglia and macrophages after mild head injury. J Neurotrauma 12:53-63
Noble, L J; Kalinyak, J E; Pitts, L H et al. (1994) Fluid-phase endocytosis of horseradish peroxidase by cerebral endothelial cells in primary culture: characterization and kinetic analysis. J Neurosci Res 38:654-63

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