The objective of this proposal is twofold; (1) to develop an in vitro co-culture system in which the interaction between two different cell types can be studied, and (2) to evaluate in vivo the role of the glial cell in the differentiation and maintenance of the Blood-Brain Barrier (BBB) properties in capillary endothelium of the brain. The in vivo studies will utilize a mouse freeze-injury model to study these processes in the regenerating cerebral microvessels.
The specific aims are to thoroughly investigate the temporal sequence and relationships of endothelial tight junction formation, early appearance of glial foot processes on the vascular baseement membrane, the completion of the glial sheath around the regenerating vessel, restoration of barrier function to tracers, the fate of endothelial cell Factor VIII and Thrombomodulin (TM) antigens during regeneration, and induction (or activation) or gamma-glutamyl transpeptidase (GGTP) ion the new endothelium. These studies will employ enzyme histochemistry for GGTP, immunocytochemistry for GGTP, TM and Factor VIII, ultrastructural studies and horse radish peroxidase tracer analysis for barrir function.
The Aims of the in vitro studies are to determine the mechanisms by which endothelial cells acquire GGTP in the presence of glial cells in co-culture, to test the glial cell's ability to induce other differentiated endothelial characteristics, and to delineate the extent to which glial cells can induce and/or activate GGTP in endothelium from extracranial sites. These studies will use radioimmunoassay, autoradiography, enzyme histochemistry and bioassay for GGTP, immunochemistry and bioassay for TM, as well as varied schemes of co-culture. The advantage this dual approach is that the in vivo studies will tell us what to look for in the glial/endothelial cell-cell interaction, and the in vitro studies will provide a place to test or validate the in vivo observations. The accomplishment of these aims will add significantly to our understanding of the microvascular regeneration process following a variety of clinical and pathological conditions where alterations in vascular integrity have resulted in varying degrees of brain damage. (Infarcts, trauma, anoxia, neoplasms, infection, etc.). Of equal importance will be the development and testing of in vitro co-culture schemes that allow the study of differentiated endothelial cell functions that are often lost in pure cell culture.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS018775-04
Application #
3398802
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1982-07-01
Project End
1990-08-31
Budget Start
1985-09-23
Budget End
1986-08-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Type
School of Medicine & Dentistry
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
De Bault, L E; Gu, J (1996) In situ hybridization, in situ transcription, and in situ polymerase chain reaction. Scanning Microsc Suppl 10:27-44;discussion 44-7
De Bault, L E; Mitro, A (1994) Species differences in the distribution of gamma-glutamyl transpeptidase in choroid plexus of lateral ventricle and microvessels of adjacent brain. Histochem J 26:447-52
Mitro, A; De Bault, L E (1994) Membrane-bound proteases of the gerbil subfornical organ and choroid plexus: an enzyme histochemical study. Acta Histochem 96:1-7
Wang, B L; Grammas, P; De Bault, L (1993) Characterization of a gamma-glutamyl transpeptidase positive subpopulation of endothelial cells in a spontaneous tube-forming clone of rat cerebral resistance-vessel endothelium. J Cell Physiol 156:531-40
Morgenstern, K; Hanson-Painton, O; Wang, B L et al. (1992) Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells. J Cell Physiol 150:104-15
Morgenstern, K; Hanson-Painton, O; De Bault, L (1991) Cell surface gamma-glutamyl transpeptidase in live cultures. Anal Biochem 192:165-72
Loudon, W G; Abraham, S R; Owen-Schaub, L B et al. (1988) Identification and selection of human lymphokine activated killer cell effectors and novel recycling intermediates by unique light-scattering properties. Cancer Res 48:2184-92
DeBault, L E; Esmon, N L; Olson, J R et al. (1986) Distribution of the thrombomodulin antigen in the rabbit vasculature. Lab Invest 54:172-8
DeBault, L E; Esmon, N L; Smith, G P et al. (1986) Localization of thrombomodulin antigen in rabbit endothelial cells in culture. An immunofluorescence and immunoelectron microscope study. Lab Invest 54:179-87