Summary
A comparison was made between endothelial cells in freshly-isolated rat brain microvessels, and following culture of the cells for 1–10 days during growth to confluence. Attention focused on tight junctions and vesicular structures, as seen in thin sections and freeze-fracture replicas. Freshly-isolated vessels had an abnormal appearance, with a profusion of luminal microvillar processes, and extensive cytoplasmic vacuolation. There were numerous vesicular profiles, reaching a density of ∼60 μm−2, and with a large proportion open to the surface, as shown by labelling with cationized ferritin at 4 °C for 5 min. Junctional zones were relatively loosely organized, with evidence for some cell: cell separation, as well as some residual tight junctional sites withinzonula adhaerens junctions. In freeze-fracture replicas, Junctional strands showed segments of tightly packed intramembrane particles, generally on the P face. After 1 day in culture, the cells appeared more normal, with no vacuolation or luminal processes. Vesicles were still numerous, some associated with junctional zones, while tight junctions were relatively sparse; freeze-fracture showed some incomplete tight junctional strands, with some of the intramembrane particles fracturing onto the E face. The double offset fibrillar nature of the strands could occasionally be seen. Cells cultured for 4 and 10 days showed a progressive increase in the completeness of the junctional zone, with more tight junctional contacts within the length of theadhaerens junction, and an aggregation of microfilaments in the underlying cytoplasm. The number of vesicular profiles declined, and they were progressively excluded from the junctional zone. These observations have relevance for studies on the physiology of the brain endotheliumin vitro, and for comparisons with thein vivo condition.
Similar content being viewed by others
References
Abbott, N. J. &Revest, P. A. (1991) Control of brain capillary permeability.Cerebrovascular and Brain Metabolism Reviews 3, 1–34.
Abbott, N. J., Hughes, C. C. W., Revest, P. A. &Greenwood, J. (1992) Development and characterization of a rat brain capillary endothelial culture: towards anin vitro blood-brain barrier.Journal of Cell Science 103, 23–37.
Barres, B. A., Hart, I. K., Coles, H. S. R., Burne, J. F., Voyvodic, J. T., Richardson, W. D. &Raff, M. C. (1992) Cell death and control of cell survival in the oligodendrocyte lineage.Cell 70, 31–46.
Bentzel, C. J., Palant, C. E. &Fromm, M. (1992) Physiological and pathological factors affecting the tight junction. InTight Junctions (edited byCereijido, M.) pp. 151–64. Ann Arbor: CRC Press.
Bowman, P. D., Du Bopis, M., Shivers, R. R. &Dorovini-Zis, K. (1992) Endothelial tight junctions. InTight Junctions (edited byCereijido, M.) pp. 305–20. Ann Arbor: CRC Press.
Bradbury, M. W. B. (1985) The blood-brain barrierin vitro.Neurochemistry International 7, 27–8.
Bradbury, M. W. B., ed (1992)Physiology and Pharmacology of the Blood-Brain Barrier. Berlin: Springer Verlag.
Brightman, M. W. (1992) Ultrastructure of brain endothelium. InPhysiology and Pharmacology of the Blood-Brain Barrier (edited byBradbury, M. W. B.) pp. 1–22. Berlin: Springer Verlag.
Brightman, M. W. &Reese, T. S. (1969) Junctions between intimately apposed cell membranes in the vertebrate brain.Journal of Cell Biology 40, 648–77.
Brightman, M. W. &Tao-Cheng, J. H. (1993) Tight junctions of brain endothelium and epithelium. InThe Blood-Brain Barrier. Cellular and Molecular Biology (edited byPardridge, W. M.) pp. 107–26. New York: Raven Press.
Broadwell, R. D. &Banks, W. A. (1993) A cell biological perspective for the transcytosis of peptides and proteins through the mammalian blood-brain fluid barriers. InThe Blood-Brain Barrier. Cellular and Molecular Biology (edited byPardridge, W. M.) pp. 165–200. New York: Raven Press.
Bundgaard, M. (1982) Ultrastructure of frog cerebral and pial microvessels and their impermeability to lanthanum ions.Brain Research 241, 57–65.
Bundgaard, M., Frøkjaer-Jensen, J. &Crone, C. (1979) Endothelial plasmalemmal vesicles as elements in a system of branching invaginations from the cell surface.Proceedings of the National Academy of Sciences (USA) 76, 6439–42.
Citi, S. (1993) The molecular organization of tight junctions.Journal of Cell Biology 121, 485–90.
Clough, G. (1982) The steady-state transport of cationized ferritin by endothelial cell vesicles.Journal of Physiology 328, 389–401.
Clough, G. &Michel, C. C. (1981) The role of vesicles in the transport of ferritin through frog endothelium.Journal of Physiology 315, 127–42.
Comber, B. L. &Stewart, P. A. (1988) Three-dimensional reconstruction of vesicles in endothelium of blood-brain barrier versus highly permeable microvessels.Anatomical Record 215, 256–61.
Davson, H., Welch, K. &Segal, M. B. (1987)The Physiology and Pathophysiology of the Cerebrospinal Fluid. Edinburgh: Churchill Livingstone.
Dehouck, M. -P., Méresse, S., Delorme, P., Fruchart, J. -C. &Cecchelli, R. (1990) An easier, reproducible, and mass-production method to study the blood-brain barrierin vitro.Journal of Neurochemistry 54, 1798–801.
Dehouck, M. P., Jolliet-Riant, P., Brée, F., Fruchart, J. -C., Cecchelli, R. &Tillement, J. P. (1992) Drug transfer across the blood-brain barrier: correlation betweenin vitro andin vivo models.Journal of Neurochemistry 58, 1790–7.
Friden, P. M. (1993) Receptor-mediated transport of peptides and proteins across the blood-brain barrier. InThe Blood-Brain Barrier. Cellular and Molecular Biology (edited byPardridge, W. M.) pp. 229–48. New York: Raven Press.
Friden, P. M., Walus, L. R., Watson, P., Doctrow, S. R., Kozarich, J. W., Backman, C., Bergman, H., Hoffer, B., Bloom, F. &Granholm, A. -C. (1993) Blood-brain barrier penetration andin vivo activity of an NGF conjugate.Science 259, 373–7.
Frøkjaer-Jensen, J. (1983) The plasmalemmal vesicular system in capillary endothelium.Progress in Applied Microcirculation 1, 17–34.
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S. A. &Tsukita, S. H. (1993) Occludin: a novel integral membrane protein localizing at tight junctions.Journal of Cell Biology 123, 1777–88.
Goldblum, S. E., Ding, X. &Washington, J. C. (1993) TNF-α induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction.American Journal of Physiology 264, 894–905.
Greenwood, J. (1992a) Experimental manipulation of the blood-brain and blood-retinal barriers. InPhysiology and Pharmacology of the Blood-Brain Barrier, (edited byBradbury, M. W. B.) pp. 460–86. Berlin: Springer Verlag.
Greenwood, J. (1992b) Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endotheliumin vitro.Journal of Neuroimmunology 39, 123–32.
Greig, N. H. (1992) Drug entry into the brain and its pharmacologic manipulation. InPhysiology and Pharmacology of the Blood-Brain Barrier (edited byBradbury, M. W. B.) pp. 487–523. Berlin: Springer-Verlag.
Joó, F. (1985) The blood-brain barrierin vitro: ten years of research on microvessels isolated from the brain.Neurochemistry International 7, 1–25.
Joó, F. (1992) The cerebral microvessels in culture, an update.Journal of Neurochemistry 58, 1–17.
Joó, F., Temesvári, P. &Dux, E. (1983) Regulation of the macromolecular transport in the brain microvessels: the role of cyclic GMP.Brain Research 278, 165–74.
Lane, N. J., Reese, T. S. &Kachar, B. (1992) Structural domains of the tight junctional intramembrane fibrils.Tissue and Cell 24, 291–300.
Laterra, J. J. &Goldstein, G. W. (1992) The blood-brain barrierin vitro and in culture. InPhysiology and Pharmacology of the Blood-Brain Barrier (edited byBradbury, M. W. B.) pp. 417–37. Berlin: Springer Verlag.
Luthert, P. J. (1992) Opening the barrier in cerebral pathology. InPhysiology and Pharmacology of the Blood-Brain Barrier (edited byBradbury, M. W. B.) pp. 439–57. Berlin: Springer Verlag.
Mandel, L. J., Bacallao, R. &Zampighi, G. (1993) Uncoupling of the molecular ‘fence’ and paracellular ‘gate’ functions in epithelial tight junctions.Nature 361, 552–5.
Méresse, S., Dehouck, M. -P., Delorme, P. M., Bensaïd, J. P., Tauber, J. -P., Delbart, C., Fruchart, J. -C. &Cecchelli, R. (1989) Bovine brain endothelial cells express tight junctions and monoamine oxidase activity in long-term culture.Journal of Neurochemistry 53, 1363–71.
Pardridge, W. M. (1983) Brain metabolism: a perspective from the blood-brain barrier.Physiological Reviews 63, 1481–535.
Pardridge, W. M., ed (1993)The Blood-Brain Barrier. Cellular and Molecular Biology. New York: Raven Press.
Pardridge, W. M., Eisenberg, J. &Cefalu, W. T. (1985) Absence of albumin receptor on brain capillariesin vivo orin vitro.American Journal of Physiology 249, E264–7.
Pardridge, W. M., Buciak, J. L., Kang, Y. -S. &Boado, R. J. (1993) Protamine-mediated transport of albumin into brain and other organs of the rat. Binding and endocytosis of protamine-albumin complex by microvascular endothelium.Journal of Clinical Investigation 92, 2224–9.
Raff, M. C., Barres, B. A., Burne, J. F., Coles, H. S., Ishizaki, Y. &Jacobson, M. D. (1993) Programmed cell death and the control of cell survival: lessons from the nervous system.Science 262, 695–700.
Ramlakhan, N. (1990) Albumin binding and endocytosis by cultured rat brain endothelium.Journal of Physiology 423, 34P.
Rist, R. J., Romero, I. A. &Abbott, N. J. (1994) The effect of a cAMP agonist and astrocyte-conditioned medium on the F-actin cytoskeleton in cultured primary and immortalized rat brain capillary endothelial cells.Journal of Physiology,480, 8–9P.
Romero, I. A., Chan, M. W. K. &Rist, R. J. (1994) The permeability of [14C] sucrose through anin vitro blood-brain barrier model, the RBE4 cell line.Journal of Physiology,480, 8P.
Rubin, L. L. (1992) Endothelial cells: adhesion and tight junctions.Current Opinion in Cell Biology 4, 830–3.
Rubin, L. L., Hall, D. E., Porter, S., Barbu, K., Cannon, C., Horner, H. C., Janatpour, M., Liaw, C. W., Manning, K., Morales, J., Tanner, L. I., Tomaselli, K. J. &Bard, F. (1991) A cell culture model of the blood-brain barrier.Journal of Cell Biology 115, 1725–35.
Rutten, M. J., Hoover, R. L. &Karnovsky, M. J. (1987) Electrical resistance and macromolecular permeability of brain endothelial monolayer culture.Brain Research 425, 301–10.
Schulze, C. &Firth, A. J. (1993) Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat.Journal of Cell Science 104, 773–82.
Simionescu, M., Ghinea, N., Fixman, A., Lasser, M., Kukes, L., Simionescu, N. &Palade, G. E. (1988) The cerebral microvasculature of the rat: structure and luminal surface properties during early development.Journal of Submicroscopic Cytology 20, 243–61.
Smith, Q. R. (1992) Methods of study. InPhysiology and Pharmacology of the Blood-Brain Barrier, (edited byBradbury, M. W. B.) pp. 23–52. Springer-Verlag: Berlin.
Vogel, A., Raines, E., Kariya, B., Rivest, M. -J. &Ross, R. (1978) Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma factors.Proceedings of the National Academy of Sciences (USA) 75, 2810–14.
Wolburg, H., Neuhaus, J., Kniesel, U., Krauss, B., Schmid, E. -M., Öcalan, M., Farrell, C. &Risau, W. (1994) Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes.Journal of Cell Science 107, 1347–57.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lane, N.J., Revest, P.A., Whytock, S. et al. Fine-structural investigation of rat brain microvascular endothelial cells: Tight junctions and vesicular structures in freshly isolated and cultured preparations. J Neurocytol 24, 347–360 (1995). https://doi.org/10.1007/BF01189062
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01189062