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Endothelial cell cultures derived from isolated cerenrbal microvessels
Brain Research, 191 (1980) 577-582 © Elsevier/North-Holland Biomedical Press
577
Endothelial cell cultures derived from isolated cerebral microvessels*
M. SPATZ, J. BEMBRY, R. F. DODSON, H. HERVONEN and M. R. MURRAY Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20205 and ( R.F.D.) Department of Cell Biology and Environmental Sciences, 7he University of Texas Health Center at Tyler, Tyler, Texas 75710 (U.S.A.)
(Accepted February 6th, 1980) Key words: endothelial cell cultures -- cerebral microvessels-- dissociated cells
The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to the capillaries of other organs. The intercellular diffusion is restricted by the tight junction separating the continuous layer of endothelial cells, while the vesicular transport is limited in the normal endothelium. Many harmful conditions such as anoxia, ischemia, metabolic and/or degenerative disturbances may alter the endothelium and thus the barrier function. Therefore the elucidation of mechanisms involved in various cerebrovascular disorders such as cerebral ischemia could be facilitated by the study of cellular components of the cerebral vessels in a living state. The present study was designed to establish a cultured cell line originating from cerebral microvessels. In this communication we will describe the establishment of a cell line culture showing the characteristic cytochemical features of cerebral endothelial cells, and which originated from dissociated cells of isolated cerebral microvessels. The brains of 2-day-old Osborne-Mendel rats (36-40) were used under sterile conditions for the separation of the microvessels from the non-vascular tissue. The cerebral hemispheres were freed of leptomeninges, minced with scissors and homogenized in buffered (10 mm Hepes) 0.1 ~ albumin-Ringer solution at pH 7.4. The fraction of microvessels was obtained by centrifugation and discontinuous sucrose gradient (1.0-1.5 m), a simple technique described previouslylk Two samples of microvessels (pellets) were pooled and washed twice with 10 ml of modified Simm's Balanced Salt Solution (BSS) containing 100 U penicillin, 100/~g streptomycin and 0.25 #g Fungizone (Gibco, Long Island, N.Y.). They were centrifuged immediately after the first wash and then washed again for 30 min in the refrigerator prior to centrifugation at * Presented in part at the Fifty-Fifth Annual Meeting of the American Association of Neuropathologists, Kansas City, Mo., 1979.
578 1000 rpm for 8 min. Then the cells of the rewashed microvessels were dissociated with 0.01 o//,,trypsin-collagenase in Hank's solution without Ca 2~ and Mg 2~ (trypsin I " 250. Gibco; collagenase type, Sigma, St. Louis, Mo.) by slow stirring for 5 min at room temperature. These dissociated cells were recentrifuged and resuspended in 5 ml feeding medium, placed in one plastic flask (75 sq. cm) and incubated in an atmosphere of air and 5 ~; CO2 at 37 °C. The feeding medium consisted of 55~,, medium 199 (with 25 mm Hepes Buffer, Earle's Salts and L-glutamine), 1 ~o BME amino acids, 1 ~ BME vitamins, 30°/o fetal calf serum (heat-inactivated), 1 °/ooantibiotic antimycotic mixture (all from Gibco) 2 o/,, of 50 ~ glucose solution (Dextrose, anhydrous, Baker, Philipsburg, N.J.) and 10 ~,~of 5 mg/ml peptone (powder, type I, Sigma) at pH 7.2. After 2 days, the feeding solution was removed and 5 ml of fresh medium was added. At 4 days, the culture was washed briefly in Simm's BSS containing 1 ~ of antibiotic-antimycotic solution and refed. This procedure was then followed on a biweekly basis, but the concentration of fetal calf serum in the medium was reduced to 20 ~ after 2 weeks of culturing the cells. The dissociated cells grew slowly as sheets and/or a network of elongated cells, covering the entire surface of the flask in about 4 weeks. At this time and/or at this stage of growth, the cells were washed with Ca 2~ and Mg ~ ~ free Hank's solution, dissociated with 0.1 ~o trypsin in Ca 2÷ and Mg 2+ free Hank's (15-25 min at 37 °C) and centrifuged at 1000 rpm for 5 rain. Then the cells were resuspended in 20 ml of feeding medium and placed in two 75 sq. cm flasks. In about two weeks the cells became confluent again and ready for the second transfer which was accomplished in the same way as the first one. However, the cells were now placed on round coverslips (7/8 in. in diameter) and kept in Petri dishes (35 mm in diameter) containing the feeding medium. They were fed at least once before the proliferating cells covered the entire coverslip's surface and were ready for investigation. Thus the histochemical and ultrastructural examinations were performed on cell cultures (150 in total) maintained from 50-70 days in vitro. The aniline blue or Giemsa stain. This culture revealed a monolayer of elongated cells interposed by small to large groups of cells appearing to be composed of more than one layer of coalescent cells. They contained oval nuclei with prominent nucleoli and showed a moderate number of mitotic figures (Fig. la). The activities of alkaline phosphatase 4, butyryl cholinesterase 10 7-glutamyl transpeptidase 1,7 and L-DOPA uptake (demonstrated by glyoxylic acid induced fluorescenceZ) which had been used as markers for cerebral capillary endothelium in explants and in vivo 1,3,12,13 were detected in these cultures, with suitable modification for dispersed cell culture, of the procedures designed for other circumstances. Alkaline phosphatase. Activity of alkaline phosphatase was present in almost all cells, demonstrable as an intense diffuse and granular bluish-purple staining reaction in the cytoplasm. However, some of the cells revealed only a slight to moderate amount of reaction product (Fig. lb). Butyryl cholinesterase. Butyryl cholinesterase activity was visible as a cytoplasmic brown granular reaction, which was more pronounced juxtanuclearly than at the
579
Fig. 1. a-e: light microscopy: tertiary cultures of cerebral endothelial cells, a ' a confluent monolayer of elongated cells with interspersed groups of cells composed of more than one layer of ceils. Note the apparent formation of branches and anastomoses. Aniline blue stain, x 130. b: alkaline phosphatase activity is localized in the cytoplasm of almost all cells but with a variable intensity, x 130. c: butyryl cholinesterase activity. The reaction product is especially heavy juxtanuclearly, x 330. d: ),-glutamyl transpeptidase activity. The most intense reactivity is seen in a group of ceils appearing to be composed of more than one layer, x 250. e: L-DOPA. Intense cytoplasmic fluorescence is seen in a granular cell as in d derived from sister cultures, x 250.
580 periphery of the cells. The enzyme was demonstrable in all the cells when a favorable technique was devised by modifying the Karnovsky-Roots method as follows: 2 h fixation, double the substrate concentration and 18 h of incubation (Fig. lc). The BuCHE activity was completely inhibited by Iso-OMPA and eserine sulfate. Reactivity was also absent when the substrate butyrylcholine iodine was substituted by acetylcholine iodine. 7-Glutamyl transpeptidase. Activity of y-glutamyl transpeptidase was demonstrated as intense red-brown reaction product primarily in the groups of cells appearing to be composed of more than one layer showing an anastomosing pattern, and in some individual cells which were surrounded by nonreactive and only slightly reactive cells (Fig. ld). The demonstration of this enzyme required also short fixation time (30-60 rain) and increased incubation time of 4-6 h as well as double concentration of the substrate when Glenner's technique was used. L-DOPA. A diffuse and slightly granular cytoplasmic fluorescence was seen in individual cells and groups of cells showing the same pattern as the one demonstrating 7glutamyl transpeptidase activity in sister cultures (Fig. le). Electron microscopy. In fixation for transmission electron microscopy the cover slips were placed in 3 ~ glutaraldehyde in 0.1 M phosphate solution. After an adequate fixation interval and a buffer rinse, postfixation was carried out in a 1 ~ osmium tetroxide/phosphate preparation. For further contrast enhancement, the tissue was soaked in 1/2 ~o uranyl acetate at 4 °C for 12 h. Following standard ethanol dehydration, the cultures were embedded in Spurr plastic*.L Ultrastructurally, these cultures revealed one cell type of numerous elongated flat cells situated in close proximity to each other and showing many intercellular 'gap' junctions (Fig. 2a and b). Their cytoplasm contained well-developed Golgi complexes, mitochondria and rough endoplasmic reticulum. Pinocytotic vesicles were present in moderate numbers and were localized in subplasmalemmal regions. The elongated nucleus occupied a central position in the cell and contained a homogeneously granular karyoplasm. A band of heterochromatin was located at the edge of the nuclear envelope. Dense organelles such as lysosomes or Weibel-Palade bodies were rarely seen in the culture material, and this was considered consistent with the metabolic state of the cells and their level of maturation. The histochemical and ultrastructural features were consistent with those in the cerebral capillaries studied in situ and in the organotypic brain cultures seen by us and others la. Although these cultures displayed some cellular differences in degree of enzymatic reactivity, they were found to be composed almost of a single cell type when examined by transmission microscopy, Therefore such variations could be functional or positional in nature and possibly related to the cellular maturity rather than to a difference in cellular origin. Even though the 7-glutamyl transpeptidase activity and LDOPA uptake could be seen in some of the dissociated brain cells of nonvascular origin, it appears unliklely that the same cells (sister cultures) would show also a marked reactivity for the nonspecific cholinesterase and alkaline phosphatase which have been considered as the best characteristic markers for the identification of cerebral capillary endothelium.
581
iii:i/ ~i: ........ii~. . . . Fig. 2. a and b: electron microscopy, a: this section through a layer of the culture reveals the homogeneity of cell type as well as the points of cellular apposition (arrows). × 24,000. b : the junctional complex shown in this field is of the typical 'gap' form commonly found throughout the preparation. × 77,000. The presence of intercellular 'gap' junctions and the absence of 'tight' junctions may be related either to the cellular immaturity 6 or to the incapacity of endothelial cells dissociated from the isolated microvessels of 2-day-old brain to f o r m ' t i g h t ' junctions in vitro. In any case further investigations are required to settle these problems. The difficulty in obtaining cerebrovascular endothelium in contrast to the relative ease described in collecting the endothelial lining of peripheral vessels 9,14 has been the greatest obstacle in establishing an endothelial culture model for elucidation of their unique cerebral function. Therefore, most of the previous observations of endothelial cell behavior were derived from organotypic cultures of nervous tissue a,12. However, recently De Bault et al. 5 described an organoid culture of cerebral microvessels which, depending on their size, gave rise to either endothelial and smooth muscle or to endothelial cells only. These cultures lacked some of the enzymatic characteristics of endothelium, but the 7-glutamyl transpeptidase could be induced by co-culturing these cells with glioma cells (personal communication). The original age of the isolated microvessels as well as the different procedure used by us for the isolation and culturing of the endothelium might have been responsible for obtaining an enzymatically more active endothelial cell line as compared to others. The availability of a relatively easy procedure for establishing and cultivating pure cerebral endothelial cells which are metabolically active provides a model for the study of their normal and altered endothelial function which may contribute to the understanding of the complex regulatory function of the blood-brain barrier.
582 1 Albert, Z., Orlowski, M., Rzucidlo, Z. and Orlowska, J., Studies on 7-glutamyl transpeptidase activity and its histochemical localization in the central nervous system, Acta Histochem., 25 (1966) 312-320. 2 Axelsson, G., Bj6rklund, A., Falck, B., Lindvall, O. and Svensson, L.-~., Glyoxylic acid condensation: a new fluorescence method for the histochemical demonstration of biogenic monoamines, Acta physiol, scand., 87 (1973) 57-62. 3 Bertler, A., Falck, B., Owman, C. H. and Rosengrenn, E., The localization of monoaminergic blood-brain barrier mechanisms, Pharmacol. Rev., 18 (1966) 369--385. 4 Burstone, M. S., Histochemical demonstration of phosphatases in frozen sections with naphtbol AS-phosphates, J. Histochem. Cytochem., 9 (1961) 146-153. 5 De Bault, L. E., Kahn, L. E., Frommes, S. P. and Cancilla, P. A., Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization, hi Vitro, 15 (1979) 473-487. 6 Delorme, P., Gayet, J. and Grignon, G., Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken, Brain Research, 22 (1970) 269-283. 7 Glenner, G. G., Folk, J. E. and McMillan, P. J., Histochemical demonstration of a 7-glutamyl transpeptidase-like activity, J. Histochem. Cytochem., 10 (I 962) 481-489. 8 Hauw, J. J., Berger, B. and Escourolle, R., Ultrastructural observations on human cerebral capillaries in organ culture, Cell Tissue Res., 1963 (1975) 133-150. 9 Jaffe, E. A., Nachman, R. L., Becker, C. G. and Minick, C. R., Culture of human endothelial ceils derived from umbilical veins. Identification by morphologic and immunologic criteria, J. clin. hlvest., 52 (1973) 2745-2756. 10 Karnovsky, M. J. and Roots, L., A 'direct-coloring' thiocholine method tbr cbolinesterase, J. Histochem. Cytochem., 12 (1964)219 221. 11 Mrsulja, B. B., Mrsulja, B. J., Fujimoto, T., Klatzo, I. and Spatz, M., Isolation of brain capillaries: a simplified technique, Brain Research, 110 (1976) 361-365. 12 Panula, P., Joo, F. and Rechardt, L., Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain, Experientia (Basel), 34 (1978) 95 96. 13 Renkawek, K., Murray, M. R., Spatz, M. and Klatzo, I., Distinctive histochemical characteristics of brain capillaries in organotypic culture, Exp. Neurol., 50 (1976) 194~206. 14 Schwartz, S. M., Selection and characterization of bovine aortic endothelial cells, In Vitro, 14 (1978) 966-980. 15 Spurr, A. R., A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastrltct. Res., 26 (1969) 31 43.
577
Endothelial cell cultures derived from isolated cerebral microvessels*
M. SPATZ, J. BEMBRY, R. F. DODSON, H. HERVONEN and M. R. MURRAY Laboratory of Neuropathology and Neuroanatomical Sciences, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20205 and ( R.F.D.) Department of Cell Biology and Environmental Sciences, 7he University of Texas Health Center at Tyler, Tyler, Texas 75710 (U.S.A.)
(Accepted February 6th, 1980) Key words: endothelial cell cultures -- cerebral microvessels-- dissociated cells
The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to the capillaries of other organs. The intercellular diffusion is restricted by the tight junction separating the continuous layer of endothelial cells, while the vesicular transport is limited in the normal endothelium. Many harmful conditions such as anoxia, ischemia, metabolic and/or degenerative disturbances may alter the endothelium and thus the barrier function. Therefore the elucidation of mechanisms involved in various cerebrovascular disorders such as cerebral ischemia could be facilitated by the study of cellular components of the cerebral vessels in a living state. The present study was designed to establish a cultured cell line originating from cerebral microvessels. In this communication we will describe the establishment of a cell line culture showing the characteristic cytochemical features of cerebral endothelial cells, and which originated from dissociated cells of isolated cerebral microvessels. The brains of 2-day-old Osborne-Mendel rats (36-40) were used under sterile conditions for the separation of the microvessels from the non-vascular tissue. The cerebral hemispheres were freed of leptomeninges, minced with scissors and homogenized in buffered (10 mm Hepes) 0.1 ~ albumin-Ringer solution at pH 7.4. The fraction of microvessels was obtained by centrifugation and discontinuous sucrose gradient (1.0-1.5 m), a simple technique described previouslylk Two samples of microvessels (pellets) were pooled and washed twice with 10 ml of modified Simm's Balanced Salt Solution (BSS) containing 100 U penicillin, 100/~g streptomycin and 0.25 #g Fungizone (Gibco, Long Island, N.Y.). They were centrifuged immediately after the first wash and then washed again for 30 min in the refrigerator prior to centrifugation at * Presented in part at the Fifty-Fifth Annual Meeting of the American Association of Neuropathologists, Kansas City, Mo., 1979.
578 1000 rpm for 8 min. Then the cells of the rewashed microvessels were dissociated with 0.01 o//,,trypsin-collagenase in Hank's solution without Ca 2~ and Mg 2~ (trypsin I " 250. Gibco; collagenase type, Sigma, St. Louis, Mo.) by slow stirring for 5 min at room temperature. These dissociated cells were recentrifuged and resuspended in 5 ml feeding medium, placed in one plastic flask (75 sq. cm) and incubated in an atmosphere of air and 5 ~; CO2 at 37 °C. The feeding medium consisted of 55~,, medium 199 (with 25 mm Hepes Buffer, Earle's Salts and L-glutamine), 1 ~o BME amino acids, 1 ~ BME vitamins, 30°/o fetal calf serum (heat-inactivated), 1 °/ooantibiotic antimycotic mixture (all from Gibco) 2 o/,, of 50 ~ glucose solution (Dextrose, anhydrous, Baker, Philipsburg, N.J.) and 10 ~,~of 5 mg/ml peptone (powder, type I, Sigma) at pH 7.2. After 2 days, the feeding solution was removed and 5 ml of fresh medium was added. At 4 days, the culture was washed briefly in Simm's BSS containing 1 ~ of antibiotic-antimycotic solution and refed. This procedure was then followed on a biweekly basis, but the concentration of fetal calf serum in the medium was reduced to 20 ~ after 2 weeks of culturing the cells. The dissociated cells grew slowly as sheets and/or a network of elongated cells, covering the entire surface of the flask in about 4 weeks. At this time and/or at this stage of growth, the cells were washed with Ca 2~ and Mg ~ ~ free Hank's solution, dissociated with 0.1 ~o trypsin in Ca 2÷ and Mg 2+ free Hank's (15-25 min at 37 °C) and centrifuged at 1000 rpm for 5 rain. Then the cells were resuspended in 20 ml of feeding medium and placed in two 75 sq. cm flasks. In about two weeks the cells became confluent again and ready for the second transfer which was accomplished in the same way as the first one. However, the cells were now placed on round coverslips (7/8 in. in diameter) and kept in Petri dishes (35 mm in diameter) containing the feeding medium. They were fed at least once before the proliferating cells covered the entire coverslip's surface and were ready for investigation. Thus the histochemical and ultrastructural examinations were performed on cell cultures (150 in total) maintained from 50-70 days in vitro. The aniline blue or Giemsa stain. This culture revealed a monolayer of elongated cells interposed by small to large groups of cells appearing to be composed of more than one layer of coalescent cells. They contained oval nuclei with prominent nucleoli and showed a moderate number of mitotic figures (Fig. la). The activities of alkaline phosphatase 4, butyryl cholinesterase 10 7-glutamyl transpeptidase 1,7 and L-DOPA uptake (demonstrated by glyoxylic acid induced fluorescenceZ) which had been used as markers for cerebral capillary endothelium in explants and in vivo 1,3,12,13 were detected in these cultures, with suitable modification for dispersed cell culture, of the procedures designed for other circumstances. Alkaline phosphatase. Activity of alkaline phosphatase was present in almost all cells, demonstrable as an intense diffuse and granular bluish-purple staining reaction in the cytoplasm. However, some of the cells revealed only a slight to moderate amount of reaction product (Fig. lb). Butyryl cholinesterase. Butyryl cholinesterase activity was visible as a cytoplasmic brown granular reaction, which was more pronounced juxtanuclearly than at the
579
Fig. 1. a-e: light microscopy: tertiary cultures of cerebral endothelial cells, a ' a confluent monolayer of elongated cells with interspersed groups of cells composed of more than one layer of ceils. Note the apparent formation of branches and anastomoses. Aniline blue stain, x 130. b: alkaline phosphatase activity is localized in the cytoplasm of almost all cells but with a variable intensity, x 130. c: butyryl cholinesterase activity. The reaction product is especially heavy juxtanuclearly, x 330. d: ),-glutamyl transpeptidase activity. The most intense reactivity is seen in a group of ceils appearing to be composed of more than one layer, x 250. e: L-DOPA. Intense cytoplasmic fluorescence is seen in a granular cell as in d derived from sister cultures, x 250.
580 periphery of the cells. The enzyme was demonstrable in all the cells when a favorable technique was devised by modifying the Karnovsky-Roots method as follows: 2 h fixation, double the substrate concentration and 18 h of incubation (Fig. lc). The BuCHE activity was completely inhibited by Iso-OMPA and eserine sulfate. Reactivity was also absent when the substrate butyrylcholine iodine was substituted by acetylcholine iodine. 7-Glutamyl transpeptidase. Activity of y-glutamyl transpeptidase was demonstrated as intense red-brown reaction product primarily in the groups of cells appearing to be composed of more than one layer showing an anastomosing pattern, and in some individual cells which were surrounded by nonreactive and only slightly reactive cells (Fig. ld). The demonstration of this enzyme required also short fixation time (30-60 rain) and increased incubation time of 4-6 h as well as double concentration of the substrate when Glenner's technique was used. L-DOPA. A diffuse and slightly granular cytoplasmic fluorescence was seen in individual cells and groups of cells showing the same pattern as the one demonstrating 7glutamyl transpeptidase activity in sister cultures (Fig. le). Electron microscopy. In fixation for transmission electron microscopy the cover slips were placed in 3 ~ glutaraldehyde in 0.1 M phosphate solution. After an adequate fixation interval and a buffer rinse, postfixation was carried out in a 1 ~ osmium tetroxide/phosphate preparation. For further contrast enhancement, the tissue was soaked in 1/2 ~o uranyl acetate at 4 °C for 12 h. Following standard ethanol dehydration, the cultures were embedded in Spurr plastic*.L Ultrastructurally, these cultures revealed one cell type of numerous elongated flat cells situated in close proximity to each other and showing many intercellular 'gap' junctions (Fig. 2a and b). Their cytoplasm contained well-developed Golgi complexes, mitochondria and rough endoplasmic reticulum. Pinocytotic vesicles were present in moderate numbers and were localized in subplasmalemmal regions. The elongated nucleus occupied a central position in the cell and contained a homogeneously granular karyoplasm. A band of heterochromatin was located at the edge of the nuclear envelope. Dense organelles such as lysosomes or Weibel-Palade bodies were rarely seen in the culture material, and this was considered consistent with the metabolic state of the cells and their level of maturation. The histochemical and ultrastructural features were consistent with those in the cerebral capillaries studied in situ and in the organotypic brain cultures seen by us and others la. Although these cultures displayed some cellular differences in degree of enzymatic reactivity, they were found to be composed almost of a single cell type when examined by transmission microscopy, Therefore such variations could be functional or positional in nature and possibly related to the cellular maturity rather than to a difference in cellular origin. Even though the 7-glutamyl transpeptidase activity and LDOPA uptake could be seen in some of the dissociated brain cells of nonvascular origin, it appears unliklely that the same cells (sister cultures) would show also a marked reactivity for the nonspecific cholinesterase and alkaline phosphatase which have been considered as the best characteristic markers for the identification of cerebral capillary endothelium.
581
iii:i/ ~i: ........ii~. . . . Fig. 2. a and b: electron microscopy, a: this section through a layer of the culture reveals the homogeneity of cell type as well as the points of cellular apposition (arrows). × 24,000. b : the junctional complex shown in this field is of the typical 'gap' form commonly found throughout the preparation. × 77,000. The presence of intercellular 'gap' junctions and the absence of 'tight' junctions may be related either to the cellular immaturity 6 or to the incapacity of endothelial cells dissociated from the isolated microvessels of 2-day-old brain to f o r m ' t i g h t ' junctions in vitro. In any case further investigations are required to settle these problems. The difficulty in obtaining cerebrovascular endothelium in contrast to the relative ease described in collecting the endothelial lining of peripheral vessels 9,14 has been the greatest obstacle in establishing an endothelial culture model for elucidation of their unique cerebral function. Therefore, most of the previous observations of endothelial cell behavior were derived from organotypic cultures of nervous tissue a,12. However, recently De Bault et al. 5 described an organoid culture of cerebral microvessels which, depending on their size, gave rise to either endothelial and smooth muscle or to endothelial cells only. These cultures lacked some of the enzymatic characteristics of endothelium, but the 7-glutamyl transpeptidase could be induced by co-culturing these cells with glioma cells (personal communication). The original age of the isolated microvessels as well as the different procedure used by us for the isolation and culturing of the endothelium might have been responsible for obtaining an enzymatically more active endothelial cell line as compared to others. The availability of a relatively easy procedure for establishing and cultivating pure cerebral endothelial cells which are metabolically active provides a model for the study of their normal and altered endothelial function which may contribute to the understanding of the complex regulatory function of the blood-brain barrier.
582 1 Albert, Z., Orlowski, M., Rzucidlo, Z. and Orlowska, J., Studies on 7-glutamyl transpeptidase activity and its histochemical localization in the central nervous system, Acta Histochem., 25 (1966) 312-320. 2 Axelsson, G., Bj6rklund, A., Falck, B., Lindvall, O. and Svensson, L.-~., Glyoxylic acid condensation: a new fluorescence method for the histochemical demonstration of biogenic monoamines, Acta physiol, scand., 87 (1973) 57-62. 3 Bertler, A., Falck, B., Owman, C. H. and Rosengrenn, E., The localization of monoaminergic blood-brain barrier mechanisms, Pharmacol. Rev., 18 (1966) 369--385. 4 Burstone, M. S., Histochemical demonstration of phosphatases in frozen sections with naphtbol AS-phosphates, J. Histochem. Cytochem., 9 (1961) 146-153. 5 De Bault, L. E., Kahn, L. E., Frommes, S. P. and Cancilla, P. A., Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization, hi Vitro, 15 (1979) 473-487. 6 Delorme, P., Gayet, J. and Grignon, G., Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken, Brain Research, 22 (1970) 269-283. 7 Glenner, G. G., Folk, J. E. and McMillan, P. J., Histochemical demonstration of a 7-glutamyl transpeptidase-like activity, J. Histochem. Cytochem., 10 (I 962) 481-489. 8 Hauw, J. J., Berger, B. and Escourolle, R., Ultrastructural observations on human cerebral capillaries in organ culture, Cell Tissue Res., 1963 (1975) 133-150. 9 Jaffe, E. A., Nachman, R. L., Becker, C. G. and Minick, C. R., Culture of human endothelial ceils derived from umbilical veins. Identification by morphologic and immunologic criteria, J. clin. hlvest., 52 (1973) 2745-2756. 10 Karnovsky, M. J. and Roots, L., A 'direct-coloring' thiocholine method tbr cbolinesterase, J. Histochem. Cytochem., 12 (1964)219 221. 11 Mrsulja, B. B., Mrsulja, B. J., Fujimoto, T., Klatzo, I. and Spatz, M., Isolation of brain capillaries: a simplified technique, Brain Research, 110 (1976) 361-365. 12 Panula, P., Joo, F. and Rechardt, L., Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain, Experientia (Basel), 34 (1978) 95 96. 13 Renkawek, K., Murray, M. R., Spatz, M. and Klatzo, I., Distinctive histochemical characteristics of brain capillaries in organotypic culture, Exp. Neurol., 50 (1976) 194~206. 14 Schwartz, S. M., Selection and characterization of bovine aortic endothelial cells, In Vitro, 14 (1978) 966-980. 15 Spurr, A. R., A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastrltct. Res., 26 (1969) 31 43.