- Email: [email protected]
Postnatal development of the endothelial system of the rat cerebellum using immunohistochemical techniques
Developmental Brain Research, 17 (1985) 253-257 Elsevier
253
BRD 60038
Short Communications
Postnatal development of the endothelial system of the rat cerebellum using immunohistochemical techniques J. P. ZANETTA, M. DONTENWILL and G. BRICCA Centre de Neurochimie du CNRS, 67084Strasbourg Cedex (France)
(Accepted July 10th, 1984) Key words: endothelial system - - rat cerebellum - - development - - immunohistochemistry
The development of the endothelial system of the rat cerebellum was studied by immunohistochemical techniques with two antibodies specific of two membrane antigens characteristic of 'mature' endothelial cells. It appears that the maturation of endothelial cells is very late in the rat cerebellum. This absence of a fully endothelial barrier in cerebella of young rats could explain the extreme sensitivity of this part of the central nervous system to various agents. Very little is known about the time course of the setting up of the functional endothelial system within the central nervous system and particularly in the cerebellum. H o w e v e r , this endothelial system is responsible for the so called b l o o d - b r a i n barrier which has a fundamental role for the normal function of the central nervous system. W e have recently isolated 10 two m e m b r a n e - b o u n d antigens from the rat liver which are specifically localized in sinusoidal cells (endothelial and K u p p f e r cells). These antigens seem to be specific for m a t u r e endothelial cells since they are absent in livers of young rats and d i s a p p e a r after partial h e p a t e c t o m y 10. These characteristics offer the opportunity using two monospecific antibodies against tl:tese antigens to follow the m a t u r a t i o n of the endothelial system in the rat cerebellum. This p a p e r reports the results of this immunohistochemical study and discusses the possible consequences of the late maturation of this system in the rat cerebellum. The antigens L1 and L2 were p r e p a r e d from rat livers as previously described 10. Briefly, the m e t h o d involved sequential extractions with saline buffer containing or not neutral detergents. The remaining
insoluble material contained m a j o r bands called LI* and L2* with respective molecular weights of 52,900 and 33,200 daltons which were isolated by p r e p a r a tive gel electrophoresis in presence of SDS. A n t i b o d ies against these antigens were raised in rabbit as previously described6,10 and their specificities were tested by immunoblotting techniques 8. F o r immunohistochemistry, rats of different ages (2,5,8,10,13,18,28 and 41 days) were fixed by intracardiac perfusion of a 4% (w/v) solution of p a r a f o r m a l d e h y d e in p h o s p h a t e saline buffer (PBS). The cerebella were excized and sections 100-/~m thickness were o b t a i n e d with an Oxford vibratome. A f t e r rinses overnight in PBS, sections were treated with the indirect i m m u n o p e r o x i dase technique as previously described. The dilution of anti-L1 and anti-L2 antibodies were 1/250 in PBS. A f t e r treatments, sections were revealed with the diaminobenzidine technique of G r a h a m and Karnovsky3. Sections were o b s e r v e d with a Leitz O r t h o p l a n microscope e q u i p e d with the O r t h o m a t system for photography. The d e v e l o p m e n t a l p a t t e r n of the immunohistochemical stain with anti-L2 antibody is shown in
Correspondence: J. P. Zanetta, Centre de Neurochimie du CNRS, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. * L1 and L2 refer to Liver 1 and Liver 2 antigens l°. They are unrelated to cerebellar antigens obtained by others (A. Faissner et al., EMBO Journal 3 (1984) 733-738; Schachner et al., Cold Spring Harbor Symposium, LVIII (1984) 557-568).
254
t
E Fig. 1. Immunohistochemical localization of L2 during the postnatal development of the rat cerebellum using the indirect immunoperoxidase staining. The following ages are represented: A: 2 days; B: 8 days; C: 13 days; D: 13 days; E: 18 days; F: 41 days. The magnification was x 375 for A, B and C and x 75 for D, E and F. The following abbreviations are used: egl, external germinative layer; M, molecular layer; P, Purkinje cell layer; G, granular layer; W, white matter.
255 Fig. 1. As expected from our previous work with endothelial cells of the liver sinusoids 1°, L2 is present in endothelial cells of the adult rat cerebellum. This is true not only for the endothelial cells of the pia mater but also for those surrounding~most blood vessels. As shown in Fig. 1F, the endothelial barrier is represented by a continuous staining for L2. At higher magnification (not shown), it appears that each cerebellar lobule is surrounded by this continuous endothelial barrier where both cell bodies and processes are entirely and strongly labeled. The pattern of staining is very similar to the 28th postnatal day. At the 18th postnatal day, the label is also continuous along the pia mater although it is less intense. At the 13th postnatal day, the staining is continuous only in the most external part of the lobuli, whereas in the internal parts it is only restricted to a :small number of endothelial cell bodies (Fig. 1). In younger animals (Fig. 1A-C), only a few number of endothelial cells bodies are labeled along the pia mater, but not their short processes.
Using anti-L1 antibody (Fig. 2A and B), virtually no staining of the pia mater is observed before the 18th postnatal day although the endothelial barrier is present as revealed by anti-L2 antibody. Only at the 28th postnatal day (Fig. 2B) is the adult pattern obtained. As we have discussed previously 10, the two antigens L1 and L2 are developmentally regulated in the sinusoids of the rat liver and are characteristic of adult rat. Furthermore, 24 h after partial hepatectomy, these antigens disappear from the endothelial cells in the region of the liver where regeneration takes place. These observations indicated that these two antigens were present only in fully differentiated endothelial cells. This is true for L1 in the rat cerebellum and only partially for L2: some endothelial cell bodies are stained in very young animals. The interesting aspect of this study is the observation that in the rat cerebellum, the endothelial barrier has a very late development, since it is not complete at the 13th postnatal day in the most internal
Fig. 2. Immunohistochemical localization of L1 using the indirect immunoperoxidase technique. Magnification × 75. A: 18-dayold rat cerebellum: note the insigificant staining of the pia mater. B: 28-day old rat cerebellum: the pia mater and blood vessels are strongly stained. Same abbreviations as in Fig. 1.
256
Fig. 3. High magnification ( x 1500) of endothelial cells of the pia mater found in 12 day old rat cerebella: staining with anti-L2 antibodies. Note the typical shape of endothelial cells at this age and the absence of a continuous endothelial barrier.
parts of the lobuli. Although this barrier seems to be complete at the 18th postnatal day (as revealed with anti-L2 antibody), it is not fully differentiated at this period since L1 is absent. This late development of the endothelial system in the rat cerebellum is not very surprising. Also for astrocytes (which are part of the b l o o d - b r a i n barrier) a specific antigen like S-1002, has a relatively late development. This retardation of the setting up of a fully mature b l o o d - b r a i n barrier in the rat cerebellum could ac1 Bohn, M. C. and Lauder, J. M., Cerebellar granule cell genesis in the hydrocortisone-treated rat, Develop. Neurosci., 3 (1980) 81-89. 2 Clos, J., Legrand, C., Legrand, J., Ghandour, M. S., Labourdette, G., Vincendon, G. and Gombos, G., Effects of thyroid state and undernutrition on S100 protein and astroglia development in rat cerebellum, Develop. Neurosci., 5 (1982) 285-292. 3 Graham, R. C. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidasc in the proxi-
count for the specific effects of various drugs and hormones on the cerebellum 1,4&7,9 of young animals. The absence of a functional b l o o d - b r a i n barrier could facilitate the contact of these drugs with developing and maturing n e u r o n s and then perturb dramatically the cerebellar development compared to the other brain regions. We are grateful to Mrs. F. Schohn for typing the manuscript.
mal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Grondin, G., Sharkey, T., Jones, M., Sculthorpe, A. and Taylor, W., Postnatal cerebellar hypoplasia and dysfunction following methylazoxymethanol acetate treatment, Proc. Soc. exp. Biol. Med., 148 (1975) 156-159. Patel, A. J., Vertes, Z. S., Lewis, P. D. and Lai, M., Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological
257 study, Brain Res., 202 (1980) 415-428. 6 Reeber, A., Vincendon, G. and Zanetta, J. P., Isolation and immunohistochemical localization of a 'Purkinje cell specific glycoprotein subunit' from rat cerebellum, Brain Res., 229 (1981) 53-56. 7 Sawasaki, Y., Yamada, N. and Nakajima, H., Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (gunn) rat with hereditary hyperbilirubinaemia, J. Neurochem., 27 (1976) 577-583. 8 Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellu-
lose sheets: procedure and some applications, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 4350-4354. 9 Yamada, N., Sawasaki, Y. and Nakajima, H., Impairment of DNA synthesis in Gunn rat cerebellum, Brain Res., 126 (1977) 295-307. 10 Zanetta, J. P., Bingen, A., Dontenwill, M. and Vincendon, G., Isolation and immunohistochemical characterization of two developmentally regulated highly insoluble antigens from the membrane of sinusoidal cells of the rat liver, Exp. Cell Res., in press.
253
BRD 60038
Short Communications
Postnatal development of the endothelial system of the rat cerebellum using immunohistochemical techniques J. P. ZANETTA, M. DONTENWILL and G. BRICCA Centre de Neurochimie du CNRS, 67084Strasbourg Cedex (France)
(Accepted July 10th, 1984) Key words: endothelial system - - rat cerebellum - - development - - immunohistochemistry
The development of the endothelial system of the rat cerebellum was studied by immunohistochemical techniques with two antibodies specific of two membrane antigens characteristic of 'mature' endothelial cells. It appears that the maturation of endothelial cells is very late in the rat cerebellum. This absence of a fully endothelial barrier in cerebella of young rats could explain the extreme sensitivity of this part of the central nervous system to various agents. Very little is known about the time course of the setting up of the functional endothelial system within the central nervous system and particularly in the cerebellum. H o w e v e r , this endothelial system is responsible for the so called b l o o d - b r a i n barrier which has a fundamental role for the normal function of the central nervous system. W e have recently isolated 10 two m e m b r a n e - b o u n d antigens from the rat liver which are specifically localized in sinusoidal cells (endothelial and K u p p f e r cells). These antigens seem to be specific for m a t u r e endothelial cells since they are absent in livers of young rats and d i s a p p e a r after partial h e p a t e c t o m y 10. These characteristics offer the opportunity using two monospecific antibodies against tl:tese antigens to follow the m a t u r a t i o n of the endothelial system in the rat cerebellum. This p a p e r reports the results of this immunohistochemical study and discusses the possible consequences of the late maturation of this system in the rat cerebellum. The antigens L1 and L2 were p r e p a r e d from rat livers as previously described 10. Briefly, the m e t h o d involved sequential extractions with saline buffer containing or not neutral detergents. The remaining
insoluble material contained m a j o r bands called LI* and L2* with respective molecular weights of 52,900 and 33,200 daltons which were isolated by p r e p a r a tive gel electrophoresis in presence of SDS. A n t i b o d ies against these antigens were raised in rabbit as previously described6,10 and their specificities were tested by immunoblotting techniques 8. F o r immunohistochemistry, rats of different ages (2,5,8,10,13,18,28 and 41 days) were fixed by intracardiac perfusion of a 4% (w/v) solution of p a r a f o r m a l d e h y d e in p h o s p h a t e saline buffer (PBS). The cerebella were excized and sections 100-/~m thickness were o b t a i n e d with an Oxford vibratome. A f t e r rinses overnight in PBS, sections were treated with the indirect i m m u n o p e r o x i dase technique as previously described. The dilution of anti-L1 and anti-L2 antibodies were 1/250 in PBS. A f t e r treatments, sections were revealed with the diaminobenzidine technique of G r a h a m and Karnovsky3. Sections were o b s e r v e d with a Leitz O r t h o p l a n microscope e q u i p e d with the O r t h o m a t system for photography. The d e v e l o p m e n t a l p a t t e r n of the immunohistochemical stain with anti-L2 antibody is shown in
Correspondence: J. P. Zanetta, Centre de Neurochimie du CNRS, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. * L1 and L2 refer to Liver 1 and Liver 2 antigens l°. They are unrelated to cerebellar antigens obtained by others (A. Faissner et al., EMBO Journal 3 (1984) 733-738; Schachner et al., Cold Spring Harbor Symposium, LVIII (1984) 557-568).
254
t
E Fig. 1. Immunohistochemical localization of L2 during the postnatal development of the rat cerebellum using the indirect immunoperoxidase staining. The following ages are represented: A: 2 days; B: 8 days; C: 13 days; D: 13 days; E: 18 days; F: 41 days. The magnification was x 375 for A, B and C and x 75 for D, E and F. The following abbreviations are used: egl, external germinative layer; M, molecular layer; P, Purkinje cell layer; G, granular layer; W, white matter.
255 Fig. 1. As expected from our previous work with endothelial cells of the liver sinusoids 1°, L2 is present in endothelial cells of the adult rat cerebellum. This is true not only for the endothelial cells of the pia mater but also for those surrounding~most blood vessels. As shown in Fig. 1F, the endothelial barrier is represented by a continuous staining for L2. At higher magnification (not shown), it appears that each cerebellar lobule is surrounded by this continuous endothelial barrier where both cell bodies and processes are entirely and strongly labeled. The pattern of staining is very similar to the 28th postnatal day. At the 18th postnatal day, the label is also continuous along the pia mater although it is less intense. At the 13th postnatal day, the staining is continuous only in the most external part of the lobuli, whereas in the internal parts it is only restricted to a :small number of endothelial cell bodies (Fig. 1). In younger animals (Fig. 1A-C), only a few number of endothelial cells bodies are labeled along the pia mater, but not their short processes.
Using anti-L1 antibody (Fig. 2A and B), virtually no staining of the pia mater is observed before the 18th postnatal day although the endothelial barrier is present as revealed by anti-L2 antibody. Only at the 28th postnatal day (Fig. 2B) is the adult pattern obtained. As we have discussed previously 10, the two antigens L1 and L2 are developmentally regulated in the sinusoids of the rat liver and are characteristic of adult rat. Furthermore, 24 h after partial hepatectomy, these antigens disappear from the endothelial cells in the region of the liver where regeneration takes place. These observations indicated that these two antigens were present only in fully differentiated endothelial cells. This is true for L1 in the rat cerebellum and only partially for L2: some endothelial cell bodies are stained in very young animals. The interesting aspect of this study is the observation that in the rat cerebellum, the endothelial barrier has a very late development, since it is not complete at the 13th postnatal day in the most internal
Fig. 2. Immunohistochemical localization of L1 using the indirect immunoperoxidase technique. Magnification × 75. A: 18-dayold rat cerebellum: note the insigificant staining of the pia mater. B: 28-day old rat cerebellum: the pia mater and blood vessels are strongly stained. Same abbreviations as in Fig. 1.
256
Fig. 3. High magnification ( x 1500) of endothelial cells of the pia mater found in 12 day old rat cerebella: staining with anti-L2 antibodies. Note the typical shape of endothelial cells at this age and the absence of a continuous endothelial barrier.
parts of the lobuli. Although this barrier seems to be complete at the 18th postnatal day (as revealed with anti-L2 antibody), it is not fully differentiated at this period since L1 is absent. This late development of the endothelial system in the rat cerebellum is not very surprising. Also for astrocytes (which are part of the b l o o d - b r a i n barrier) a specific antigen like S-1002, has a relatively late development. This retardation of the setting up of a fully mature b l o o d - b r a i n barrier in the rat cerebellum could ac1 Bohn, M. C. and Lauder, J. M., Cerebellar granule cell genesis in the hydrocortisone-treated rat, Develop. Neurosci., 3 (1980) 81-89. 2 Clos, J., Legrand, C., Legrand, J., Ghandour, M. S., Labourdette, G., Vincendon, G. and Gombos, G., Effects of thyroid state and undernutrition on S100 protein and astroglia development in rat cerebellum, Develop. Neurosci., 5 (1982) 285-292. 3 Graham, R. C. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidasc in the proxi-
count for the specific effects of various drugs and hormones on the cerebellum 1,4&7,9 of young animals. The absence of a functional b l o o d - b r a i n barrier could facilitate the contact of these drugs with developing and maturing n e u r o n s and then perturb dramatically the cerebellar development compared to the other brain regions. We are grateful to Mrs. F. Schohn for typing the manuscript.
mal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Grondin, G., Sharkey, T., Jones, M., Sculthorpe, A. and Taylor, W., Postnatal cerebellar hypoplasia and dysfunction following methylazoxymethanol acetate treatment, Proc. Soc. exp. Biol. Med., 148 (1975) 156-159. Patel, A. J., Vertes, Z. S., Lewis, P. D. and Lai, M., Effect of chlorpromazine on cell proliferation in the developing rat brain. A combined biochemical and morphological
257 study, Brain Res., 202 (1980) 415-428. 6 Reeber, A., Vincendon, G. and Zanetta, J. P., Isolation and immunohistochemical localization of a 'Purkinje cell specific glycoprotein subunit' from rat cerebellum, Brain Res., 229 (1981) 53-56. 7 Sawasaki, Y., Yamada, N. and Nakajima, H., Developmental features of cerebellar hypoplasia and brain bilirubin levels in a mutant (gunn) rat with hereditary hyperbilirubinaemia, J. Neurochem., 27 (1976) 577-583. 8 Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellu-
lose sheets: procedure and some applications, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 4350-4354. 9 Yamada, N., Sawasaki, Y. and Nakajima, H., Impairment of DNA synthesis in Gunn rat cerebellum, Brain Res., 126 (1977) 295-307. 10 Zanetta, J. P., Bingen, A., Dontenwill, M. and Vincendon, G., Isolation and immunohistochemical characterization of two developmentally regulated highly insoluble antigens from the membrane of sinusoidal cells of the rat liver, Exp. Cell Res., in press.