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Distribution of the endothelial constitutive nitric oxide synthase in the developing rat brain: an immunohistochemical study
Brain Research 788 Ž1998. 43–48
Research report
Distribution of the endothelial constitutive nitric oxide synthase in the developing rat brain: an immunohistochemical study Ingolf Topel, Andreas Stanarius, Gerald Wolf ¨
)
Institute of Medical Neurobiology, Otto-Õon-Guericke-UniÕersity of Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany Accepted 9 December 1997
Abstract The present study deals with the distribution of endothelial constitutive nitric oxide synthase ŽecNOS. in the developing rat brain using optimized protocols for preparation and fixation and the tyramide-signal-amplification technique. The immunostaining patterns of a monoclonal antibody against ecNOS are compared with results obtained with a rat pan-endothelial marker, the monoclonal RECA-1 antibody. It is shown that ecNOS is present in the endothelial lining of all types of blood vessels and the choroid plexuses of the rat brain from the beginning of vasculogenesis at embryonic day 11 until adulthood Ž75 weeks.. The same is true for RECA-1 immunoreactivity, that was demonstrated in the developmental brain vasculature for the first time. Both antigens expressed identical immunostaining patterns. At all investigated stages of brain development neither ecNOS negative blood vessels nor ecNOS positive non-endothelial cells, e.g., neurons, were found. The data indicate that ecNOS is involved in the embryonic angiogenesis and the regulation of hemodynamic functions of brain vasculature throughout the individual life. q 1998 Elsevier Science B.V. Keywords: Endothelial nitric oxide synthase; Brain development; Vasculature; Rat; Immunohistochemistry; Tyramide-signal amplification
1. Introduction Nitric oxide ŽNO. is a free radical gas involved in the regulation of blood flow, in neurotransmission and immunological defense Žfor review see Refs. w10,35x.. Nitric oxide synthase ŽNOS. catalyzes the formation of NO from L-arginine. In the central nervous system, NOS exists constitutively in two isoforms that are distinct gene products: neuronal NOS ŽnNOS. described to be predominantly located in neurons and the endothelial constitutive NOS ŽecNOS., which has been specified from bovine aortic endothelial cells. In the adult rat brain, ecNOS protein has been found in endothelial cells of vasculature and choroid plexus w28x. The expression of constitutive NOS isoforms is developmentally regulated w14,20x, but no data exist about the distribution patterns of ecNOS in rat brain blood vessels during embryonic development. Progressive ecNOS contents in brain vessels have been described by Northington et al. w20x for the embryonic sheep, and Ursell and Mayes w32x have found an increase in ecNOS immunoreactivity in
)
Corresponding author. Fax.: q49-391-6714365
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 5 0 6 - 0
the rat heart during ontogenesis. Here, we demonstrate by means of immunohistochemistry the occurrence and the distribution patterns of ecNOS during rat brain development. The tyramide-signal-amplification ŽTSA. method w1,5x was used to improve the outcome of the immunostaining procedure as detailed in a parallel study w28x. Data obtained have been compared with immunoreactivity patterns of a rat pan-endothelial cell-specific marker, the RECA-1 antibody w8x.
2. Materials and methods The study was conducted on Wistar rats ŽSchonwalde, ¨ Germany.. The animals were housed under temperature controlled conditions at 218C, 12 h light and 12 h darkness with free access to food and water. The age of the animals was determined by the conceptional day as embryonic day 0 ŽED 0. and the day of delivery as day 0 of post-natal life ŽP 0.. Brains of at least three animals of each of the following ages were studied: ED 10, 11, 12, 13, 15, 17, and P 0, 5, 20, as well as adult rats aging 16 and 75 weeks. Pregnant rats were deeply anaesthetized with ether and the fetuses
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I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
Fig. 1. Parietal cortex of the adult rat brain, RECA-1 immunohistochemistry. The endothelium of the entire vasculature is labeled. Bar s 90 m m.
Fig. 2. Parietal cortex of the adult rat brain, ecNOS immunohistochemistry. ŽA. The endothelial lining of arteries, veins and capillaries is immunopositive. No other structures are labeled. Arrow: artery; arrowhead: vein. ŽB. Capillaries at high magnification: the immunostaining is particularly intense in the perinuclear region. Arrow: nucleus. Bars s 90 m m in ŽA. and 15 m m in ŽB..
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
were removed. The gestational age of the fetuses was confirmed by respective morphological signs according to Baker et al. w3x. Embryonic brains were prepared quickly and shock-frozen in y208C isopentane, whereas all postnatal stages were first transcardially perfused with a solution of 0.9% NaCl in phosphate buffered saline ŽPBS, pH 7.4. under deep ether anesthesia. Sections were cut at 20 m m on a freezing microtome ŽJung 2800, LEICA, Germany. and mounted on Superfrost-plus glass slides ŽMenzel-Glaser, Germany.. After air-drying the specimens ¨ were pre-fixed in acetone at room temperature for 10 s and stored at y208C until use. Immunostaining for ecNOS was carried out with a monoclonal antibody from Transduction Laboratories ŽLexington, USA. that was selected from a panel of commercially available antibodies by specificity control experiments with bovine aortic tissue Žfor details see, Ref. w28x.. In the same study, the suitability of the TSA method was tested for the application of this ecNOS antibody and optimized. Briefly, the sections were fixed in acetone at y208C for 10 min followed by a mixture of 4% paraformaldehyde and 0.4% glutaraldehyde in PBS at room temperature for 10 min. After a threefold rinsing with Tris-phosphate buffered saline Ž46.7 ml 0.2 M PBS, 1.21 g
45
Tris diluted in 950 ml of distilled water; TPBS, pH 7.4. containing 0.3% Triton ŽTPBS–T. the sections were incubated with 3% normal horse serum ŽNHS. diluted in the same buffer for 1 h. Then incubation followed with the monoclonal ecNOS antibody diluted 1:10,000 in TPBS–T with 3% NHS at room temperature overnight. The following steps were carried out using the Vectastain ABC-Elite Kit ŽVector Laboratories, Burlingame, USA.: after an extensive wash, the sections were incubated with a secondary antibody Žgoat-anti-mouse IgG. at a dilution of 1:200 in TPBS–T for 1 h, followed by 0.25% avidin–biotin reagent for 1 h. Thereafter, the sections were incubated with 0.01% biotinylated tyramide ŽDuPont NEN, Boston, USA. and 0.005% H 2 O 2 in TPBS–T for 1 h followed by 0.125% avidin–biotin reagent in TPBS–T for 1 h again. Each incubation step was preceded by a thorough wash with TPBS–T. The sections were visualized with 0.05% 3,3X-diaminobenzidine ŽDAB, Sigma Deishagen, Germany. and 0.02% H 2 O 2 in TPBS under microscopical control, washed in TPBS, counterstained with Mayer’s hematoxylin and then mounted with DePeX-mountant. For control experiments, the primary antisera were omitted or substituted with nonimmune serum.
Fig. 3. Dorsal rhombencephalon at ED 12. Stem vessels invading the brain parenchyma from the leptomeningeal plexus are immunopositive. ŽA. RECA-1 immunohistochemistry. ŽB. ecNOS immunohistochemistry. Arrows: leptomeningeal plexus; curved arrows: stem vessels. Bars s 45 m m in ŽA. and ŽB..
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I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
For the pan-endothelial staining, the sections were fixed as mentioned above. After three washes with TPBS–T, the slices were pre-incubated with 3% NHS in TPBS–T for 1 h followed by incubation with the monoclonal mouse RECA-1 antibody ŽCamon, Wiesbaden, Germany. at a dilution of 1:10,000 in TPBS–T with 3% NHS at room temperature overnight. The following steps were carried out using the Vectastain ABC-Elite Kit according to the manufacturer. Control experiments and the visualization were carried out in the same way as mentioned for ecNOS.
3. Results Adult brains examined by RECA-1 immunohistochemistry showed, throughout the brain, a distinct labeling of endothelial lining of the vasculature ranging from large vessels to capillaries ŽFig. 1., including those of choroid plexus. No other cells were stained, indicating that the RECA-1 antibody reveals selectively the total of endothelial cells. Control experiments where the primary antibody was omitted or substituted with nonimmune serum failed to show any labeling. Beyond that we could show that the
RECA-1 antigen is expressed by brain endothelial cells from the beginning of brain vasculogenesis at ED 11 until adulthood Ž75 weeks of age.. In adult rat brains ecNOS immunoreactivity was localized to endothelial cells of all of the blood vessels and matched strictly the distribution pattern of RECA-1 staining. Immunostaining of ecNOS was most prominent in endothelial cells of arteries and arterioles, but it was also seen in veins, venules and capillaries ŽFig. 2A.. At higher magnification, the staining appeared to be particularly intense in the perinuclear region ŽFig. 2B.. Neither neurons nor glial cells showed any detectable staining. Again, control experiments where the ecNOS antibody was omitted or substituted with nonimmune serum failed to show any labeling. In developing rat brains, the endothelium of all blood vessels detectable with the RECA-1 antibody also expressed ecNOS immunopositivity. The developing vasculature was termed according to the nomenclature established by Baer w2x. At ED 10, no immunolabeled structures could be observed after immunostaining with the RECA-1 or the ecNOS antibody, apparently because neither blood vessels nor any endothelial cells developed in the prosencephal vesicle at this time. The onset of ecNOS immunolabeling in the brain was at ED 11, when the endothelium of the leptomeningeal plexus Žundifferentiated capillary plexus. was found to be selectively stained with both of the antibodies. However, within the brain parenchyma no labeled structures were detectable at this stage of brain development. Labeled stem vessels radially invading the brain parenchyma from the leptomeningeal plexus were first seen at ED 12 ŽFig. 3A and B.. Immunostained capillaries of the choroid plexus and the tangential sprouts of the radially penetrating stem vessels appeared at ED 13 ŽFig. 4.. The highest density of capillary branches in brain tissue was observed at P 20. There were no evident differences in distribution patterns of ecNOS containing structures between animals aging 16 and 75 weeks.
4. Discussion
Fig. 4. Dorsal rhombencephalon at ED 13, ecNOS immunohistochemistry. Note the tangential sprouting Žarrows. of the labeled stem vessels. Bar s90 m m.
The results of ecNOS immunohistochemistry in adult rat brain are generally congruent with those of other authors w30x. Our results, obtained with optimized fixation and preparation procedures w28x, were confirmed by in situ hybridization with an ecNOS specific probe w26x. Our results corroborate also those of Duijvestijn et al. w8x showing that the RECA-1 antigen, derived from lymph node homogenates, may serve as a pan-endothelial marker in adult rat brain and, as presented in this study for the first time, in the developing brain as well. In no case we have seen any distinct ecNOS immunoreactivity in neurons, as reported by others using the same monoclonal ecNOS antibody w6,7,21x. This discrepancy
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
might be explained by the fact that the outcome of ecNOS immunoreactivity depends largely on tissue fixation and staining protocols w11,33,36x, as pointed out in detail in our parallel study based on the aortic tissue for respective control experiments w28x. In the developing rat brain, we detected ecNOS protein in the endothelium of every blood vessel arising. These findings are supported by Western and Northern blot analysis by Keilhoff et al. w14x. Blood vessels negative for ecNOS could not be found. On the other hand, no other structures were found to be labeled in brain parenchyma. Northington et al. w20x obtained similar results in developing sheep brains. But we could not confirm observations whereupon at early stages of brain development only a few of the vessels are ecNOS immunopositive w18x. Mito et al. w17x have described immunopositive spots on spouting blood vessels in embryonic human brain tissue after application of antisera to laminin, fibronectine and type IV collagen, suggesting that these structures represent very early stages of angiogenesis. Using the ecNOS and RECA-1 antibodies in embryonic rat tissue, these spots could not be observed, possibly because both of the antigens are expressed not before the precursors of the endothelial cells are sufficiently differentiated. NO has been shown to contribute to the basal cerebral tone and the maintenance of the cerebral blood flow in adult and newborn subjects w9,25x. The same seems to be true for the premature brain w12x. NO leads to a dilation of large blood vessels by relaxation of the smooth muscle cells in the vessel wall w19x, and several studies showed that NO released from the microvascular endothelium serves as an important regulator of small artery and arteriolar tone in cerebral circulation w31,34x. Since capillaries lack smooth muscle cells other functions have to be assumed. So it is known that NO attenuates the expression of intercellular adhesion molecule-1 ŽICAM-1. and vascular cell adhesion molecule-1 ŽVCAM-1. in vitro w4,29x, which modulate the endothelial surface for rolling and adhesion of circulating blood cells. Mitchell and Tyml w16x have hypothesized that low levels of NO modify capillary blood flow by modulating local hemoconcentration and leukocyte adhesion, and that higher levels may cause, therefore, an increase in microvascular blood flow. These considerations have been supported by intravital microscopy w13x. Besides that, NO was shown to alter the microvascular permeability in the intact and inflamed vasculature w15x and, thus, to affect the blood brain barrier w27x. Moreover, NO produced by endothelial cells is involved in regulation of vascular sprouting and capillary organization. That was observed in tumor vasculogenesis w23x and in vitro capillarization w22x. Our results support the assumption that NO released from endothelium promotes, in combination with growth factors, the angiogenesis w38x rather than it has an inhibitory effect on neo-vascularization w24,37x. Considered together, our results indicate that ecNOS is within the rat brain exclusively expressed in the endothe-
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lial cells of the entire vasculature including capillaries. NO released by ecNOS is likely to play a significant role in the regulation of brain blood flow and, possibly for the vasculogenesis in early brain development.
Acknowledgements This work was supported by grants of Ministerium fur ¨ Bildung, Wissenschaft, Forschung und Technologie Ž01 ZZ 9505,B6. and Deutsche Forschungsgemeinschaft ŽWo 474r10-1.. We wish to thank Ms. M. Mockel for skillful ¨ technical assistance and Mrs. M. Stein for photographic work.
References w1x J.C. Adams, Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains, J. Histochem. Cytochem. 40 Ž1992. 1457–1463. w2x T. Baer, Morphometric evaluation of capillaries in different laminae of rat cerebral cortex by automatic image analysis: changes during development and aging, Adv. Neurol. 20 Ž1978. 1–9. w3x H.J. Baker, L. Lindsey, S. Weisbroth, The Laboratory Rat, Vol. II, Research Applications, Academic Press, San Diego, CA, 1980. w4x E. Biffl, E. Moore, F.A. Moore, C.C. Barnett Jr., Nitric oxide reduces endothelial expression of intercellular adhesion molecule ŽICAM.-1, J. Surg. Res. 63 Ž1996. 328–332. w5x M.N. Bobrow, T.D. Harris, K.J. Shaughnessy, G.J. Litt, Catalyzed reporter deposition, a novel method of signal amplification, J. Immunol. Meth. 125 Ž1989. 279–285. w6x J.L. Dinerman, T.M. Dawson, M.J. Schell, A. Snowman, S.H. Snyder, Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: Implications for synaptic plasticity, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 4214–4218. w7x C.A. Doyle, P. Slater, Localization of neuronal and endothelial nitric oxide synthase isoforms in human hippocampus, Neuroscience 76 Ž1997. 387–395. w8x A.M. Duijvestijn, H. van Goor, F. Klatter, G.D. Majoor, E. van Bussel, J.C. van Breda Vriesman, Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody, Lab. Invest. 66 Ž1992. 459–466. w9x F.M. Faraci, D.D. Heistad, Does basal production of nitric oxide contribute to regulation of brain-fluid balance?, Am. J. Physiol. 262 Ž1992. H340–H344. w10x U. Forstermann, H. Kleinert, Nitric oxide synthase: expression and ¨ expressional control of the three isoforms, Naunyn-Schmiedeberg’s Arch. Pharmacol. 352 Ž1995. 351–364. w11x T. Gonzalez-Hernandez, M.A. Perez de la Cruz, B. MantolanSarmiento, Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against non-neuronal NOS antisera, J. Histochem. Cytochem. 44 Ž1996. 1399–1413. w12x P. Hardy, D.R. Varma, S. Chemtob, Control of cerebral and ocular blood flow autoregulation in neonates, Pediat. Clin. N. Am. 44 Ž1997. 137–152. w13x S. Kanwar, P. Kubes, Nitric oxide is an anti-adhesive molecule for leukocytes, New Horizons 3 Ž1995. 93–104. w14x G. Keilhoff, B. Seidel, H. Noack, W. Tischmeyer, D. Stanek, G. Wolf, Patterns of nitric oxide synthase at the messenger RNA and protein levels during early rat brain development, Neuroscience 75 Ž1996. 1193–1201.
48
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
w15x P. Kubes, Nitric oxide affects microvascular permeability in the intact and inflamed vasculature, Microcirculation 2 Ž1995. 235–244. w16x D. Mitchell, K. Tyml, Nitric oxide release in rat skeletal muscle capillary, Am. J. Physiol. 270 Ž1996. H1693–H1703. w17x T. Mito, H. Konomi, S. Houdou, S. Takashima, Immunohistochemical study of the vasculature in the developing brain, Pediat. Neurol. 7 Ž1991. 18–22. w18x T. Miyawaki, O. Sohma, M. Mizuguchi, S. Takashima, Development of nitric oxide synthase in endothelial cells in the human cerebrum, Dev. Brain Res. 89 Ž1995. 161–166. w19x C. Nathan, Q.-W. Xie, Nitric oxide synthases: roles, tolls, and controls, Cell 78 Ž1994. 1360–1362. w20x F.J. Northington, R.C. Koehler, R.J. Traystman, L.J. Martin, Nitric oxide synthase 1 and nitric oxide synthase 3 protein expression is regionally and temporally regulated in fetal brain, Dev. Brain. Res. 95 Ž1996. 1–14. w21x T.J. O’Dell, P.L. Huang, T.M. Dawson, J.L. Dinerman, S.H. Snyder, E.R. Kandel, M.C. Fishman, Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS, Science 265 Ž1994. 542–546. w22x A. Papapetropulus, K.M. Desai, R.D. Rudic, B. Mayer, R. Zhang, M.P. Ruiz-Torres, G. Garcia-Gardena, J.A. Madri, W.C. Sessa, Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro, Am. J. Pathol. 150 Ž1997. 1835–1844. w23x H.I. Peterson, Modification of tumour blood flow—a review, Int. J. Radiat. Biol. 60 Ž1991. 201–210. w24x E. Pilipi-Synetos, E. Sakkouia, G. Haralobopoulos, P. Andriopoulou, P. Peristeris, M.E. Maragoudakis, Evidence that nitric oxide is an endogenous anti-angiogenic mediator, Br. J. Pharmacol. 111 Ž1993. 894–904. w25x W.I. Rosenblum, H. Nishimura, G.H. Nelson, Endothelium-dependent L-Arg- and L-NMMA-sensitive mechanisms regulate tone of brain microvessels, Am. J. Physiol. 259 Ž1990. H1396–H1401. w26x B. Seidel, A. Stanarius, G. Wolf, Differential expression of neuronal and endothelial nitric oxide synthase in blood vessels, Neurosci. Lett. 239 Ž1997. 109–112. w27x A. Shukla, M. Dikshit, R.C. Srimal, Nitric oxide-dependent blood–
w28x
w29x
w30x
w31x w32x
w33x
w34x
w35x w36x
w37x
w38x
brain barrier permeability alteration in the rat brain, Experientia 52 Ž1996. 136–140. A. Stanarius, I. Topel, S. Schulz, H. Noack, G. Wolf, Immunocyto¨ chemistry of endothelial nitric oxide synthase in the rat brain: a light and electron microscopical study using the tyramide signal amplification technique, Acta Histochem. 99 Ž1997. 411–419. M. Takahashi, U. Ikeda, J.-I. Masuyama, H. Funayama, S. Kano, K. Shimada, Nitric oxide attenuates adhesion molecule expression in human endothelial cells, Cytokine 8 Ž1996. 817–821. H. Tomimoto, I. Akiguchi, H. Wakita, S. Nakamura, J. Kimura, Histochemical demonstration of membranous localization of endothelial cells of the rat brain, Brain Res. 667 Ž1994. 107–110. J.G. Umans, R. Levi, Nitric oxide in the regulation of blood flow and arterial pressure, Ann. Rev. Physiol. 57 Ž1995. 771–790. P.C. Ursell, M. Mayes, Endothelial isoform of nitric oxide synthase in rat heart increases during development, Anat. Rec. 246 Ž1996. 465–465. R.R. Vaid, B.K. Yee, J.N.P. Rawlins, S. Totterdell, NADPH-diaphorase reactive pyramidal neurons in Ammon’s horn and the subiculum of the rat hippocampal formation, Brain Res. 733 Ž1996. 31–40. E.P. Wei, R. Kukreja, H.A. Kontos, Effects in cats of inhibition of nitric oxide synthesis on cerebral vasodilation and endothelium-derived relaxing factor from acetylcholine, Stroke 23 Ž1992. 1623– 1629. G. Wolf, Nitric oxide and nitric oxide synthase: biology, pathology, localization, Histol. Histopathol. 12 Ž1997. 251–261. J. Worl, W.L. Neuhuber, ¨ M. Wiesand, B. Mayer, K.R. Greskotter, ¨ Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation, Histochemistry 102 Ž1994. 353–364. W. Yang, J. Ando, R. Korenaga, T. Toyo-oka, A. Kamija, Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells, Biochem. Biophys. Res. Commun. 203 Ž1994. 1160–1167. M. Ziche, L. Morbidelli, E. Masini, S. Amerini, H.J. Granger, C.A. Maggi, T. Geppetti, F. Ledda, Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P, J. Clin. Invest. 94 Ž1994. 2036–2044.
Research report
Distribution of the endothelial constitutive nitric oxide synthase in the developing rat brain: an immunohistochemical study Ingolf Topel, Andreas Stanarius, Gerald Wolf ¨
)
Institute of Medical Neurobiology, Otto-Õon-Guericke-UniÕersity of Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany Accepted 9 December 1997
Abstract The present study deals with the distribution of endothelial constitutive nitric oxide synthase ŽecNOS. in the developing rat brain using optimized protocols for preparation and fixation and the tyramide-signal-amplification technique. The immunostaining patterns of a monoclonal antibody against ecNOS are compared with results obtained with a rat pan-endothelial marker, the monoclonal RECA-1 antibody. It is shown that ecNOS is present in the endothelial lining of all types of blood vessels and the choroid plexuses of the rat brain from the beginning of vasculogenesis at embryonic day 11 until adulthood Ž75 weeks.. The same is true for RECA-1 immunoreactivity, that was demonstrated in the developmental brain vasculature for the first time. Both antigens expressed identical immunostaining patterns. At all investigated stages of brain development neither ecNOS negative blood vessels nor ecNOS positive non-endothelial cells, e.g., neurons, were found. The data indicate that ecNOS is involved in the embryonic angiogenesis and the regulation of hemodynamic functions of brain vasculature throughout the individual life. q 1998 Elsevier Science B.V. Keywords: Endothelial nitric oxide synthase; Brain development; Vasculature; Rat; Immunohistochemistry; Tyramide-signal amplification
1. Introduction Nitric oxide ŽNO. is a free radical gas involved in the regulation of blood flow, in neurotransmission and immunological defense Žfor review see Refs. w10,35x.. Nitric oxide synthase ŽNOS. catalyzes the formation of NO from L-arginine. In the central nervous system, NOS exists constitutively in two isoforms that are distinct gene products: neuronal NOS ŽnNOS. described to be predominantly located in neurons and the endothelial constitutive NOS ŽecNOS., which has been specified from bovine aortic endothelial cells. In the adult rat brain, ecNOS protein has been found in endothelial cells of vasculature and choroid plexus w28x. The expression of constitutive NOS isoforms is developmentally regulated w14,20x, but no data exist about the distribution patterns of ecNOS in rat brain blood vessels during embryonic development. Progressive ecNOS contents in brain vessels have been described by Northington et al. w20x for the embryonic sheep, and Ursell and Mayes w32x have found an increase in ecNOS immunoreactivity in
)
Corresponding author. Fax.: q49-391-6714365
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 5 0 6 - 0
the rat heart during ontogenesis. Here, we demonstrate by means of immunohistochemistry the occurrence and the distribution patterns of ecNOS during rat brain development. The tyramide-signal-amplification ŽTSA. method w1,5x was used to improve the outcome of the immunostaining procedure as detailed in a parallel study w28x. Data obtained have been compared with immunoreactivity patterns of a rat pan-endothelial cell-specific marker, the RECA-1 antibody w8x.
2. Materials and methods The study was conducted on Wistar rats ŽSchonwalde, ¨ Germany.. The animals were housed under temperature controlled conditions at 218C, 12 h light and 12 h darkness with free access to food and water. The age of the animals was determined by the conceptional day as embryonic day 0 ŽED 0. and the day of delivery as day 0 of post-natal life ŽP 0.. Brains of at least three animals of each of the following ages were studied: ED 10, 11, 12, 13, 15, 17, and P 0, 5, 20, as well as adult rats aging 16 and 75 weeks. Pregnant rats were deeply anaesthetized with ether and the fetuses
44
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
Fig. 1. Parietal cortex of the adult rat brain, RECA-1 immunohistochemistry. The endothelium of the entire vasculature is labeled. Bar s 90 m m.
Fig. 2. Parietal cortex of the adult rat brain, ecNOS immunohistochemistry. ŽA. The endothelial lining of arteries, veins and capillaries is immunopositive. No other structures are labeled. Arrow: artery; arrowhead: vein. ŽB. Capillaries at high magnification: the immunostaining is particularly intense in the perinuclear region. Arrow: nucleus. Bars s 90 m m in ŽA. and 15 m m in ŽB..
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
were removed. The gestational age of the fetuses was confirmed by respective morphological signs according to Baker et al. w3x. Embryonic brains were prepared quickly and shock-frozen in y208C isopentane, whereas all postnatal stages were first transcardially perfused with a solution of 0.9% NaCl in phosphate buffered saline ŽPBS, pH 7.4. under deep ether anesthesia. Sections were cut at 20 m m on a freezing microtome ŽJung 2800, LEICA, Germany. and mounted on Superfrost-plus glass slides ŽMenzel-Glaser, Germany.. After air-drying the specimens ¨ were pre-fixed in acetone at room temperature for 10 s and stored at y208C until use. Immunostaining for ecNOS was carried out with a monoclonal antibody from Transduction Laboratories ŽLexington, USA. that was selected from a panel of commercially available antibodies by specificity control experiments with bovine aortic tissue Žfor details see, Ref. w28x.. In the same study, the suitability of the TSA method was tested for the application of this ecNOS antibody and optimized. Briefly, the sections were fixed in acetone at y208C for 10 min followed by a mixture of 4% paraformaldehyde and 0.4% glutaraldehyde in PBS at room temperature for 10 min. After a threefold rinsing with Tris-phosphate buffered saline Ž46.7 ml 0.2 M PBS, 1.21 g
45
Tris diluted in 950 ml of distilled water; TPBS, pH 7.4. containing 0.3% Triton ŽTPBS–T. the sections were incubated with 3% normal horse serum ŽNHS. diluted in the same buffer for 1 h. Then incubation followed with the monoclonal ecNOS antibody diluted 1:10,000 in TPBS–T with 3% NHS at room temperature overnight. The following steps were carried out using the Vectastain ABC-Elite Kit ŽVector Laboratories, Burlingame, USA.: after an extensive wash, the sections were incubated with a secondary antibody Žgoat-anti-mouse IgG. at a dilution of 1:200 in TPBS–T for 1 h, followed by 0.25% avidin–biotin reagent for 1 h. Thereafter, the sections were incubated with 0.01% biotinylated tyramide ŽDuPont NEN, Boston, USA. and 0.005% H 2 O 2 in TPBS–T for 1 h followed by 0.125% avidin–biotin reagent in TPBS–T for 1 h again. Each incubation step was preceded by a thorough wash with TPBS–T. The sections were visualized with 0.05% 3,3X-diaminobenzidine ŽDAB, Sigma Deishagen, Germany. and 0.02% H 2 O 2 in TPBS under microscopical control, washed in TPBS, counterstained with Mayer’s hematoxylin and then mounted with DePeX-mountant. For control experiments, the primary antisera were omitted or substituted with nonimmune serum.
Fig. 3. Dorsal rhombencephalon at ED 12. Stem vessels invading the brain parenchyma from the leptomeningeal plexus are immunopositive. ŽA. RECA-1 immunohistochemistry. ŽB. ecNOS immunohistochemistry. Arrows: leptomeningeal plexus; curved arrows: stem vessels. Bars s 45 m m in ŽA. and ŽB..
46
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
For the pan-endothelial staining, the sections were fixed as mentioned above. After three washes with TPBS–T, the slices were pre-incubated with 3% NHS in TPBS–T for 1 h followed by incubation with the monoclonal mouse RECA-1 antibody ŽCamon, Wiesbaden, Germany. at a dilution of 1:10,000 in TPBS–T with 3% NHS at room temperature overnight. The following steps were carried out using the Vectastain ABC-Elite Kit according to the manufacturer. Control experiments and the visualization were carried out in the same way as mentioned for ecNOS.
3. Results Adult brains examined by RECA-1 immunohistochemistry showed, throughout the brain, a distinct labeling of endothelial lining of the vasculature ranging from large vessels to capillaries ŽFig. 1., including those of choroid plexus. No other cells were stained, indicating that the RECA-1 antibody reveals selectively the total of endothelial cells. Control experiments where the primary antibody was omitted or substituted with nonimmune serum failed to show any labeling. Beyond that we could show that the
RECA-1 antigen is expressed by brain endothelial cells from the beginning of brain vasculogenesis at ED 11 until adulthood Ž75 weeks of age.. In adult rat brains ecNOS immunoreactivity was localized to endothelial cells of all of the blood vessels and matched strictly the distribution pattern of RECA-1 staining. Immunostaining of ecNOS was most prominent in endothelial cells of arteries and arterioles, but it was also seen in veins, venules and capillaries ŽFig. 2A.. At higher magnification, the staining appeared to be particularly intense in the perinuclear region ŽFig. 2B.. Neither neurons nor glial cells showed any detectable staining. Again, control experiments where the ecNOS antibody was omitted or substituted with nonimmune serum failed to show any labeling. In developing rat brains, the endothelium of all blood vessels detectable with the RECA-1 antibody also expressed ecNOS immunopositivity. The developing vasculature was termed according to the nomenclature established by Baer w2x. At ED 10, no immunolabeled structures could be observed after immunostaining with the RECA-1 or the ecNOS antibody, apparently because neither blood vessels nor any endothelial cells developed in the prosencephal vesicle at this time. The onset of ecNOS immunolabeling in the brain was at ED 11, when the endothelium of the leptomeningeal plexus Žundifferentiated capillary plexus. was found to be selectively stained with both of the antibodies. However, within the brain parenchyma no labeled structures were detectable at this stage of brain development. Labeled stem vessels radially invading the brain parenchyma from the leptomeningeal plexus were first seen at ED 12 ŽFig. 3A and B.. Immunostained capillaries of the choroid plexus and the tangential sprouts of the radially penetrating stem vessels appeared at ED 13 ŽFig. 4.. The highest density of capillary branches in brain tissue was observed at P 20. There were no evident differences in distribution patterns of ecNOS containing structures between animals aging 16 and 75 weeks.
4. Discussion
Fig. 4. Dorsal rhombencephalon at ED 13, ecNOS immunohistochemistry. Note the tangential sprouting Žarrows. of the labeled stem vessels. Bar s90 m m.
The results of ecNOS immunohistochemistry in adult rat brain are generally congruent with those of other authors w30x. Our results, obtained with optimized fixation and preparation procedures w28x, were confirmed by in situ hybridization with an ecNOS specific probe w26x. Our results corroborate also those of Duijvestijn et al. w8x showing that the RECA-1 antigen, derived from lymph node homogenates, may serve as a pan-endothelial marker in adult rat brain and, as presented in this study for the first time, in the developing brain as well. In no case we have seen any distinct ecNOS immunoreactivity in neurons, as reported by others using the same monoclonal ecNOS antibody w6,7,21x. This discrepancy
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
might be explained by the fact that the outcome of ecNOS immunoreactivity depends largely on tissue fixation and staining protocols w11,33,36x, as pointed out in detail in our parallel study based on the aortic tissue for respective control experiments w28x. In the developing rat brain, we detected ecNOS protein in the endothelium of every blood vessel arising. These findings are supported by Western and Northern blot analysis by Keilhoff et al. w14x. Blood vessels negative for ecNOS could not be found. On the other hand, no other structures were found to be labeled in brain parenchyma. Northington et al. w20x obtained similar results in developing sheep brains. But we could not confirm observations whereupon at early stages of brain development only a few of the vessels are ecNOS immunopositive w18x. Mito et al. w17x have described immunopositive spots on spouting blood vessels in embryonic human brain tissue after application of antisera to laminin, fibronectine and type IV collagen, suggesting that these structures represent very early stages of angiogenesis. Using the ecNOS and RECA-1 antibodies in embryonic rat tissue, these spots could not be observed, possibly because both of the antigens are expressed not before the precursors of the endothelial cells are sufficiently differentiated. NO has been shown to contribute to the basal cerebral tone and the maintenance of the cerebral blood flow in adult and newborn subjects w9,25x. The same seems to be true for the premature brain w12x. NO leads to a dilation of large blood vessels by relaxation of the smooth muscle cells in the vessel wall w19x, and several studies showed that NO released from the microvascular endothelium serves as an important regulator of small artery and arteriolar tone in cerebral circulation w31,34x. Since capillaries lack smooth muscle cells other functions have to be assumed. So it is known that NO attenuates the expression of intercellular adhesion molecule-1 ŽICAM-1. and vascular cell adhesion molecule-1 ŽVCAM-1. in vitro w4,29x, which modulate the endothelial surface for rolling and adhesion of circulating blood cells. Mitchell and Tyml w16x have hypothesized that low levels of NO modify capillary blood flow by modulating local hemoconcentration and leukocyte adhesion, and that higher levels may cause, therefore, an increase in microvascular blood flow. These considerations have been supported by intravital microscopy w13x. Besides that, NO was shown to alter the microvascular permeability in the intact and inflamed vasculature w15x and, thus, to affect the blood brain barrier w27x. Moreover, NO produced by endothelial cells is involved in regulation of vascular sprouting and capillary organization. That was observed in tumor vasculogenesis w23x and in vitro capillarization w22x. Our results support the assumption that NO released from endothelium promotes, in combination with growth factors, the angiogenesis w38x rather than it has an inhibitory effect on neo-vascularization w24,37x. Considered together, our results indicate that ecNOS is within the rat brain exclusively expressed in the endothe-
47
lial cells of the entire vasculature including capillaries. NO released by ecNOS is likely to play a significant role in the regulation of brain blood flow and, possibly for the vasculogenesis in early brain development.
Acknowledgements This work was supported by grants of Ministerium fur ¨ Bildung, Wissenschaft, Forschung und Technologie Ž01 ZZ 9505,B6. and Deutsche Forschungsgemeinschaft ŽWo 474r10-1.. We wish to thank Ms. M. Mockel for skillful ¨ technical assistance and Mrs. M. Stein for photographic work.
References w1x J.C. Adams, Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains, J. Histochem. Cytochem. 40 Ž1992. 1457–1463. w2x T. Baer, Morphometric evaluation of capillaries in different laminae of rat cerebral cortex by automatic image analysis: changes during development and aging, Adv. Neurol. 20 Ž1978. 1–9. w3x H.J. Baker, L. Lindsey, S. Weisbroth, The Laboratory Rat, Vol. II, Research Applications, Academic Press, San Diego, CA, 1980. w4x E. Biffl, E. Moore, F.A. Moore, C.C. Barnett Jr., Nitric oxide reduces endothelial expression of intercellular adhesion molecule ŽICAM.-1, J. Surg. Res. 63 Ž1996. 328–332. w5x M.N. Bobrow, T.D. Harris, K.J. Shaughnessy, G.J. Litt, Catalyzed reporter deposition, a novel method of signal amplification, J. Immunol. Meth. 125 Ž1989. 279–285. w6x J.L. Dinerman, T.M. Dawson, M.J. Schell, A. Snowman, S.H. Snyder, Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: Implications for synaptic plasticity, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 4214–4218. w7x C.A. Doyle, P. Slater, Localization of neuronal and endothelial nitric oxide synthase isoforms in human hippocampus, Neuroscience 76 Ž1997. 387–395. w8x A.M. Duijvestijn, H. van Goor, F. Klatter, G.D. Majoor, E. van Bussel, J.C. van Breda Vriesman, Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody, Lab. Invest. 66 Ž1992. 459–466. w9x F.M. Faraci, D.D. Heistad, Does basal production of nitric oxide contribute to regulation of brain-fluid balance?, Am. J. Physiol. 262 Ž1992. H340–H344. w10x U. Forstermann, H. Kleinert, Nitric oxide synthase: expression and ¨ expressional control of the three isoforms, Naunyn-Schmiedeberg’s Arch. Pharmacol. 352 Ž1995. 351–364. w11x T. Gonzalez-Hernandez, M.A. Perez de la Cruz, B. MantolanSarmiento, Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against non-neuronal NOS antisera, J. Histochem. Cytochem. 44 Ž1996. 1399–1413. w12x P. Hardy, D.R. Varma, S. Chemtob, Control of cerebral and ocular blood flow autoregulation in neonates, Pediat. Clin. N. Am. 44 Ž1997. 137–152. w13x S. Kanwar, P. Kubes, Nitric oxide is an anti-adhesive molecule for leukocytes, New Horizons 3 Ž1995. 93–104. w14x G. Keilhoff, B. Seidel, H. Noack, W. Tischmeyer, D. Stanek, G. Wolf, Patterns of nitric oxide synthase at the messenger RNA and protein levels during early rat brain development, Neuroscience 75 Ž1996. 1193–1201.
48
I. Topel ¨ et al.r Brain Research 788 (1998) 43–48
w15x P. Kubes, Nitric oxide affects microvascular permeability in the intact and inflamed vasculature, Microcirculation 2 Ž1995. 235–244. w16x D. Mitchell, K. Tyml, Nitric oxide release in rat skeletal muscle capillary, Am. J. Physiol. 270 Ž1996. H1693–H1703. w17x T. Mito, H. Konomi, S. Houdou, S. Takashima, Immunohistochemical study of the vasculature in the developing brain, Pediat. Neurol. 7 Ž1991. 18–22. w18x T. Miyawaki, O. Sohma, M. Mizuguchi, S. Takashima, Development of nitric oxide synthase in endothelial cells in the human cerebrum, Dev. Brain Res. 89 Ž1995. 161–166. w19x C. Nathan, Q.-W. Xie, Nitric oxide synthases: roles, tolls, and controls, Cell 78 Ž1994. 1360–1362. w20x F.J. Northington, R.C. Koehler, R.J. Traystman, L.J. Martin, Nitric oxide synthase 1 and nitric oxide synthase 3 protein expression is regionally and temporally regulated in fetal brain, Dev. Brain. Res. 95 Ž1996. 1–14. w21x T.J. O’Dell, P.L. Huang, T.M. Dawson, J.L. Dinerman, S.H. Snyder, E.R. Kandel, M.C. Fishman, Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS, Science 265 Ž1994. 542–546. w22x A. Papapetropulus, K.M. Desai, R.D. Rudic, B. Mayer, R. Zhang, M.P. Ruiz-Torres, G. Garcia-Gardena, J.A. Madri, W.C. Sessa, Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro, Am. J. Pathol. 150 Ž1997. 1835–1844. w23x H.I. Peterson, Modification of tumour blood flow—a review, Int. J. Radiat. Biol. 60 Ž1991. 201–210. w24x E. Pilipi-Synetos, E. Sakkouia, G. Haralobopoulos, P. Andriopoulou, P. Peristeris, M.E. Maragoudakis, Evidence that nitric oxide is an endogenous anti-angiogenic mediator, Br. J. Pharmacol. 111 Ž1993. 894–904. w25x W.I. Rosenblum, H. Nishimura, G.H. Nelson, Endothelium-dependent L-Arg- and L-NMMA-sensitive mechanisms regulate tone of brain microvessels, Am. J. Physiol. 259 Ž1990. H1396–H1401. w26x B. Seidel, A. Stanarius, G. Wolf, Differential expression of neuronal and endothelial nitric oxide synthase in blood vessels, Neurosci. Lett. 239 Ž1997. 109–112. w27x A. Shukla, M. Dikshit, R.C. Srimal, Nitric oxide-dependent blood–
w28x
w29x
w30x
w31x w32x
w33x
w34x
w35x w36x
w37x
w38x
brain barrier permeability alteration in the rat brain, Experientia 52 Ž1996. 136–140. A. Stanarius, I. Topel, S. Schulz, H. Noack, G. Wolf, Immunocyto¨ chemistry of endothelial nitric oxide synthase in the rat brain: a light and electron microscopical study using the tyramide signal amplification technique, Acta Histochem. 99 Ž1997. 411–419. M. Takahashi, U. Ikeda, J.-I. Masuyama, H. Funayama, S. Kano, K. Shimada, Nitric oxide attenuates adhesion molecule expression in human endothelial cells, Cytokine 8 Ž1996. 817–821. H. Tomimoto, I. Akiguchi, H. Wakita, S. Nakamura, J. Kimura, Histochemical demonstration of membranous localization of endothelial cells of the rat brain, Brain Res. 667 Ž1994. 107–110. J.G. Umans, R. Levi, Nitric oxide in the regulation of blood flow and arterial pressure, Ann. Rev. Physiol. 57 Ž1995. 771–790. P.C. Ursell, M. Mayes, Endothelial isoform of nitric oxide synthase in rat heart increases during development, Anat. Rec. 246 Ž1996. 465–465. R.R. Vaid, B.K. Yee, J.N.P. Rawlins, S. Totterdell, NADPH-diaphorase reactive pyramidal neurons in Ammon’s horn and the subiculum of the rat hippocampal formation, Brain Res. 733 Ž1996. 31–40. E.P. Wei, R. Kukreja, H.A. Kontos, Effects in cats of inhibition of nitric oxide synthesis on cerebral vasodilation and endothelium-derived relaxing factor from acetylcholine, Stroke 23 Ž1992. 1623– 1629. G. Wolf, Nitric oxide and nitric oxide synthase: biology, pathology, localization, Histol. Histopathol. 12 Ž1997. 251–261. J. Worl, W.L. Neuhuber, ¨ M. Wiesand, B. Mayer, K.R. Greskotter, ¨ Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation, Histochemistry 102 Ž1994. 353–364. W. Yang, J. Ando, R. Korenaga, T. Toyo-oka, A. Kamija, Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells, Biochem. Biophys. Res. Commun. 203 Ž1994. 1160–1167. M. Ziche, L. Morbidelli, E. Masini, S. Amerini, H.J. Granger, C.A. Maggi, T. Geppetti, F. Ledda, Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P, J. Clin. Invest. 94 Ž1994. 2036–2044.