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Distribution of NADPH-diaphorase in the cerebral blood vessels of rats: a histochemical study
Neuroscience Letters, 156 (1993) 105 108 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
105
NSL 09605
Distribution of NADPH-diaphorase in the cerebral blood vessels of rats: a histochemical study Hidekazu Tomimoto, Ichiro Akiguchi, Hideaki Wakita, Shinichi N a k a m u r a and Jun Kimura Department of Neurology, Faculty of Medicine, Kyoto Universit); Kyoto (Japan) (Received 23 December 1992; Revised version received 23 March 1993; Accepted 26 March 1993)
Key words: NADPH-diaphorase; Cerebral blood vessel; Histochemistry; Nitric oxide NADPH-diaphorase (neuronal nitric oxide synthase) activity in the cerebral blood vessels was investigated by light and electron microscopic histochemistry to elucidate the sites of nitric oxide production. Networks of adventitial nerves containing NADPH-diaphorase were distributed in all parts of the circle of Willis. However, NADPH-diaphorase activity in adventitial nerves was much sparser in the region of the posterior cerebral artery, and absent in the pial arteries smaller than 100/lm in diameter. Endothelial cells were intensely stained in arteries and arterioles. These results support the hypothesis that vascular tone is regulated by nitric oxide, which is derived from endothelial ceils and adventitial nerves.
Nitric oxide (NO) is a free radical gas which is synthesized by nitric oxide synthase (NOS) from e-arginine in the presence of NADPH [8], and is involved in vasodilation by acting on sites of neurotransmission [3] and on endothelial cells [9]. Recent evidence indicates that NADPH-diaphorase is neuronal NOS, and a close similarity has been shown between neuronal and endothelial NOS [4]. Although neuronal/endothelial NOS in blood vessels has been demonstrated in endothelial cells and adventitia [2], the histochemical localization of NADPHdiaphorase activity in endothelial cells has not been described. Under deep anesthesia with sodium pentobarbital (100 mg/kg), 14 Wistar rats (250-350 g) were perfusion fixed with 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4), and postfixed in 4% PFA in 0.1 M PB (pH 7.4) for 8 to 12 h. The anterior, middle and posterior cerebral arteries (ACA, MCA, PCA) as well as the basilar artery (BA) attached to the pial membrane were removed in 10 rats, and incubated in 0.1 M PB (pH 7.4) containing 0.3% Triton X100, 0.5 mg/ml N A D P H and 0.1 mg/ml nitroblue tetrazolium at 37°C for 1 h [10]. Serial cryostat sections were cut in 4 rats and treated in the same way. For electron microscopy, blood vessels were flat-embedded in Spurr and cut in a ultramicrotome. Nerve fibers containing NADPH-diaphorase were Correspondence: H. Tomimoto, Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
found in the walls of all parts of the circle of Willis, including the branches of the ACA, MCA, PCA and BA; however, few were found in the PCA (Fig. 1). The fibers running along the axis of blood vessels tended to be thick periadventitial nerve bundles, while those encircling the vessels were fine and varicose, and located in the adventitia (Figs. 1 and 2g). They formed a plexus in the tunica adventitia of major pial arteries, became less dense distally, and were absent in smaller vessels less than 100 pm in diameter (Fig. 2a). Larger vessels in the neural parenchyma were occasionally surrounded by NADPH-diaphorase containing nerve fibers (Fig. 2b). Endothelial cells in the trunks of pial arteries were stained densely showing patches of dense reaction products (Fig. 2c,d). These patches were present in arteries and arterioles 20 pm in diameter or larger. Trace staining was shown in the small pial arteries and veins regardless of size (Fig. 2e,f). Larger vessels in the neural parenchyma were stained similarly, but the reaction products were not definitely obtained in the capillaries (Fig. 2h). Under electron microscopy, pale granular precipitates were observed in endothelial cells (Fig. 3d). Cell components in the tunica media such as fibroblasts and smooth muscle cells were negative for NADPH-diaphorase. Reaction products in the adventitial nerves could not be determined definitely because they were present in small amounts and had a low electron density. In the absence of NADPH, no staining was observed (Fig. 3a,c). These results provide for the first time a detailed distribution of NADPH-diaphorase in endothelial cells and
106
Fig. 1. Photomicrographs of whole mount vessels of the circle of Willis after NADPH-diaphorase histochemical staining, a: ACA, b: MCA, c: PCA, d: BA. Original photographs were taken at x66.
a d v e n t i t i a l nerves in cerebral arteries. The N A D P H - d i a p h o r a s e d i s t r i b u t i o n was p a t c h y in the e n d o t h e l i a l cell layer, dense in the arterial t r u n k s a n d trace in the capillaries and veins. T h e results c o r r e s p o n d to those o f previous reports [5] which showed activity o f this enzyme in p a r t i c u l a t e a n d p a r t l y cytosolic fractions o f e n d o t h e l i a l cells. A t present, a large b o d y o f evidence indicates that N O represents v a s c u l a r e n d o t h e l i u m - d e r i v e d relaxing factor in the b l o o d vessels [7]. Thus, the d i s t r i b u t i o n o f e n d o t h e l i a l N A D P H - d i a p h o r a s e observed here suggests that v a s c u l a r t o n e c o n t r o l by N O [6] is effective in cerebral arteries a n d arterioles. N e t w o r k s o f fibers c o n t a i n i n g N A D P H - d i a p h o r a s e d i s t r i b u t e d exclusively in the arteries which form the circle o f Willis and their b r a n c h e s larger t h a n 1 0 0 / l m in d i a m e t e r except in the P C A region. Since N A D P H - d i a p h o r a s e has been shown to be n e u r o n a l N O S , N A D P H d i a p h o r a s e positive fibers seem to be N O - c o n t a i n i n g nerves. Indeed, the c o l o c a l i z a t i o n o f N A D P H - d i a p h o rase a n d n e u r o n a l / e n d o t h e l i a l N O S has been extensively d e m o n s t r a t e d in n e u r o n a l structures including the myenteric plexus o f the gut [1] a n d the b r a i n [2]. L o c a l i z a t i o n o f N A D P H - d i a p h o r a s e positive nerves in the adventitia
argues against these a x o n s being c o n c o m i t a n t l y present, b u t r a t h e r suggests a role in n e u r o t r a n s m i s s i o n and consequent r e g u l a t i o n o f arteries especially in the the circle o f Willis except the P C A .
1 Belai, A., Schmidt, H.H.H.W., Hoyl, C.H.V.. Hasaall, C.J.S., Saffrey, M.J., Moss, J., F6rstermann, U., Murad, F. and Burnstock, G., Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut, Neurosci. Lett., 143 11992) 60 64. 2 Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3 11. 3 Bult. H., Boeckxstaens, G.E., Pelckmans, RA.~ Jordaens, F.H., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature. 345 (1990) 346 347. 4 Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H, Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797 7801. 5 F6rstermann, U., Pollack, J.S., Schmidt, H.H.H.W., Heller, M. and Murad. F., Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activily is present in the particulate and cytosolic fractions of bovine aortic endothelial cells, Proc. Natl. Acad. Sci. USA, 88 (19911 1788 1792.
107
Fig. 2. Photomicrographs of whole mount vessels in the M C A region (a,c,e,f), a semithin section (1/lm thick) of BA embedded in Spurr (d) and cryostat sections (b,g,h) after NADPH-diaphorase histochemical staining. Note the sparse distribution of NADPH-diaphorase positive fibers in the pial arteries with a diameter of 100/lm (*) (a). Arrowheads indicate patches of reaction products in endothelial cells (c,d). Veins were slightly stained (e,f; arrowheads). The ACA and a larger vessel in the corpus callosum are shown in (g) and (h), respectively. The arrow and arrowheads in (g) indicate a NADPH-diaphorase positive adventitial nerve and endothelial cells, respectively. The stars and arrows in (h) indicate NADPH-diaphorase positive neurons and capillaries which were not stained, respectively. Original photographs were taken at x50 in (a,f), xl00 in (b,c,g,h), ×500 in (d) and ×10 in (e).
108
Fig. 3. Photomicrographs of the BA (a,b) and the endothelial cells (c,d), which were stained for NADPH-diaphorase and embedded in Spurr. Note the reaction products in the endothelial cells in (b) and (d). As a control, specimens which underwent NADPH-diaphorase histochemistry without N A D P H are shown in (a) and (c). Original photographs were taken at ×50 in (a! and ibt, and x211,000 in (c,d).
6 Furchgott, R.F. and Zawadzki, J.VL The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373 376. 7 Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci., 14 (1991) 60 67. 8 Knowles, R.G., Palacios, M., Palmer, R.M. and Moncada, S., Kinetic characteristics of nitric oxide synthase from rat brain, Biochem. J., 269 (19901 207 210.
9 Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (19871 524 526. 10 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience, 46 (1991) 755 784.
105
NSL 09605
Distribution of NADPH-diaphorase in the cerebral blood vessels of rats: a histochemical study Hidekazu Tomimoto, Ichiro Akiguchi, Hideaki Wakita, Shinichi N a k a m u r a and Jun Kimura Department of Neurology, Faculty of Medicine, Kyoto Universit); Kyoto (Japan) (Received 23 December 1992; Revised version received 23 March 1993; Accepted 26 March 1993)
Key words: NADPH-diaphorase; Cerebral blood vessel; Histochemistry; Nitric oxide NADPH-diaphorase (neuronal nitric oxide synthase) activity in the cerebral blood vessels was investigated by light and electron microscopic histochemistry to elucidate the sites of nitric oxide production. Networks of adventitial nerves containing NADPH-diaphorase were distributed in all parts of the circle of Willis. However, NADPH-diaphorase activity in adventitial nerves was much sparser in the region of the posterior cerebral artery, and absent in the pial arteries smaller than 100/lm in diameter. Endothelial cells were intensely stained in arteries and arterioles. These results support the hypothesis that vascular tone is regulated by nitric oxide, which is derived from endothelial ceils and adventitial nerves.
Nitric oxide (NO) is a free radical gas which is synthesized by nitric oxide synthase (NOS) from e-arginine in the presence of NADPH [8], and is involved in vasodilation by acting on sites of neurotransmission [3] and on endothelial cells [9]. Recent evidence indicates that NADPH-diaphorase is neuronal NOS, and a close similarity has been shown between neuronal and endothelial NOS [4]. Although neuronal/endothelial NOS in blood vessels has been demonstrated in endothelial cells and adventitia [2], the histochemical localization of NADPHdiaphorase activity in endothelial cells has not been described. Under deep anesthesia with sodium pentobarbital (100 mg/kg), 14 Wistar rats (250-350 g) were perfusion fixed with 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4), and postfixed in 4% PFA in 0.1 M PB (pH 7.4) for 8 to 12 h. The anterior, middle and posterior cerebral arteries (ACA, MCA, PCA) as well as the basilar artery (BA) attached to the pial membrane were removed in 10 rats, and incubated in 0.1 M PB (pH 7.4) containing 0.3% Triton X100, 0.5 mg/ml N A D P H and 0.1 mg/ml nitroblue tetrazolium at 37°C for 1 h [10]. Serial cryostat sections were cut in 4 rats and treated in the same way. For electron microscopy, blood vessels were flat-embedded in Spurr and cut in a ultramicrotome. Nerve fibers containing NADPH-diaphorase were Correspondence: H. Tomimoto, Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
found in the walls of all parts of the circle of Willis, including the branches of the ACA, MCA, PCA and BA; however, few were found in the PCA (Fig. 1). The fibers running along the axis of blood vessels tended to be thick periadventitial nerve bundles, while those encircling the vessels were fine and varicose, and located in the adventitia (Figs. 1 and 2g). They formed a plexus in the tunica adventitia of major pial arteries, became less dense distally, and were absent in smaller vessels less than 100 pm in diameter (Fig. 2a). Larger vessels in the neural parenchyma were occasionally surrounded by NADPH-diaphorase containing nerve fibers (Fig. 2b). Endothelial cells in the trunks of pial arteries were stained densely showing patches of dense reaction products (Fig. 2c,d). These patches were present in arteries and arterioles 20 pm in diameter or larger. Trace staining was shown in the small pial arteries and veins regardless of size (Fig. 2e,f). Larger vessels in the neural parenchyma were stained similarly, but the reaction products were not definitely obtained in the capillaries (Fig. 2h). Under electron microscopy, pale granular precipitates were observed in endothelial cells (Fig. 3d). Cell components in the tunica media such as fibroblasts and smooth muscle cells were negative for NADPH-diaphorase. Reaction products in the adventitial nerves could not be determined definitely because they were present in small amounts and had a low electron density. In the absence of NADPH, no staining was observed (Fig. 3a,c). These results provide for the first time a detailed distribution of NADPH-diaphorase in endothelial cells and
106
Fig. 1. Photomicrographs of whole mount vessels of the circle of Willis after NADPH-diaphorase histochemical staining, a: ACA, b: MCA, c: PCA, d: BA. Original photographs were taken at x66.
a d v e n t i t i a l nerves in cerebral arteries. The N A D P H - d i a p h o r a s e d i s t r i b u t i o n was p a t c h y in the e n d o t h e l i a l cell layer, dense in the arterial t r u n k s a n d trace in the capillaries and veins. T h e results c o r r e s p o n d to those o f previous reports [5] which showed activity o f this enzyme in p a r t i c u l a t e a n d p a r t l y cytosolic fractions o f e n d o t h e l i a l cells. A t present, a large b o d y o f evidence indicates that N O represents v a s c u l a r e n d o t h e l i u m - d e r i v e d relaxing factor in the b l o o d vessels [7]. Thus, the d i s t r i b u t i o n o f e n d o t h e l i a l N A D P H - d i a p h o r a s e observed here suggests that v a s c u l a r t o n e c o n t r o l by N O [6] is effective in cerebral arteries a n d arterioles. N e t w o r k s o f fibers c o n t a i n i n g N A D P H - d i a p h o r a s e d i s t r i b u t e d exclusively in the arteries which form the circle o f Willis and their b r a n c h e s larger t h a n 1 0 0 / l m in d i a m e t e r except in the P C A region. Since N A D P H - d i a p h o r a s e has been shown to be n e u r o n a l N O S , N A D P H d i a p h o r a s e positive fibers seem to be N O - c o n t a i n i n g nerves. Indeed, the c o l o c a l i z a t i o n o f N A D P H - d i a p h o rase a n d n e u r o n a l / e n d o t h e l i a l N O S has been extensively d e m o n s t r a t e d in n e u r o n a l structures including the myenteric plexus o f the gut [1] a n d the b r a i n [2]. L o c a l i z a t i o n o f N A D P H - d i a p h o r a s e positive nerves in the adventitia
argues against these a x o n s being c o n c o m i t a n t l y present, b u t r a t h e r suggests a role in n e u r o t r a n s m i s s i o n and consequent r e g u l a t i o n o f arteries especially in the the circle o f Willis except the P C A .
1 Belai, A., Schmidt, H.H.H.W., Hoyl, C.H.V.. Hasaall, C.J.S., Saffrey, M.J., Moss, J., F6rstermann, U., Murad, F. and Burnstock, G., Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut, Neurosci. Lett., 143 11992) 60 64. 2 Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3 11. 3 Bult. H., Boeckxstaens, G.E., Pelckmans, RA.~ Jordaens, F.H., Van Maercke, Y.M. and Herman, A.G., Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter, Nature. 345 (1990) 346 347. 4 Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H, Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797 7801. 5 F6rstermann, U., Pollack, J.S., Schmidt, H.H.H.W., Heller, M. and Murad. F., Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activily is present in the particulate and cytosolic fractions of bovine aortic endothelial cells, Proc. Natl. Acad. Sci. USA, 88 (19911 1788 1792.
107
Fig. 2. Photomicrographs of whole mount vessels in the M C A region (a,c,e,f), a semithin section (1/lm thick) of BA embedded in Spurr (d) and cryostat sections (b,g,h) after NADPH-diaphorase histochemical staining. Note the sparse distribution of NADPH-diaphorase positive fibers in the pial arteries with a diameter of 100/lm (*) (a). Arrowheads indicate patches of reaction products in endothelial cells (c,d). Veins were slightly stained (e,f; arrowheads). The ACA and a larger vessel in the corpus callosum are shown in (g) and (h), respectively. The arrow and arrowheads in (g) indicate a NADPH-diaphorase positive adventitial nerve and endothelial cells, respectively. The stars and arrows in (h) indicate NADPH-diaphorase positive neurons and capillaries which were not stained, respectively. Original photographs were taken at x50 in (a,f), xl00 in (b,c,g,h), ×500 in (d) and ×10 in (e).
108
Fig. 3. Photomicrographs of the BA (a,b) and the endothelial cells (c,d), which were stained for NADPH-diaphorase and embedded in Spurr. Note the reaction products in the endothelial cells in (b) and (d). As a control, specimens which underwent NADPH-diaphorase histochemistry without N A D P H are shown in (a) and (c). Original photographs were taken at ×50 in (a! and ibt, and x211,000 in (c,d).
6 Furchgott, R.F. and Zawadzki, J.VL The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 288 (1980) 373 376. 7 Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci., 14 (1991) 60 67. 8 Knowles, R.G., Palacios, M., Palmer, R.M. and Moncada, S., Kinetic characteristics of nitric oxide synthase from rat brain, Biochem. J., 269 (19901 207 210.
9 Palmer, R.M.J., Ferrige, A.G. and Moncada, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature, 327 (19871 524 526. 10 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience, 46 (1991) 755 784.