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Distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase staining in intraparenchymal blood vessels of the rat brain
ELSEVIER
Neuroscience Letters 196 (1995) 113-115
NEUROSCIEHCE LETTERS
Distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase staining in intraparenchymal blood vessels of the rat brain T.A. Lovick a,*, B.J. K e y b aDepartment of Physiology, The Medical School, BirminghamBI5 277, UK bDepartment of Pharmacology, The Medical School, BirminghamB15 2T1], UK Received 14 June 1995; revised version received 7 July 1995; accepted 7 July 1995
Abstract
The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase (nitric oxide synthase, NOS) in the endothelial lining of intraparenchymal blood vessels was examined in sections of rat brain prepared from 500ktm thick slices of brain fixed by immersion or from blocks of tissue taken from whole brains fixed by vascular perfusion. In immersion-fixed tissue, a network of stained vessels, many as small as 3/~m in diameter was seen throughout the grey and white matter. In tissue fixed by perfusion small calibre vessels less than 5/tm were less prevalent. The results indicate that NOS is normally present in the endothelial lining throughout the cerebrovas~zulartree, including the capillaries. Endothelium-derived nitric oxide could have a more widespread role in the regulation of cerebral blood flow than considered previously.
Keywords: Nitric oxide synthase; Cerebral blood vessels; Cerebral capillaries; Vascular endothelium
Endothelium-derived nitric oxide (NO) or a related compound plays an important role in the regulation of cerebral blood flow. It is now well established that endothelium-derived NO produces relaxation of large cerebral arteries and parenchymal vessels (see Refs. [1,3] for reviews). The role of NO :in intraparenchymal vessels is less clear. Studies on isolaled or cultured intraparenchymal arterioles have demonstrated a vasorelaxant action of NO [4,5]. However, histochemical studies have indicated that nitric oxide synthase (NOS) is distributed unevenly throughout the cerebral vascular tree. NOS could be detected in only a proportion of the larger intraparenchymal arterioles. Moreover, the enzyme was absent in vessels supplying white matter and in all vessels less than 20/~m in diameter [2,7,8]. In these studies, the brains were fixed by vascular perfusion. The vascular endothelium is highly sensitive to shear stress. Thus it is possible that fixation by perfusion could damage or even displace some of the endothelial lining, particularly in smaller vessels. We have therefore re-examined the distribution of NADPHdependent diaphorase staining in tissue in which the in* Corresponding author, Faz[: +44 121 4146924.
tegrity of the vascular endothelium has not been compromised by perfusion of fixative. The results suggest that the distribution of NADPH-dependent diaphorase within the intraparenchymal vasculature is more extensive than reported previously. Experiments were carried out on 6 adults rats 300350 g body weight. After induction of anaesthesia with urethane (1.5 g/kg i.p.), two rats were decapitated and the brain rapidly removed into chilled artificial cerebrospinal fluid (CSF). The brain was then transected in the coronal plane at the level of the rostral forebrain and glued to the stage of a Vibroslice (Oxford Instruments). Slices of fresh tissue, 500/tm thick were cut through the midbrain and forebrain and placed flat on pieces of filter paper moistened with CSE The filter paper was then gently submerged in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 3 h. The slices were then transferred to 0.1 M PB containing 15% sucrose and stored overnight at 4°C. In the remaining four rats a cannula was inserted retrogradely into the descending aorta distal to the level of the renal arteries. The jugular veins were transected bilaterally and 100 ml of heparinised (100 units/ml) 165 mM NaC1 at
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 8 2 6 - F
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T.A. Lovick, B.J. Key / Neuroscience Letters 196 (1995) 113-115
37°C followed by 200 ml of 4% paraformaldehyde in 0.1 M PB at room temperature, was infused over a period of 10-15 min. The brain was then removed and post-fixed for 1 h at 4°C before being transferred to 15% sucrose in 0.1 M PB at 4°C overnight. Sections 40-gm thick, from the fixed slices or from trimmed blocks of perfusion-fixed brain, were cut on a freezing microtome and collected in 0.1 M PB. The sections were transferred to a freshly prepared solution containing 1 mg/ml NADPH and 0.1 mg/ml nitroblue tetrazolium in 0.1 M PB containing 0.3% Triton-X I00. They were incubated in the dark at 37°C for 60-75 min, rinsed several times in PB and then mounted onto gelatinised slides. Some sections were counterstained with Neutral Red. After coverslipping with Hystomount (Hughes and Hughes) the sections were examined in an Olympus BH-2 microscope and selected material photographed using Kodak TMAX 400 film. NADPH-dependent diaphorase-positive blood vessels were present throughout the brain and formed a network throughout the neuropil in both perfusion- and immersion-fixed material. Stained neuronal perikarya and processes were also located throughout the neuropil and showed a heterogeneous distribution similar to that reported previously [9]. Four regions were selected for detailed study because they provided respectively, (a) examples of cortical tissue containing large numbers of NADPH-dependent diaphorase-positive neuronal processes but only a few labelled neuronal perikarya (parietal cortex), (b) a fibre tract with very few neuronal processes and no stained perikarya (internal capsule), (c) a region with a dense population of NADPH-dependent diaphorase-stained perikarya surrounded by a meshwork of stained fibres (periaqueductal grey matter) and (d) a region almost devoid of stained perikarya but containing many stained fibres (mesencephalic reticular formation) [9]. In the immersion-fixed material, staining was seen in all regions in intraparenchymal vessels ranging from large penetrating arteries 40/tm in diameter, to vessels as small as 3/tm in diameter (Fig. 1). Of the four regions studied, the highest density of micro-vessels, i.e. those less than 10/~m in diameter, was present in the parietal cortex where they formed a dense network throughout the neuropil (Fig. 1A). The lowest density of vessels was found in the internal capsule (Fig. 1B). In some sections several orders of branching of a single vessel could be followed. In every case the staining appeared to be continuous as far as the smallest vessels (Fig. 1). Densely stained neuronal processes were frequently seen in close proximity to vessels greater than 20/tm in diameter and in many cases fine fibres and discrete regions of punctate staining were associated with vessels as small as 10/tm in diameter (Fig. 1A,C). In sections taken from perfusion-fixed brains diaphorase-stained blood vessels and neuronal elements were present in all the regions examined. However, whilst the
Fig. 1. (A,B) NADPH-dependentdiaphorase staining in intraparenchymal blood vessels in sections prepared from immersion-fixed material. (A) Extensive network of stained vessels within lamina III of the parietal cortex. Fine beaded neural processeswere also present (arrows). (B) Stained blood vessels within the internal capsule. Note paucity of vessels and lack of stained neural processes. The fields shown in (A) and (B) were taken from the same histological section. (C) Small arteriole in the mesencephalic reticular formation. Arrows show areas of punctate neuronal staining in close association with the vessel. Scale bars: (A,B) 20/.tm; (C) 100/~m. neuronal staining appeared to be identical to that seen in the immersion-fixed sections, there appeared to be fewer small diameter stained blood vessels in perfusion-fixed sections. Comparisons of camera lucida drawings of the vascular architecture in immersion- and perfusion-fixed
T.A. Lovick, B.J. Key / Neuroscience Letters 196 (1995) 113-115
tissue revealed only a patchy occurrence o f stained vessels less than 5 / z m in diameter in the latter material. Thus perfusion o f the fixative may have resulted in a loss of endothelial lining from the smaller vessels. Alternatively, fixation by perfusion may have caused inactivation o f NOS in the smaller vessels which led to a loss o f N A D P H - d e p e n d e n t diaphorase staining. These factors may explain the paucity o f diaphorase-staining in intraparenchymal vessels which has been reported in previous studies using perfusion-fixed material [2,7,8]. The staining procedure used in the present study does not permit differentiation between different types of small calibre vessel. However, other authors have considered all intraparenchymal vesse]Is less than 7 / z m in diameter to be capillaries [6]. Based on this criterion, the extensive staining of small vessels seen in the present study indicates that N A D P H - d e p e n d e n t diaphorase is present within the endothelial lining throughout vascular tree, including the capillaries. This morphological finding has important functional implications since it suggests that nitric oxidemediated regulation of cerebral blood flow by the vascular endothelium is more widespread than previously thought. This work was supported by the Medical Research Council. We wish to thank Dr V.V. Stezhka for the preparation of brain slices and Mrs. S. Ethell for photographic services.
115
[1] Faraci, EM. and Brian, J.E., Nitric oxide and the cerebral circulation, Stroke, 25 (1994) 692-703. [2] Gabbot, P.L.A. and Bacon, S.J., Histochemical localization of NADPH-dependent diaphorase (nitric oxide synthase) activity in vascular endothelial cells in the rat brain, Neuroscienee, 37 (1993) 79-95. [3] Iadecola, C., Pellegrino, D.A., Moskowitz, M.A. and Lassen, N.A., Nitric oxide synthase inhibition and cerebrovascular regulation, J. Cerebral Blood Flow Metab., 14 (1994) 175-192. [4] Kim, P., Franco, L., Sasaki, T., Kirini, T., Takamura, K. and Nonamura, Y., Observation of myogenic rhythmic contractions of brain intraparenchymal arterioles and responsiveness to nitric oxide, J. Cerebral Blood Flow Metab., 13 (Suppl. 1) (1993) XVIII-3. [5] Kimura, M., Dietrich, H.H. and Dacey, R.G., Nitric oxide regulates cerebral arteriolar tone in rats, Stroke, 25 (1994) 22272233. [6] Rackl, A., Pawlik, G. and Bing, R.J,, Cerebral capillary topography and red cell flow in vivo. In J. Ceros-Navarro and E. Fritschka (Eds.) Cerebral Mierocirculation and Metabolism, Raven Press, New York, 1981, pp. 17-21. [7l Tomimoto, H., Akigudu, I., Wakita, H., Nakamura, S. and Kimura, J., Distribution of NADPH-diaphorase in the cerebral blood vessels of rats: a histochemical study, Neurosci. Lett., 156 (1993) 105-108. [8] Tomimoto, H., Akiguchi, I., Akita, H., Nakamura, S. and Kimura, J., Histochemical demonstration of membranous localisation of endothelial nitric oxide synthase in endothelial ceils of the rat brain, Brain Res., 667 (1994) 107-110. [9l Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755784.
Neuroscience Letters 196 (1995) 113-115
NEUROSCIEHCE LETTERS
Distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase staining in intraparenchymal blood vessels of the rat brain T.A. Lovick a,*, B.J. K e y b aDepartment of Physiology, The Medical School, BirminghamBI5 277, UK bDepartment of Pharmacology, The Medical School, BirminghamB15 2T1], UK Received 14 June 1995; revised version received 7 July 1995; accepted 7 July 1995
Abstract
The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase (nitric oxide synthase, NOS) in the endothelial lining of intraparenchymal blood vessels was examined in sections of rat brain prepared from 500ktm thick slices of brain fixed by immersion or from blocks of tissue taken from whole brains fixed by vascular perfusion. In immersion-fixed tissue, a network of stained vessels, many as small as 3/~m in diameter was seen throughout the grey and white matter. In tissue fixed by perfusion small calibre vessels less than 5/tm were less prevalent. The results indicate that NOS is normally present in the endothelial lining throughout the cerebrovas~zulartree, including the capillaries. Endothelium-derived nitric oxide could have a more widespread role in the regulation of cerebral blood flow than considered previously.
Keywords: Nitric oxide synthase; Cerebral blood vessels; Cerebral capillaries; Vascular endothelium
Endothelium-derived nitric oxide (NO) or a related compound plays an important role in the regulation of cerebral blood flow. It is now well established that endothelium-derived NO produces relaxation of large cerebral arteries and parenchymal vessels (see Refs. [1,3] for reviews). The role of NO :in intraparenchymal vessels is less clear. Studies on isolaled or cultured intraparenchymal arterioles have demonstrated a vasorelaxant action of NO [4,5]. However, histochemical studies have indicated that nitric oxide synthase (NOS) is distributed unevenly throughout the cerebral vascular tree. NOS could be detected in only a proportion of the larger intraparenchymal arterioles. Moreover, the enzyme was absent in vessels supplying white matter and in all vessels less than 20/~m in diameter [2,7,8]. In these studies, the brains were fixed by vascular perfusion. The vascular endothelium is highly sensitive to shear stress. Thus it is possible that fixation by perfusion could damage or even displace some of the endothelial lining, particularly in smaller vessels. We have therefore re-examined the distribution of NADPHdependent diaphorase staining in tissue in which the in* Corresponding author, Faz[: +44 121 4146924.
tegrity of the vascular endothelium has not been compromised by perfusion of fixative. The results suggest that the distribution of NADPH-dependent diaphorase within the intraparenchymal vasculature is more extensive than reported previously. Experiments were carried out on 6 adults rats 300350 g body weight. After induction of anaesthesia with urethane (1.5 g/kg i.p.), two rats were decapitated and the brain rapidly removed into chilled artificial cerebrospinal fluid (CSF). The brain was then transected in the coronal plane at the level of the rostral forebrain and glued to the stage of a Vibroslice (Oxford Instruments). Slices of fresh tissue, 500/tm thick were cut through the midbrain and forebrain and placed flat on pieces of filter paper moistened with CSE The filter paper was then gently submerged in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature for 3 h. The slices were then transferred to 0.1 M PB containing 15% sucrose and stored overnight at 4°C. In the remaining four rats a cannula was inserted retrogradely into the descending aorta distal to the level of the renal arteries. The jugular veins were transected bilaterally and 100 ml of heparinised (100 units/ml) 165 mM NaC1 at
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1 8 2 6 - F
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T.A. Lovick, B.J. Key / Neuroscience Letters 196 (1995) 113-115
37°C followed by 200 ml of 4% paraformaldehyde in 0.1 M PB at room temperature, was infused over a period of 10-15 min. The brain was then removed and post-fixed for 1 h at 4°C before being transferred to 15% sucrose in 0.1 M PB at 4°C overnight. Sections 40-gm thick, from the fixed slices or from trimmed blocks of perfusion-fixed brain, were cut on a freezing microtome and collected in 0.1 M PB. The sections were transferred to a freshly prepared solution containing 1 mg/ml NADPH and 0.1 mg/ml nitroblue tetrazolium in 0.1 M PB containing 0.3% Triton-X I00. They were incubated in the dark at 37°C for 60-75 min, rinsed several times in PB and then mounted onto gelatinised slides. Some sections were counterstained with Neutral Red. After coverslipping with Hystomount (Hughes and Hughes) the sections were examined in an Olympus BH-2 microscope and selected material photographed using Kodak TMAX 400 film. NADPH-dependent diaphorase-positive blood vessels were present throughout the brain and formed a network throughout the neuropil in both perfusion- and immersion-fixed material. Stained neuronal perikarya and processes were also located throughout the neuropil and showed a heterogeneous distribution similar to that reported previously [9]. Four regions were selected for detailed study because they provided respectively, (a) examples of cortical tissue containing large numbers of NADPH-dependent diaphorase-positive neuronal processes but only a few labelled neuronal perikarya (parietal cortex), (b) a fibre tract with very few neuronal processes and no stained perikarya (internal capsule), (c) a region with a dense population of NADPH-dependent diaphorase-stained perikarya surrounded by a meshwork of stained fibres (periaqueductal grey matter) and (d) a region almost devoid of stained perikarya but containing many stained fibres (mesencephalic reticular formation) [9]. In the immersion-fixed material, staining was seen in all regions in intraparenchymal vessels ranging from large penetrating arteries 40/tm in diameter, to vessels as small as 3/tm in diameter (Fig. 1). Of the four regions studied, the highest density of micro-vessels, i.e. those less than 10/~m in diameter, was present in the parietal cortex where they formed a dense network throughout the neuropil (Fig. 1A). The lowest density of vessels was found in the internal capsule (Fig. 1B). In some sections several orders of branching of a single vessel could be followed. In every case the staining appeared to be continuous as far as the smallest vessels (Fig. 1). Densely stained neuronal processes were frequently seen in close proximity to vessels greater than 20/tm in diameter and in many cases fine fibres and discrete regions of punctate staining were associated with vessels as small as 10/tm in diameter (Fig. 1A,C). In sections taken from perfusion-fixed brains diaphorase-stained blood vessels and neuronal elements were present in all the regions examined. However, whilst the
Fig. 1. (A,B) NADPH-dependentdiaphorase staining in intraparenchymal blood vessels in sections prepared from immersion-fixed material. (A) Extensive network of stained vessels within lamina III of the parietal cortex. Fine beaded neural processeswere also present (arrows). (B) Stained blood vessels within the internal capsule. Note paucity of vessels and lack of stained neural processes. The fields shown in (A) and (B) were taken from the same histological section. (C) Small arteriole in the mesencephalic reticular formation. Arrows show areas of punctate neuronal staining in close association with the vessel. Scale bars: (A,B) 20/.tm; (C) 100/~m. neuronal staining appeared to be identical to that seen in the immersion-fixed sections, there appeared to be fewer small diameter stained blood vessels in perfusion-fixed sections. Comparisons of camera lucida drawings of the vascular architecture in immersion- and perfusion-fixed
T.A. Lovick, B.J. Key / Neuroscience Letters 196 (1995) 113-115
tissue revealed only a patchy occurrence o f stained vessels less than 5 / z m in diameter in the latter material. Thus perfusion o f the fixative may have resulted in a loss of endothelial lining from the smaller vessels. Alternatively, fixation by perfusion may have caused inactivation o f NOS in the smaller vessels which led to a loss o f N A D P H - d e p e n d e n t diaphorase staining. These factors may explain the paucity o f diaphorase-staining in intraparenchymal vessels which has been reported in previous studies using perfusion-fixed material [2,7,8]. The staining procedure used in the present study does not permit differentiation between different types of small calibre vessel. However, other authors have considered all intraparenchymal vesse]Is less than 7 / z m in diameter to be capillaries [6]. Based on this criterion, the extensive staining of small vessels seen in the present study indicates that N A D P H - d e p e n d e n t diaphorase is present within the endothelial lining throughout vascular tree, including the capillaries. This morphological finding has important functional implications since it suggests that nitric oxidemediated regulation of cerebral blood flow by the vascular endothelium is more widespread than previously thought. This work was supported by the Medical Research Council. We wish to thank Dr V.V. Stezhka for the preparation of brain slices and Mrs. S. Ethell for photographic services.
115
[1] Faraci, EM. and Brian, J.E., Nitric oxide and the cerebral circulation, Stroke, 25 (1994) 692-703. [2] Gabbot, P.L.A. and Bacon, S.J., Histochemical localization of NADPH-dependent diaphorase (nitric oxide synthase) activity in vascular endothelial cells in the rat brain, Neuroscienee, 37 (1993) 79-95. [3] Iadecola, C., Pellegrino, D.A., Moskowitz, M.A. and Lassen, N.A., Nitric oxide synthase inhibition and cerebrovascular regulation, J. Cerebral Blood Flow Metab., 14 (1994) 175-192. [4] Kim, P., Franco, L., Sasaki, T., Kirini, T., Takamura, K. and Nonamura, Y., Observation of myogenic rhythmic contractions of brain intraparenchymal arterioles and responsiveness to nitric oxide, J. Cerebral Blood Flow Metab., 13 (Suppl. 1) (1993) XVIII-3. [5] Kimura, M., Dietrich, H.H. and Dacey, R.G., Nitric oxide regulates cerebral arteriolar tone in rats, Stroke, 25 (1994) 22272233. [6] Rackl, A., Pawlik, G. and Bing, R.J,, Cerebral capillary topography and red cell flow in vivo. In J. Ceros-Navarro and E. Fritschka (Eds.) Cerebral Mierocirculation and Metabolism, Raven Press, New York, 1981, pp. 17-21. [7l Tomimoto, H., Akigudu, I., Wakita, H., Nakamura, S. and Kimura, J., Distribution of NADPH-diaphorase in the cerebral blood vessels of rats: a histochemical study, Neurosci. Lett., 156 (1993) 105-108. [8] Tomimoto, H., Akiguchi, I., Akita, H., Nakamura, S. and Kimura, J., Histochemical demonstration of membranous localisation of endothelial nitric oxide synthase in endothelial ceils of the rat brain, Brain Res., 667 (1994) 107-110. [9l Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755784.