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A Silver Impregnation Method for Study of Cerebral Microcirculation Using Confocal, Light, and Electron Microscopy
MICROVASCULAR RESEARCH
51, 116– 120 (1996)
Article No. 0012
TECHNICAL REPORT A Silver Impregnation Method for Study of Cerebral Microcirculation Using Confocal, Light, and Electron Microscopy KATSUKUNI FUJIMOTO,* MAKI MURAKAMI-HISAICHI,* CHIKAKO TOKUDA,† AND FUMIHIKO KAJIYA† Departments of *Anatomy and †Medical Electronics and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-01 Japan Received April 11, 1995
INTRODUCTION The presented method was designed to examine the cerebral microvasculature and cerebral tissue simultaneously with light, confocal laser scanning, and transmission electron microscopes. The morphology of the microvasculature in the brain is a reliable marker of local cerebral metabolism. The most common method of threedimensional observation of the microvascular bed is done by examining corrosion casts with a scanning electron microscope (Anderson and Anderson, 1978; Duvernoy et al., 1981). Corrosion casts provide a panoramic three-dimensional view of the microvessels but do not allow study of the relationship between the nervous tissue and the microcirculatory bed. Rats were perfused with solutions containing silver nitrate to visualize the contour of the vascular endothelial cells. We have modified the method described by Adamson (1993) in order to apply it to light, confocal laser scanning, and electron microscopic studies. The techniques described in this report provide a simultaneous three-dimensional visualization of the microcirculation and the surrounding nervous tissue. MATERIALS AND METHODS Rats (Jcl;ICR) were housed under a 12-hr light/dark cycle and were provided with food and water ad libitum. All animals used in this study were housed and handled according to the guidelines established by the National Institutes of Health. Five adult rats of both sexes (250– 350 g of body weight) were anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg, intraperitoneal injection). The heart was exposed and the ascending aorta was cannulated with a 16-G needle and connected to an infusion pump, with the flow rate being set at 60 ml/min. The 116 0026-2862/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Light micrograph of the cerebral cortex. A, arteriole; V, venule. Bar, 500 mm.
whole circulatory system was flushed with a 0.1 M cacodylate buffer (pH 7.4) containing 10 m M sodium nitroprusside and 5% glucose for 10 min. Immediately after perfusion was begun, the cardiac auricle was cut to provide an outflow from the circulation. A mixture of glutaraldehyde (2.5%) and paraformaldehyde (2.0%) in the same buffer was perfused for 5 min. Subsequent procedures followed Adamson’s method with a 0.5% silver nitrate in 5% aqueous glucose solution. Finally, the fixatives were perfused 20 min to complete the fixation. The brain and spinal cord were dissected out and immersed in a fresh fixative for an additional 60 min at 47. The tissues were cut with a Microslicer (Dosaka, Tokyo) at intervals of 100 to 200 m m. Sections were infiltrated with graded aqueous glycerol solutions and mounted in pure glycerol for light and confocal laser microscopy. Some representative sections were prepared for observation with a transmission electron microscope (Hitachi, H-7100). Confocal laser scanning microscopy. The specimens were imaged on a confocal laser scanning microscope (LSM-10, Zeiss, Germany), equipped with an argon/ helium laser and a camera. They were mainly illuminated with an argon laser (568nm wavelength), and separate filter blocks were used. Z-series images for threedimensional reconstruction of the cerebral microcirculation were acquired by laser imaging at 2- to 15- mm intervals. After computer-assisted digitizing, these images were stored on a hard disk. Three-dimensional figures of the microvascular network of a selected region can be viewed as colored overlay images on a monitor.
m4278$1928
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TECHNICAL REPORT
FIG. 2. Transmission electron micrograph of the cerebral cortex stained by intravascular perfusion of silver nitrate solution. L, capillary lumen; N, nucleus of nerve cell; P, nucleus of pericyte; EN, nucleus of endothelial cell. Arrowheads indicate silver deposits along the interendothelial cleft. Asterisk indicate perivascular edema of astroglial cells. Bar, 10 mm.
RESULTS AND DISCUSSION There are broad differences in local neural activity, cellular organization, blood flow, neurotransmitters, endocrine activity, glucose utilization, and other metabolic processes within the central nervous system. In the present study, the rat vasculature was perfused with solutions containing silver nitrate (Fig. 1) to visualize the endothelial cell clefts. The adluminal en surface contour of endothelial cells was identified by the dark lines produced by silver grains between adjacent endothelial cells. In the large vessels, the outlines of medial smooth muscle cells were identified with this concentration of silver nitrate. Thus, arteriole and venule could be easily differentiated according to the orientation of medial smooth muscle cells. The silver impregnation method has been applied for more than a century to reveal the presence of endothelial clefts (Arnold, 1875) and was revised by Romeis in 1948. Recently, the shape of the endothelium in vessels of various diameters (Adamson, 1993) and in tissue culture (Furie et al., 1984) has been observed by this method. It is usually assumed that silver impregnation will destroy the fine structure and thus hamper electron microscopic observations. The present method provides satisfactory results for fine structural studies with a transmission electron microscope,
m4278$1928
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FIG. 3. LSM picture from rat cerebral cortex stained by the silver impregnation method. The specimen is 100-mm thick. This colored overlay image is composed of 10 optical slices of 2-mm thickness each. The color wedge in the top left filed represents distance of the specimen from cover glass; for example, orangecolored zone was obtained from an optical slice which was close to the objective. Bar, 50 mm.
since all lumens from the arteries through the capillaries to the veins are kept patent by the flushing of sodium nitroprusside solutions. This is followed by perfusion of the appropriate fixative before staining by silver nitrate perfusion (Fig. 2). Satisfactory results for light microscopic observation were also achieved with sections stained by Klu¨ver-Barrera’s method. In the present study, 100- to 200-m m thick tissue sections from the brain were observed by confocal laser scanning electron microscope. Flairless three-dimensional color images were composed by overlaying of 10 to 20 optical slices obtained at 2- to 15- mm intervals (Fig. 3). With voxel processing, the relationship between microvessels and microvascular patterns can be studied concurrently in three dimensions. Although scanning electron microscopy of corrosion casts can easily generate three-dimensional views of the microvasculature, the procedure is timeconsuming and sacrifices surrounding tissue. The present method provides images comparable to scanning electron microscopy of corrosion-cast preparations while preserving surrounding tissue. Thus, the relationship between microvessels and surrounding histology can be examine in three dimensions. Laser scanning microscopy of silver-impregnated specimens is an alternative to corrosion-cast preparations for studying microvascular architecture.
m4278$1928
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TECHNICAL REPORT
ACKNOWLEDGMENTS Part of this study was supported by a project grant from Kawasaki Medical School (VII-5-709). The LSM used in this study was purchased with funds provided by the Ministry of Education, Science and Culture of Japan through a grant-in-aid (1991) to Kawasaki Medical School.
REFERENCES Adamson, R. H. (1993). Microvascular endothelial cell shape and size in situ. Microvasc. Res. 46, 77– 88. Anderson, B. G., and Anderson, W. D. (1978). Scanning electron microscopy of corrosion casts: Intracranial and abdominal microvasculature in domestic animals. Am. J. Anat. 153, 523 – 536. ¨ ber das halten der Wandungen der Blutgefa¨sse bei der Emigration weisser Blutko¨rper. Arnold, J. (1875). U Arch. Pathol. Anat. 62, 87– 103. Duvernoy, H. M., Delon, S., and Vannson, J. L. (1981). Cortical blood vessels of the human brain. Brain Res. Bull. 7, 519 – 579. Furie, M. B., Cramer, E. B., Naprstek, B. L., and Meek, G. A. (1984). Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current. J. Cell Biol. 98, 1033– 1041. Romeis, B. (1948). ‘‘Mikroskopische Technik,’’ S. 469. Leibniz, Verlag, Munich.
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR
51, 116– 120 (1996)
Article No. 0012
TECHNICAL REPORT A Silver Impregnation Method for Study of Cerebral Microcirculation Using Confocal, Light, and Electron Microscopy KATSUKUNI FUJIMOTO,* MAKI MURAKAMI-HISAICHI,* CHIKAKO TOKUDA,† AND FUMIHIKO KAJIYA† Departments of *Anatomy and †Medical Electronics and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-01 Japan Received April 11, 1995
INTRODUCTION The presented method was designed to examine the cerebral microvasculature and cerebral tissue simultaneously with light, confocal laser scanning, and transmission electron microscopes. The morphology of the microvasculature in the brain is a reliable marker of local cerebral metabolism. The most common method of threedimensional observation of the microvascular bed is done by examining corrosion casts with a scanning electron microscope (Anderson and Anderson, 1978; Duvernoy et al., 1981). Corrosion casts provide a panoramic three-dimensional view of the microvessels but do not allow study of the relationship between the nervous tissue and the microcirculatory bed. Rats were perfused with solutions containing silver nitrate to visualize the contour of the vascular endothelial cells. We have modified the method described by Adamson (1993) in order to apply it to light, confocal laser scanning, and electron microscopic studies. The techniques described in this report provide a simultaneous three-dimensional visualization of the microcirculation and the surrounding nervous tissue. MATERIALS AND METHODS Rats (Jcl;ICR) were housed under a 12-hr light/dark cycle and were provided with food and water ad libitum. All animals used in this study were housed and handled according to the guidelines established by the National Institutes of Health. Five adult rats of both sexes (250– 350 g of body weight) were anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg, intraperitoneal injection). The heart was exposed and the ascending aorta was cannulated with a 16-G needle and connected to an infusion pump, with the flow rate being set at 60 ml/min. The 116 0026-2862/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR
TECHNICAL REPORT
117
FIG. 1. Light micrograph of the cerebral cortex. A, arteriole; V, venule. Bar, 500 mm.
whole circulatory system was flushed with a 0.1 M cacodylate buffer (pH 7.4) containing 10 m M sodium nitroprusside and 5% glucose for 10 min. Immediately after perfusion was begun, the cardiac auricle was cut to provide an outflow from the circulation. A mixture of glutaraldehyde (2.5%) and paraformaldehyde (2.0%) in the same buffer was perfused for 5 min. Subsequent procedures followed Adamson’s method with a 0.5% silver nitrate in 5% aqueous glucose solution. Finally, the fixatives were perfused 20 min to complete the fixation. The brain and spinal cord were dissected out and immersed in a fresh fixative for an additional 60 min at 47. The tissues were cut with a Microslicer (Dosaka, Tokyo) at intervals of 100 to 200 m m. Sections were infiltrated with graded aqueous glycerol solutions and mounted in pure glycerol for light and confocal laser microscopy. Some representative sections were prepared for observation with a transmission electron microscope (Hitachi, H-7100). Confocal laser scanning microscopy. The specimens were imaged on a confocal laser scanning microscope (LSM-10, Zeiss, Germany), equipped with an argon/ helium laser and a camera. They were mainly illuminated with an argon laser (568nm wavelength), and separate filter blocks were used. Z-series images for threedimensional reconstruction of the cerebral microcirculation were acquired by laser imaging at 2- to 15- mm intervals. After computer-assisted digitizing, these images were stored on a hard disk. Three-dimensional figures of the microvascular network of a selected region can be viewed as colored overlay images on a monitor.
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR
118
TECHNICAL REPORT
FIG. 2. Transmission electron micrograph of the cerebral cortex stained by intravascular perfusion of silver nitrate solution. L, capillary lumen; N, nucleus of nerve cell; P, nucleus of pericyte; EN, nucleus of endothelial cell. Arrowheads indicate silver deposits along the interendothelial cleft. Asterisk indicate perivascular edema of astroglial cells. Bar, 10 mm.
RESULTS AND DISCUSSION There are broad differences in local neural activity, cellular organization, blood flow, neurotransmitters, endocrine activity, glucose utilization, and other metabolic processes within the central nervous system. In the present study, the rat vasculature was perfused with solutions containing silver nitrate (Fig. 1) to visualize the endothelial cell clefts. The adluminal en surface contour of endothelial cells was identified by the dark lines produced by silver grains between adjacent endothelial cells. In the large vessels, the outlines of medial smooth muscle cells were identified with this concentration of silver nitrate. Thus, arteriole and venule could be easily differentiated according to the orientation of medial smooth muscle cells. The silver impregnation method has been applied for more than a century to reveal the presence of endothelial clefts (Arnold, 1875) and was revised by Romeis in 1948. Recently, the shape of the endothelium in vessels of various diameters (Adamson, 1993) and in tissue culture (Furie et al., 1984) has been observed by this method. It is usually assumed that silver impregnation will destroy the fine structure and thus hamper electron microscopic observations. The present method provides satisfactory results for fine structural studies with a transmission electron microscope,
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR
TECHNICAL REPORT
119
FIG. 3. LSM picture from rat cerebral cortex stained by the silver impregnation method. The specimen is 100-mm thick. This colored overlay image is composed of 10 optical slices of 2-mm thickness each. The color wedge in the top left filed represents distance of the specimen from cover glass; for example, orangecolored zone was obtained from an optical slice which was close to the objective. Bar, 50 mm.
since all lumens from the arteries through the capillaries to the veins are kept patent by the flushing of sodium nitroprusside solutions. This is followed by perfusion of the appropriate fixative before staining by silver nitrate perfusion (Fig. 2). Satisfactory results for light microscopic observation were also achieved with sections stained by Klu¨ver-Barrera’s method. In the present study, 100- to 200-m m thick tissue sections from the brain were observed by confocal laser scanning electron microscope. Flairless three-dimensional color images were composed by overlaying of 10 to 20 optical slices obtained at 2- to 15- mm intervals (Fig. 3). With voxel processing, the relationship between microvessels and microvascular patterns can be studied concurrently in three dimensions. Although scanning electron microscopy of corrosion casts can easily generate three-dimensional views of the microvasculature, the procedure is timeconsuming and sacrifices surrounding tissue. The present method provides images comparable to scanning electron microscopy of corrosion-cast preparations while preserving surrounding tissue. Thus, the relationship between microvessels and surrounding histology can be examine in three dimensions. Laser scanning microscopy of silver-impregnated specimens is an alternative to corrosion-cast preparations for studying microvascular architecture.
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR
120
TECHNICAL REPORT
ACKNOWLEDGMENTS Part of this study was supported by a project grant from Kawasaki Medical School (VII-5-709). The LSM used in this study was purchased with funds provided by the Ministry of Education, Science and Culture of Japan through a grant-in-aid (1991) to Kawasaki Medical School.
REFERENCES Adamson, R. H. (1993). Microvascular endothelial cell shape and size in situ. Microvasc. Res. 46, 77– 88. Anderson, B. G., and Anderson, W. D. (1978). Scanning electron microscopy of corrosion casts: Intracranial and abdominal microvasculature in domestic animals. Am. J. Anat. 153, 523 – 536. ¨ ber das halten der Wandungen der Blutgefa¨sse bei der Emigration weisser Blutko¨rper. Arnold, J. (1875). U Arch. Pathol. Anat. 62, 87– 103. Duvernoy, H. M., Delon, S., and Vannson, J. L. (1981). Cortical blood vessels of the human brain. Brain Res. Bull. 7, 519 – 579. Furie, M. B., Cramer, E. B., Naprstek, B. L., and Meek, G. A. (1984). Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current. J. Cell Biol. 98, 1033– 1041. Romeis, B. (1948). ‘‘Mikroskopische Technik,’’ S. 469. Leibniz, Verlag, Munich.
m4278$1928
12-13-95 05:47:26
mvras
AP: MVR