=========ANNALS
or ANATOMY =========
Endothelial cell helix in small arterioles of human ureters. A study by scanning electron microscopy (SEM) K. Spanel-Borowski*, P. Kuhri and W. Kuhnel Institut ffir Anatomie der Medizinischen Universitat zu Lubeck, Ratzeburger Allee 160, D -W - 2400 Lubeck 1, Deutschland
Summary. Microvessel corrosion casts of human ureters were prepared. Portions of casts were longitudinally cut by use of a pulsed Eximer laser beam in order to get insight into microvessel branches studied in SEM. Larger arterioles (> 30 !lm in diameter) showed oval imprints of endothelial cell nuclei along the vessel axis. Imprints were found in furrows parallel to resin ridges. Smaller arterioles « !lm in diameter) displayed imprints of nuclei aligned in a helix around the vessel axis. With regard to capillaries, one nucleus imprint was spaced far apart from the other. Venules demonstrated a reticular network of thick, attenuated resin ridges and of small meshes which consisted of round or oval nucleus imprints. It is concluded that smaller arterioles form an endothelial cells helix. Key words: Arteriole - Microvessel endothelial cell culture - SEM
Introduction Peripheral resistance and hence blood flow are regulated by arterioles which change their diameters either by relaxation or contraction of wall components (Joyner and Davis 1987; Meininger 1987; Slaaf et al. 1987). Contraction of arterioles causes endothelial cells to change from a flat to a cylindrical shape (van Citters et al. 1962; Greensmith and Duling 1984; Walmsley et al. 1982). Such remodelled endothelial cells contribute to the occurrence of endothelial cell ridges buckling into the lumen in an orientation parallel to the vessel axis. These ridges are composed not only of endothelial cells, but in addition contain wavy elastic lamina and interpolated portions of juxtaposed vascular smooth muscle cells.
* Address to which proofs are mailed: Prof. Dr. K. SpanelBorowski, Anatomisches Institut, Universitiit Basel, PestalozzistraBe 20, CH-4056 Basel
)~I SEMP'Ett~
Ann. Anat. (1992) 174: 213-216 Gustav Fischer Verlag Jena
While endothelial cell ridges represent the passive inner annulus, the adjacent portion of the arteriolar wall is considered to be the active outer annulus due to force-generating capacity of circurnferentially arranged myofilaments of smooth muscle cells (Sleek and Duling 1986). The vasoconstrictive force appears to be responsible for incomplete occlusion in large arterioles or complete occlusion in small arterioles, respectively (van Citters 1966). Yet complete occlusion cannot be sufficiently explained by way of muscular force in view of the concept that smooth muscle cells from vessels of different sizes are mechanically similar (Davis and Gore 1989). The final determinant of complete occlusion has been assumed to be related to the inner annulus (Carlson et al. 1982) and probably to the surface architecture of endothelial cells and their array. Since it is impossible to study the inner lining of a microvascular tree by use of SEM, differences of structure were examined in microvascular corrosion casts of human ureters. A pulsed Eximer-Iaser beam was successfully used to dissect the casts.
Materials and methods In the scope of a thesis carried out to study the blood supply of the lamina propria (Kuhri 1990), twelve ureters were obtained during autopsy. The urogenital tract of male or female human bodies was removed 12-36 h of death, and the ureter dissected with the supplying arteries, i.e. ureteric artery, testicular artery or ovaric artery, respectively. The arteries were cannulated and perfused manually with 20 ml 0.9% NaCI for approximately 5 minutes. When the colour of the vasculature had turned white, polymethylmethacrylate (Mercox®, Norwald KG, Hamburg, FRG) was injected manually. The resin was prepared at room temperature prior to use. 100 ml polymethylmethacrylate, the basic component, was mixed with 5 ml hardener for 30 seconds. 15-20 ml resin mixture was injected within 5 minutes. The whole ureters were removed and kept in distilled water for 24 h. For maceration, 1 M NaOH was applied for two weeks, upon which 1 M HCI and 1 M
NaOH, were applied for one week each . Two turns and multiple rinses with distilled water were carried out. The microcorrosion casts were kept in water and frozen at -20 °C. The frozen tissue was cut transversely by use of a razor blade drawn through a flame . The sliced specimens were freeze-dried at -40°C. They were mounted parallel to the ureter axis on SEM specimen holders, sputtered with gold and viewed in a Philips SEM 505 at 15 kY. In order to evaluate how blood vessels branched from the adventital coat of the ureter to its lamina propria the sputtered specimens were processed further. Longitudinal cuts were obtained by use of an Eximer-laser beam (Questek Inc., Billerica, MA, USA). The procedure was controlled under a stereornicroscope equipped with an ultraviolet filter. 351 nm wave length was applied and density of energy kept stable at 1.5 J/cm2 • The laser beam,
focused to a diameter of 70 /lm and its pulse frequency adjusted to 20 Hz, was operated by a hand-driven micromanipulator (Brinkmann, Mannheim, FRG). 150-200 /lm thick sections were cut, and each exposed surface section was sputter coated and examined at different stages of processing. By an anterograde and retrograde evaluation, the architecture of individual rnicrovessels became evident.
Results Each studied segment of the microvessel corrosion cast had characteristic features (Figs. 1-5). They were considered to
Fig. 1. The arteriole of second order has a diameter of 40- 50 /lm (not completely shown). Oval imprints of endothelial cell nuclei (large arrows) are found within furrows (small arrows) along the axis of the rnicrocorrsosion cast. SEM, X2,100. Bar: 10 /lm. Fig. 2. The ateriole of third order measures 20 /lID in diameter. Furrows (arrows) form a helix around the axis of the rnicrocorrosion cast. They contain imprints of endothelial cell nuclei hardly to be discerned at this magnification. Broad ridges occur parallel to furrows. SEM. X2,300. Bar: 10 /lm. Fig. 3. The arteriole of fourth order displaya diameter of 12-14 /!ill. Oval imprints of endothelial cell nuclei (arrows) are oriented in a helix the pitch of which is larger than in figure 2. Furrows and ridges are less evident probably due to vasodilation. A cast of a capillary is apparent at the bottom of the figure. SEM. x2,100. Bar: 10 /lm.
214
Fig. 4. A capillary loop of a microcorrsosion cast shows a diameter of 5-6 !lm. Imprints of endothelial cell nuclei are randomly located. SEM. X 2,300. Bar: 10 !lm. Fig. 5. The venule of a microcorrosion cast has a diameter of 110 !lm (not completely shown). Imprints of endothelial cell nuclei (arrows) are of round or oval shape. The imprints represent the small meshes of a reticular network composed of attenuated, thick resin ridges. SEM. X 1,300. Bar: 10 !lm.
represent the replica of in vivo occurrence. Nuclei of endothelial cells appeared as oval surface imprints found in arterioles and capillaries. Round imprints were also seen in venules. Imprints were apparent in furrows of arterioles and "meshes" of venules (see below). Furrows were preferentially oriented along the axis of larger arterioles (> 30 !lm in diameter). Yet the furrows aligned like a helix around the axis of smaller arterioles « 30!-lm in diameter). The pitch of the helix seemed to change with the state of contraction considering height of ridges or depth of furrows, respectively, as a parameter of contraction. Contraction was caused presumably by rigor mortis. In venules, small "meshes" were surrounded by a reticular network of attenuated, thick resin ridges.
inner diameters during induced contraction (Carlson et al. 1982). Endothelial cell ridges appear at one location, and, with further contraction, spread around the circumference of the lumen (Greensmith and Duling 1984). Vascular smooth muscle cells remained in our microcorrosion casts occasionally, and such the muscle cells were always arranged circumferentially to the axis of arterioles of all sizes (not shown). This is in agreement with the observations of Beacham et al. (1976), Walmsley et al. (1982) and Davis and Gore (1989) and in disagreement with those of Rhodin (1980). It is our conclusion that in smaller arterioles the endothelial cells are arranged in the form of a helix.
Acknowledgements. We thank Dr. K. Ley for reading and Mrs. R. Miinzinger for typing the manuscript. The study was supported by the Deutsche Forschungsgemeinschaft, grant. no. Sp 232/4-1.
Discussion Hodde and Nowell (1980) were the first to use a continuous Argon laser beam to cut microvessel corrosion casts. The problems they faced due to high or low energy release were overcome in the present by using a pulsed Eximer laser beam. Endothelial cell imprint patterns as reported by Hodde and Nowell (1980) are confrrmed by our findings, i.e. longitudinal array of imprints in arterioles, and the mesh-like meandering appearance of oval or round imprints in venules. In contrast to larger arterioles, smaller arterioles have a helical orientation of endothelial cells. In retrospect, this phenomenon is to be seen with figures of casted smaller arterioles published by Hodde and Nowell (1980) and Greensmith and Duling (1984), although the helical structure was not explicitly recognized. As noticed with cross sections, dilated arterioles from the mesentery show irregular
References Beacham WS, Konishi A, Hunt CC (1976) Observations on the microcirculatory bed in rat mesocecum using differential interference contrast microscopy in vivo and electron microscopy. Am J Anat 146: 385-426. Carlson EC, Burrows ME, Johnson PC (1982) Electron microscopic studies of cat mesenteric arterioles: A structure-function analysis. Microvasc Res 24: 123-141 Davis, MJ, Gore RW (1989) Length-tension relationship of vascular smooth muscle in single arterioles. Am J Physiol 256: H630-640 Greensmith, JE, Duling BR (1984) Morphology of the constricted arteriolar wall: physiological implications. Am J Physiol 247: H687-698
215
Hodde KC, Howell JA (1980) SEM of micro-corrosion casts. Scan Electron Microsc II: 88-106 Joyner WL, Davis MJ (1987) Pressure profile along the microvascular network and its control. Fed Proc 46: 266-269 Kuhri P (1990) Die GefaBversorgung der menschlichen Harnleiterwand. Bine korrosionsanatomische Untersuchung. Dissertation an der Vorklinisch-Naturwissenschaftlichen Universitat der Medizinischen Universitat zu Lubeck, Lubeck Meininger GA (1987) Responses of sequentially branching macro- and microvessels during reactive hyperemia in skeletal muscle. Microvasc Res 34: 29-45 Meininger, GA, Mack, CA, Fehr KL, Bohlen G (1987) Myogenic vasoregulation overrides metabolic control in resting rat skeletal muscle. Circ Res 60: 861-870 Rhodin JAG (1980) Architecture of the vessel wall. In: Bohr DF, Somlyo AP, Sparks Jr HU (eds) Handbook of Physiology. Sec 2. The Cardiovascular System. Vol 2.
Vascular Smooth Muscle. Am Physiol Soc, pp 16-17, Bethesda, Maryland Slaaf, DW, Reneman RS, Wiederhielm CA (1987) Pressure regulation in muscle of unanesthetized bats. Microvasc Res 33: 315-326 Sleek GE, Duling BR (1986) Coordination of mural elements and myofilaments during arteriolar constriction. Circ Res 59: 620-627 Van Citters RL (1966) Occlusion of lumina in small arterioles during vasoconstriction. Circ Res 18: 199-204 Van Citters RL, Wagner BM, Rushmer RF (1962) Architecture of small arteries during vasoconstriction. Circ Res 10: 668-675 Walmsley JG, Gore RW, Dacey Jr RG, Damon ON, Duling BR (1982) Quantitative morphology of arterioles from the hamster cheek pouch related to mechanical analysis. Microvasc Res 24: 249-271 Accepted October 25, 1991
216
or ANATOMY =========
Endothelial cell helix in small arterioles of human ureters. A study by scanning electron microscopy (SEM) K. Spanel-Borowski*, P. Kuhri and W. Kuhnel Institut ffir Anatomie der Medizinischen Universitat zu Lubeck, Ratzeburger Allee 160, D -W - 2400 Lubeck 1, Deutschland
Summary. Microvessel corrosion casts of human ureters were prepared. Portions of casts were longitudinally cut by use of a pulsed Eximer laser beam in order to get insight into microvessel branches studied in SEM. Larger arterioles (> 30 !lm in diameter) showed oval imprints of endothelial cell nuclei along the vessel axis. Imprints were found in furrows parallel to resin ridges. Smaller arterioles « !lm in diameter) displayed imprints of nuclei aligned in a helix around the vessel axis. With regard to capillaries, one nucleus imprint was spaced far apart from the other. Venules demonstrated a reticular network of thick, attenuated resin ridges and of small meshes which consisted of round or oval nucleus imprints. It is concluded that smaller arterioles form an endothelial cells helix. Key words: Arteriole - Microvessel endothelial cell culture - SEM
Introduction Peripheral resistance and hence blood flow are regulated by arterioles which change their diameters either by relaxation or contraction of wall components (Joyner and Davis 1987; Meininger 1987; Slaaf et al. 1987). Contraction of arterioles causes endothelial cells to change from a flat to a cylindrical shape (van Citters et al. 1962; Greensmith and Duling 1984; Walmsley et al. 1982). Such remodelled endothelial cells contribute to the occurrence of endothelial cell ridges buckling into the lumen in an orientation parallel to the vessel axis. These ridges are composed not only of endothelial cells, but in addition contain wavy elastic lamina and interpolated portions of juxtaposed vascular smooth muscle cells.
* Address to which proofs are mailed: Prof. Dr. K. SpanelBorowski, Anatomisches Institut, Universitiit Basel, PestalozzistraBe 20, CH-4056 Basel
)~I SEMP'Ett~
Ann. Anat. (1992) 174: 213-216 Gustav Fischer Verlag Jena
While endothelial cell ridges represent the passive inner annulus, the adjacent portion of the arteriolar wall is considered to be the active outer annulus due to force-generating capacity of circurnferentially arranged myofilaments of smooth muscle cells (Sleek and Duling 1986). The vasoconstrictive force appears to be responsible for incomplete occlusion in large arterioles or complete occlusion in small arterioles, respectively (van Citters 1966). Yet complete occlusion cannot be sufficiently explained by way of muscular force in view of the concept that smooth muscle cells from vessels of different sizes are mechanically similar (Davis and Gore 1989). The final determinant of complete occlusion has been assumed to be related to the inner annulus (Carlson et al. 1982) and probably to the surface architecture of endothelial cells and their array. Since it is impossible to study the inner lining of a microvascular tree by use of SEM, differences of structure were examined in microvascular corrosion casts of human ureters. A pulsed Eximer-Iaser beam was successfully used to dissect the casts.
Materials and methods In the scope of a thesis carried out to study the blood supply of the lamina propria (Kuhri 1990), twelve ureters were obtained during autopsy. The urogenital tract of male or female human bodies was removed 12-36 h of death, and the ureter dissected with the supplying arteries, i.e. ureteric artery, testicular artery or ovaric artery, respectively. The arteries were cannulated and perfused manually with 20 ml 0.9% NaCI for approximately 5 minutes. When the colour of the vasculature had turned white, polymethylmethacrylate (Mercox®, Norwald KG, Hamburg, FRG) was injected manually. The resin was prepared at room temperature prior to use. 100 ml polymethylmethacrylate, the basic component, was mixed with 5 ml hardener for 30 seconds. 15-20 ml resin mixture was injected within 5 minutes. The whole ureters were removed and kept in distilled water for 24 h. For maceration, 1 M NaOH was applied for two weeks, upon which 1 M HCI and 1 M
NaOH, were applied for one week each . Two turns and multiple rinses with distilled water were carried out. The microcorrosion casts were kept in water and frozen at -20 °C. The frozen tissue was cut transversely by use of a razor blade drawn through a flame . The sliced specimens were freeze-dried at -40°C. They were mounted parallel to the ureter axis on SEM specimen holders, sputtered with gold and viewed in a Philips SEM 505 at 15 kY. In order to evaluate how blood vessels branched from the adventital coat of the ureter to its lamina propria the sputtered specimens were processed further. Longitudinal cuts were obtained by use of an Eximer-laser beam (Questek Inc., Billerica, MA, USA). The procedure was controlled under a stereornicroscope equipped with an ultraviolet filter. 351 nm wave length was applied and density of energy kept stable at 1.5 J/cm2 • The laser beam,
focused to a diameter of 70 /lm and its pulse frequency adjusted to 20 Hz, was operated by a hand-driven micromanipulator (Brinkmann, Mannheim, FRG). 150-200 /lm thick sections were cut, and each exposed surface section was sputter coated and examined at different stages of processing. By an anterograde and retrograde evaluation, the architecture of individual rnicrovessels became evident.
Results Each studied segment of the microvessel corrosion cast had characteristic features (Figs. 1-5). They were considered to
Fig. 1. The arteriole of second order has a diameter of 40- 50 /lm (not completely shown). Oval imprints of endothelial cell nuclei (large arrows) are found within furrows (small arrows) along the axis of the rnicrocorrsosion cast. SEM, X2,100. Bar: 10 /lm. Fig. 2. The ateriole of third order measures 20 /lID in diameter. Furrows (arrows) form a helix around the axis of the rnicrocorrosion cast. They contain imprints of endothelial cell nuclei hardly to be discerned at this magnification. Broad ridges occur parallel to furrows. SEM. X2,300. Bar: 10 /lm. Fig. 3. The arteriole of fourth order displaya diameter of 12-14 /!ill. Oval imprints of endothelial cell nuclei (arrows) are oriented in a helix the pitch of which is larger than in figure 2. Furrows and ridges are less evident probably due to vasodilation. A cast of a capillary is apparent at the bottom of the figure. SEM. x2,100. Bar: 10 /lm.
214
Fig. 4. A capillary loop of a microcorrsosion cast shows a diameter of 5-6 !lm. Imprints of endothelial cell nuclei are randomly located. SEM. X 2,300. Bar: 10 !lm. Fig. 5. The venule of a microcorrosion cast has a diameter of 110 !lm (not completely shown). Imprints of endothelial cell nuclei (arrows) are of round or oval shape. The imprints represent the small meshes of a reticular network composed of attenuated, thick resin ridges. SEM. X 1,300. Bar: 10 !lm.
represent the replica of in vivo occurrence. Nuclei of endothelial cells appeared as oval surface imprints found in arterioles and capillaries. Round imprints were also seen in venules. Imprints were apparent in furrows of arterioles and "meshes" of venules (see below). Furrows were preferentially oriented along the axis of larger arterioles (> 30 !lm in diameter). Yet the furrows aligned like a helix around the axis of smaller arterioles « 30!-lm in diameter). The pitch of the helix seemed to change with the state of contraction considering height of ridges or depth of furrows, respectively, as a parameter of contraction. Contraction was caused presumably by rigor mortis. In venules, small "meshes" were surrounded by a reticular network of attenuated, thick resin ridges.
inner diameters during induced contraction (Carlson et al. 1982). Endothelial cell ridges appear at one location, and, with further contraction, spread around the circumference of the lumen (Greensmith and Duling 1984). Vascular smooth muscle cells remained in our microcorrosion casts occasionally, and such the muscle cells were always arranged circumferentially to the axis of arterioles of all sizes (not shown). This is in agreement with the observations of Beacham et al. (1976), Walmsley et al. (1982) and Davis and Gore (1989) and in disagreement with those of Rhodin (1980). It is our conclusion that in smaller arterioles the endothelial cells are arranged in the form of a helix.
Acknowledgements. We thank Dr. K. Ley for reading and Mrs. R. Miinzinger for typing the manuscript. The study was supported by the Deutsche Forschungsgemeinschaft, grant. no. Sp 232/4-1.
Discussion Hodde and Nowell (1980) were the first to use a continuous Argon laser beam to cut microvessel corrosion casts. The problems they faced due to high or low energy release were overcome in the present by using a pulsed Eximer laser beam. Endothelial cell imprint patterns as reported by Hodde and Nowell (1980) are confrrmed by our findings, i.e. longitudinal array of imprints in arterioles, and the mesh-like meandering appearance of oval or round imprints in venules. In contrast to larger arterioles, smaller arterioles have a helical orientation of endothelial cells. In retrospect, this phenomenon is to be seen with figures of casted smaller arterioles published by Hodde and Nowell (1980) and Greensmith and Duling (1984), although the helical structure was not explicitly recognized. As noticed with cross sections, dilated arterioles from the mesentery show irregular
References Beacham WS, Konishi A, Hunt CC (1976) Observations on the microcirculatory bed in rat mesocecum using differential interference contrast microscopy in vivo and electron microscopy. Am J Anat 146: 385-426. Carlson EC, Burrows ME, Johnson PC (1982) Electron microscopic studies of cat mesenteric arterioles: A structure-function analysis. Microvasc Res 24: 123-141 Davis, MJ, Gore RW (1989) Length-tension relationship of vascular smooth muscle in single arterioles. Am J Physiol 256: H630-640 Greensmith, JE, Duling BR (1984) Morphology of the constricted arteriolar wall: physiological implications. Am J Physiol 247: H687-698
215
Hodde KC, Howell JA (1980) SEM of micro-corrosion casts. Scan Electron Microsc II: 88-106 Joyner WL, Davis MJ (1987) Pressure profile along the microvascular network and its control. Fed Proc 46: 266-269 Kuhri P (1990) Die GefaBversorgung der menschlichen Harnleiterwand. Bine korrosionsanatomische Untersuchung. Dissertation an der Vorklinisch-Naturwissenschaftlichen Universitat der Medizinischen Universitat zu Lubeck, Lubeck Meininger GA (1987) Responses of sequentially branching macro- and microvessels during reactive hyperemia in skeletal muscle. Microvasc Res 34: 29-45 Meininger, GA, Mack, CA, Fehr KL, Bohlen G (1987) Myogenic vasoregulation overrides metabolic control in resting rat skeletal muscle. Circ Res 60: 861-870 Rhodin JAG (1980) Architecture of the vessel wall. In: Bohr DF, Somlyo AP, Sparks Jr HU (eds) Handbook of Physiology. Sec 2. The Cardiovascular System. Vol 2.
Vascular Smooth Muscle. Am Physiol Soc, pp 16-17, Bethesda, Maryland Slaaf, DW, Reneman RS, Wiederhielm CA (1987) Pressure regulation in muscle of unanesthetized bats. Microvasc Res 33: 315-326 Sleek GE, Duling BR (1986) Coordination of mural elements and myofilaments during arteriolar constriction. Circ Res 59: 620-627 Van Citters RL (1966) Occlusion of lumina in small arterioles during vasoconstriction. Circ Res 18: 199-204 Van Citters RL, Wagner BM, Rushmer RF (1962) Architecture of small arteries during vasoconstriction. Circ Res 10: 668-675 Walmsley JG, Gore RW, Dacey Jr RG, Damon ON, Duling BR (1982) Quantitative morphology of arterioles from the hamster cheek pouch related to mechanical analysis. Microvasc Res 24: 249-271 Accepted October 25, 1991
216
