Scanning electron microscopy of an elastic fiber network which forms the internal elastic lamina in canine saphenous vein.код для вставкиСкачать
THE ANATOMICALRECORD 198:581-593 (1980) Scanning Electron Microscopy of an Elastic Fiber Network Which Forms the Internal Elastic Lamina in Canine Saphenous Vein ROBERT S.CRISSMAN, JAMES N. ROSS, JR., AND TERRANCE DAVIS Departments of Anatomy, Physiology, and Surgery, Medical College of Ohio. Toledo, Ohio 43699 ABSTRACT Scanning electron microscopy (SEM) was used to study the arrangement of elastic fibers in the canine saphenous vein as the basis for further studies of veins used in by-pass grafting operations. The elastic fiber arrangement in distended and non-distended veins was examined in both immersion-fixed and perfusion-fixed vessels. Transmission electron microscope (TEM) observation of the SEM samples confirmed the identity of these fibrillar structures as elastic fibers. In addition, specific stains for elastic fibers (Verhoeffs iron hematoxylin and orcein) were used. The elastic fibers forming the internal elastic lamina were arranged in a fishnet-like pattern. Large-diameter fibers, running longitudinally along the vascular wall, were interconnected by smaller oblique fibers. Together the fibers formed an elastic cylindrical network between the endothelium and the smooth muscle cells. The thicker longitudinal fibers were the same diameter in distended and non-distended veins. By contrast, the oblique fibers were thinner and more complexly branched in distended veins. The architecture of the elastic fiber network contributes to vascular flexibility and allows circumferential distension. The interconnecting oblique fibers presumably serve to distribute internal pressure equally around the venous wall. The architecture of the internal elastic lamina (IEL) in veins of animals has been largely overlooked. More attention is devoted to the IEL of human veins, but descriptions in current textbooks are quite diverse. In large veins, such as the external liliac, superior mesenteric, or portal veins, the IEL is interpreted as being a thin, fenestrated sheet of elastic tissue (Weiss and Greep, '77). In small- to medium-sized veins the IEL is described as absent, ill-defined, discontinuous, or simply represented by scattered elastic fibers (Rhodin, '74; Bloom and Fawcett, '75; Weiss and Greep, '77). The use of saphenous veins for arterial by-pass grafting surgery has stimulated interest in the normal structure of these vessels in human and in canine models. The structural modifications resulting from a greater than normal intraluminal pressure are of particular importance (Brody et al., '72; Szilagyi et al., '73). The arrangement of the elastic fibers within the normal venous wall and the IEL, however, has remained ambiguous. The morphology of elastic fibers in vessels is comparable to that of elastic fibers elsewhere 0003-276X/80/[email protected] 1980 ALAN R. LISS, INC. throughout the body (Gotte et al., '72); Hunter and Finlay, '73; Ross, '73; Sandberg, '76; Kewley et al., '77). These fibers are specifically revealed by elastic stains such as Verhoeff s iron hematoxylin (Brissie et al., '74; Spicer et al., '75) and orcein (Nakamura et al., '77). The fine structure of the IEL in arteries has been described by several investigators (Parker, '58; Pease and Paule, '60; Pease and Molinari, '60). These investigators postulated that the IEL anastomosed with fibers of the media to form a continuous network throughout the vascular wall. This concept was supported by scanning electron microscopic (SEM) observations of elastic fibers interconnecting adjacent lamellae, forming three-dimensional networks of anastornosing elastic fibers in both the aorta and the pulmonary trunk (Smith, '76). These findings were in accordance with the conclusions of previous investigators (Burton, '54; Wolinsky and Glagov, '64, '67) that vascular distensibility was based on the structural configuration of the smooth muscle cells, colReceived December 10, 1 9 7 9 accepted April 16, 1980, 582 R.S. CRISSMAN, J.N. ROSS, JR., AND T. DAVIS lagenous and elastic fibers in the vascular wall. Azuma and Hasegawa ('73) postulated, on the basis of distensibility characteristics and histologicalfeatures of several large veins, that elastic fibers throughout the venous wall formed a lattice-like arrangement. However, no mention was made of the configuration of the IEL. In the present study, SEM has demonstrated that the scattered elastic fibers between the endothelium and smooth muscle cells of canine saphenous veins form an organized, lattice-like icternal elastic lamina. An accurate knowledge of architecture of the IEL aids in understanding the distensibility characteristics of veins. MATERIAL AND METHODS Distended and non-distended saphenous veins from seven healthy adult dogs were observed by SEM and transmission electron microscopy (TEM). Distended veins were removed from two dogs after perfusion fixation. The saphenous vein was carefully dissected free from its surrounding connective tissue bed and the side branches tied off. Perfusion at 110 mm Hg was carried out via a large cannula inserted into the abdominal aorta; an initial flush of 1.5 liters of heparinized normal saline was followed by 4 liters of 0.2 N cacodylate-buffered fixative (1%paraformaldehyde and 1.25% glutaraldehyde). Non-distended vessels were procured from five dogs undergoing saphenous vein by-pass grafting operations. In this procedure, the veins were isolated surgically and the side branches tied off while maintaining blood flow through the main vessel. The proximal and distal ends were then clamped and severed. The vessels were immediately flushed with heparinized solutions of normal saline or lactated Ringer's. Venospasm in seven vessels was prevented by transient distension with the heparinized rinse solution in three short pulses of 20C250 mm Hg pressure. Then the veins were fixed by immersion. The remaining three veins were periodically moistened with Z?? xylocaine solution during dissection and removal to prevent smooth muscle contraction. (Thistechnique has previously been reported to be the least traumatic to the vessel [Ross et al., '771.) All manipulation was carried out as gently and quickly as possible (1-2 minutes from time of removal from the dog to fixation) to minimize trauma. The veins were rapidly placed in cold, cacodylate-buffered aldehyde fixative for 2 hours and then post-osmicated for 1hour. During this period the luminal surface was exposed by hemisecting the vein along the longitudinal axis. The tissue was then prepared for SEM by dehydration in graded acetone solutions and critical point-dried in CO,. The specimens were mounted with silver print paint and coated with palladium gold in a sputter coater. All SEM observations were carried out in regions of the luminal surface that were free from side branches and valves. Several immersion-fixed specimens were observed by SEM to have regions that were devoid of endothelium. Endothelial desquamation was capricious and apparently resulted from occasional mechanical abrasion during flushing or hydraulic pressure to prevent venospasm, since desquamation was not observed in perfusion-fixed vessels. Selected SEM samples were embedded in Epon-Araldite for TEM. One-micronsections of these blocks were examined by light microscopy to confirm the identification of denuded regions by SEM. Thin sections of the same areas were stained for elastic fibers using orcein and Verhoeffs stains (Nakamura et al., '77; and Brissie et al., '74) and studdied by TEM. RESULTS Small areas of the luminal surfaces of some non-distended, immersion-fixed veins were devoid of endothelium. The remainder of the endothelial lining in these specimens appeared to be intact. Along the borders of these desquamated regions, longitudinally oriented fibers were observed lying between the endothelium and the transversely oriented smooth muscle cells (Fig. 1).In larger regions denuded of endothelium, these longitudinally oriented fibers appeared closely packed on the adluminal surfaces of the smooth muscle cells (Fig. 2). The number of fibers present and the spacing between them varied from region to region, Careful tracing of individual fibers demonstrated that they were interconnected. These fibers correspond to the layer of elastic fibers adjacent to the luminal surface a t the light microscopic level (Fig. 2, inset). In some instances, these fibers were partially concealed by the underlying smooth muscle cells (Fig. 3). Occasionally a fiber appeared to be formed by the coalescence of several small fibers into one main fiber trunk (Fig. 4).After a short distance (%3 mm) this main trunk split into several small, diverging fibers. The typical arrangement of these fibers was that of a continuously branching network (Fig. 5). Most of the 583 INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN fibers ran parallel to the longitudinal axis of the vein. These fibers in the non-distended state (as represented by immersion fixation) were 1-2 pm in diameter. Fibers of the same size or slightly smaller branched obliquely from the main fiber and merged with adjacent fibers. Individual fibers never appeared to terminate. Instead, an anastomosing network was formed by oblique branches merging with longitudinal fibers. This continuous network resembled the meshwork of fishnet lying on the adluminal surface of the smooth muscle cells. Occasionally a fiber disappeared from view underneath a smooth muscle cell (Fig. 6). At low magnification the fibers appeared to be sharply defined with smooth surfaces, although small surface irregularities were noted at higher magnification (Fig. 7). These fibers were also observed in distended veins from perfusion-fixed dogs, even though the endothelial lining of these vessels was intact. The extreme thinness of the stretched endothelial cells in these specimens revealed linear elevations caused by longitudinally oriented fibers beneath the endothelium (Fig. 8).These fibers, while thinner and separated by greater intervals, formed a network similar to that observed in immersion-fixed veins. After perfusion fixation the main longitudinally oriented fibers were between 1.2 and 1.8 pm in diameter. The oblique fibers appeared thinner in diameter ( O . S O . 8 pm) and more complexly branched than those seen in denuded regions of immersion-fixed veins. Transmission electron microscopy of the venous segments that were previously examined by SEM revealed electron-lucent structures immediately beneath the gold coating on the surface of smooth muscle cells (Fig. 9). In cross sections of non-distended veins, these structures were 1.2-2.0 Wm in diameter and resembled the amorphous component of elastic fibers (Fig. 10). However, the gold coat was removed on sections stained with Verhoeff's iron hematoxylin. To determine that the densely stained fibers were not residual masses of gold coating, thin sections were stained with orcein (Fig. 11). In these sections the gold coating was intact and the fibers were positively stained with orcein. Elastic fibers were also demonstrable by the latter procedure immediately subjacent to the very thin endothelium of perfusion-fixedtissue (Fig. 12). The microfibrillar component of the elastic fibers could be visualized in this perfused tissue in TEM. These fibers stained positively with orcein and could be divided into TABLE I . Measurements of elastic fiber diameters as observed in distended and non-distended veins by SEM and TEM Immersion-fixed Perfusion-fixed Fiber size SEM TEM SEM TEM Large Small 1.&2.0 1.2-2.0 0.5-0.8 1.s1.8 O.SO.8 O.bl.2 O.SO.6 - All measurements are in micrometers (pm). A minimum of 50 fibers wasmeasuredineach category. The onlyexceptionto thls is the TEM of small elastic fibers in the non-distended, immersion-fixed vessels There were only 14 fibers found in this size category. Note the close Correlation of size between the SEM and TEM measurements. The thickness of the gold coat as measured from the TEM samples is subtracted from the fiber measurements In SEM micrographs two categories on the basis of size. Larger fibers were between 0.8 and 1.2pm in diameter, while the smaller fibers were between 0.3 and 0.6pm in diameter (Fig. 13). When the vein was sectioned parallel to the longitudinal axis of the vein, elastic fibers were observed passing deep t o the smooth muscle cells adjacent to the luminal surface in some places (Fig. 14). Measurements of the diameters of elastic fibers in SEM and TEM specimens of either distended or non-distended veins were summarized in Table 1. A minimum of 50 fibers were measured in each category. Note the close correlation in size obtained in measurements by both SEM and TEM. DISCUSSION The fortuitous endothelial damage incurred during by-pass grafting surgery on canine saphenous veins has helped elucidate the architecture of the venous IEL. The position of the IEL and the ultrastructure of the elastic fibers was not altered by the surgical technique, as confirmed by our comparison of SEM and TEM micrographs from tissues fixed by either immersion or perfusion. The round structures on the adluminal surface observed by TEM had the characteristics of elastic fibers and corresponded in size and location with the longitudinal fibers observed in the same specimens by SEM. The TEM morphology of these fibers corresponds to that of elastic fibers, as established by other investigators (Ross, '73; Sandberg, '76). Microfibrils were not consistently visualized in tissues processed for SEM, but the amorphous core stained normally with uranyl acetate and lead citrate. Positive staining of elastic fibers with orcein and Verhoeff s iron hematoxylin is further proof of their identity. On this basis, we suggest that the microfibrillar component is still present but is some- 584 R.S. CRISSMAN, J.N. ROSS, JR., AND T. DAVIS how altered or obscured during processing for SEM. The morphology of elastic fibers, as documented by SEM in the present study, is consistent with that reported by other investigators (Gotte et al., '72; Hunber and Finlay, '73; Smith, '76; Kewley et al., '77). New insights into the arrangement of elastic fibers within the venous wall were provided by the well-known advantages of SEM for the examination of large surface areas. Elastic fibers formed a cylindrical meshwork between the endothelium and the adluminal surface of circularly oriented smooth muscle cells in canine saphenous veins. This meshwork was continuous around the entire vascular wall. On the basis of location, this network formed the IEL upon which the endothelium rests. In contrast to an "ill-defined"IEL of scattered elastic fibers (Rhodin, '74), this medium- sized vein has a distinct IEL formed by a network of elastic fibers. The presence of branching elastic fibers within the vessel wall was not unusual, since branching is characteristic of elastic fibers. It was surprising, however that these branching fibers within the IEL were arranged as a single-layered cylindrical lattice around the lumen. The scattered elastic fibers observed in this study by both light and transmission electron microscopy anastomosed with each other to form a cylindrical network. This network had fibers oriented both longitudinally and obliquely with respect to the main axis of the vein. No striking differences in diameter were noted between these fibers in non-distended, immersion-fixed veins. However, in the distended, perfusion-fixed veins, the longitudinal fibers were thicker than the oblique fibers. In addition, the distended oblique fibers appeared to be more complexly branched. This indicates that the oblique fibers were stretched more than the longitudinal fibers during normal venous distension. With reduction of the distension pressure, the fibers shorten and become thicker. This observation readily explains the differencesin diameter of these fibers from distended and non-distended veins. The anastomosing network of fibers within the lamellae of rabbit aorta and pulmonary trunk (Smith, '76) was similar to that of the IEL of canine saphenous veins. The major differences were the number of fibers present, the diameter of the fibers, and the complex branching of the oblique fibers in saphenous veins. In non-distended veins, the largest longitudinal elastic fibers were equivalent in diameter to the smallest longitudinal fibers in the un- stretched pulmonary trunk and aorta. By extrapolation from the work by Wolinsky and Glagov ('67), it may be hypothesized that the fewer fibers and smaller fiber diameters result from lower tension within the saphenous wall. Termination of elastic fibers in the IEL was not observed, even though an extensive search was made, suggesting that these fibers anastomose with adjacent fibers without physical termination or discontinuity. Sometimes the longitudinal fibers arising in the IEL disappear from view beneath the transversely arranged smooth muscle cells. This corresponds to our TEM observations of elastic fibers near the luminal surface coursing deep to the smooth muscle cells. Parker ('58) also reported elastic strands extending into the tunica media from the IEL in rabbit aorta. Our observation supports the TEM findings of other investigators (Pease and Paule, '60; Pease and Molinari, '60) and more recent SEM observations (Smith '761, which suggest that a continuous, three-dimensional network of elastic fibers occurs throughout the vascular wall. Veins are typically characterized by their capacity to accommodate large volumes of blood without an accompanying rise in intraluminal pressure. This has been attributed to the arrangement of three of the main vascular components: smooth muscle, collagenous fibers, and elastic fibers (Burton, '54; Wolinsky and Glagov, '64; Azuma and Hasegawa, '73). Distensibility of the elastic fiber component may be explained in terms of the configuration of the elastic fiber network. It would appear that the oblique fibers were stretched more than the longitudinal fibers under normal intraluminal distension pressure, allowing greater circumferential distension. The thicker longitudinal fibers may not be stretched until greater distending pressures occur. Azuma and Hasegawa ('73) reported that in several large veins (axillary, jugular, inferior vena cava, internal iliac veins), the circumferential distensibility was significantly greater than the corresponding longitudinal distensibility in the normal range of pressures. It is probable that intrinsic pressures greater than normal would not only distend veins circumferentially but would also lengthen them by stretching the thick longitudinal fibers. This is supported by the work of Azuma and Hasegawa ('731, who report greater longitudinal distensibility at above-normal distension pressures. Such a condition is characteristically seen in the distended, tortuous, lengthened vessels of varicose veins (Abramson, '74). INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN The delicate oblique fibers stretch more readily, as previously noted, due to their small size. As distension occurs, the longitudinal fibers separate, causing the oblique fibers to stretch and become truely oblique. This also reveals their more complex branching pattern. In addition, their oblique course would accommodate further circumferential distension. In this way, the stretch and separation of the oblique interconnecting fibers of the network may act to distribute the distension pressure uniformly around the circumference of the vascular wall. The location of the IEL network adjacent to the luminal surface may also aid in distributing distension pressure uniformly around the venous wall. It is suggested that the IEL network functions as a unit, expanding outward against the smooth muscle cells and collagenous fibers located peripheral to the IEL. Likewise, any contraction pressure against the luminal contents produced by the individual smooth muscle cells (lying transversely across several main longitudinal elastic fibers) is presumably distributed uniformly around the circumference of the vessel by the network of the IEL. The organization of the IEL as a cylindrical network is similar t o that postulated for the arrangement of elastic fibers within the outer layers of the venous wall (Azuma and Hasegawa ’73).However, we did not observe the IEL to contain longitudinal gaps, as suggested by the previous workers, nor did we observe the longitudinal and circumferential elastic fibers to be arranged a t right angles to each other. Our observations are limited to the single layer of elastic fibers adjacent to the lumen, whereas the previous investigators’ hypothesis is based on the distensibility characteristics of the entire vascular wall. The arrangement of the scattered elastic fibers embedded within the other regions of the venous wall remains to be studied. ACKNOWLEDGMENT This research was supported by the American Heart Association, Northwestern Ohio Chapter. LITERATURE CITED Abramson, D.I. (1974) Primary varicosities. In: Vascular Disorders of the Extremities, 2nd ed. Harper and Row, 585 New York, pp. 509-520. Azuma, T., and M. Hasegawa (1973) Distensibility of the vein: From the architectural point of view. Biorheology, 10:469479. Bloom, W., and D.W. Fawcett (1975) Veins. In: Textbook of Histology. W.B. Saunders Co., Philadelphia, pp. 40%413. Brissie, R.M., S.S. Spicer, B.J. Hall, and N.T. Thompson (1974) Ultrastructural staining of thin sections with iron hematoxylin. J. Histochem. Cytochem., 22:895907. Brody, W.R., W.W. Angell, and J.C. Kosek (1972)Histologic fate of the venous coronary artery bypass in dogs. Am. J. Pathol., 66:lll-129. Burton, A.C. (1954) Relation of structure of function of the tissues of the wall of blood vessels. Physiol. Rev., 34:619642. Gotte, L., M. Mammi, and G. Pezzin (1972)Scanning electron microscope observations on elastin. Connective Tissue Res., 1:61-67. Hunter, J.A.A., and B. Finlay (1973)Identification of elastic tissue in human skin viewed in the scanning electron microscope. J. Microsc., 98:41-47. Kewley, M.A., F.S. Steven, and G. Williams (1977) The presence of fine elastin fibrils within the elastin fiber observed by scanning electron microscopy. J. Anat., 123:129-134. Nakamura,H.,C. Kanai, andV.Mizuhira (1977)Anelectron stain for elastic fibers using orcein. J. Histochem. Cytochem., 25:30&308. Parker, F. (19581 An electron microscope study of coronary arteries. Am. J. Anat., 103:247-273. Pease, D.C., and S. Molinari (1960) Electron microscopy of muscular arteries; pial vessels of the cat and monkey. J. Ultrastruct. Res., 3:447-468. Pease, D.C., and W.J. Paule (1960) Electron microscopy of elasticarteries; the thoracic aorta of the rat. J. Ultrastruct. Res., 3:469483. Rhcdin, J.A.G. (1974) Veins. In: Histology Text and Atlas. Oxford University Press, New York, pp. 362-366. Ross, J.N., Jr., R.S. Crissman, and S.M. Dosick (1977)Endothelial alterations in veins prepared for by-pass grafting: Electron microscopic analysis. Trans. 3rd Ann. Meeting Society Biomat’l and 9th Ann. Internat. Biomat’l Symp., 1 :35. Ross, R. (1973) The elastic fiber. A review. J. Histochem. Cytochem., 31 :199-208. Sandberg, L.B. (1976) Elastin structure in health and disease. In: International Review of Connective Tissue Research, 7159-210. Smith, P. (1976) A comparison of the orientation of elastin fibers in the elastic laminae of the pulmonary trunk and aorta of rabbits using the scanning electron microscope. Lab. Invest., 3 5 5 2 5 5 2 9 . Spicer, S.S., R.M. Brissie, and N.T. Thompson (1975) Variability ofdermal elastin visualized ultrastructurally with iron hematoxylin. Am. J. Pathol., 79:481-492. Szilagyi, D.E., J.P. Elliott, J.H. Hageman, R.F. Smith, and C.A. Dall’Olmo (1973) Biologic fate of autogenous vein implants as arterial substitutes. Ann. Surg., 178:23%246. Weiss, L., and R.O. Greep (1977) Veins. In: Histology. McGraw-Hill Book Co., New York, pp. 415420. Wolinsky, H., and S. Glagov (1964) Structural basis for the static mechanical properties of the aortic media. Circ. Res., XN:[email protected] Wolinsky, H., and S. Glagov (1967)A lamellar unit of aortic medial structure and function in mammals. Circ. Res., ss: 9 9111. 586 R.S. CRISSMAN, J.N. ROSS, JR.,AND T.DAVIS INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN Figs. 1-14. All scanning electron micrographs were taken with the longitudinal axis of the vein placed parallel to the vertical axis of the micrograph. In this way one looks down the “barrel” of the vessel. The transmission electron micrographs were taken from sections perpendicular to the longitudinal axis ofthe vessel. These transverse sections were then rotated to place the luminal surface on the right side of the micrograph. Fig. 1. Edge of endothelial, denuded region ofnon-distended saphenous vein. The endothelial cells (El have been partially removed, revealing underlying longitudinally arranged fibers (F) lying on the adluminal surface of transversely oriented smooth muscle cells (SM). SEM; 1,400 x . Fig. 2. Denuded luminal surface of non-distended saphenous vein. The endothelium has been completely removed to expose numerous longitudinally arranged elastic fibers. These fibers (arrow) were branched and closely packed together. They lie on the adluminal surface of the circularly oriented smooth muscle cells. The elastic fibers form an anastomosing network across the entire adluminal surface. SEM; 500 X . Inset: Cross section of non-distended saphenous vein. A layer of cross-sectional elastic fibers (arrow) was located adjacent to the luminal surface. Close inspection reveals branching fibers. Larger elastic fibers are seen scattered in the tunica media (MI. Weigert’s resorcin-fuchsin. LM, 300 x . Fig. 3. Denuded luminal surface of non-distended saphenous veins. The branching fibers (F) appeared to be partially embedded in the adluminal surface of underlying smooth muscle cells (SM) in some regions of immersion-fixed tissue. SEM; 1,500 x , Fig. 4. Denuded luminal surface ofnon-distended saphenous vein. The branching of the fibers was variable. In this instance the main longitudinal fiber (F) was formed by the coalescence of several smaller fibers. After approximately Z3 mm, the fiber diverged into several smaller fibers. SEM, 1,600 x . 587 588 R.S. CRISSMAN, J.N. ROSS, JR.,AND T. DAVIS INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN Fig. 5. Denuded luminal surface of saphenous vein. A continuous anastomosing network of elastic fibers was the typical fibrous arrangement found. In non-distended vessels, large fibers (FJran parallel to the longitudinal axis of the vessel. Obliquely oriented fibers IOFJ interconnected the adjacent longitudinal fibers. SEM; 2,100 x . Fig. 6. Partially denuded luminal surface of non-distended saphenous vein. Some endothelial cells IE) remain. Fibers (F) sometimes disappeared underneath (or externally to) smooth muscle cells (arrows). Individual fibers were never observed to terminate. SEM; 1,200 X. Fig. 7. Denuded luminal surface of non-distended saphenous vein. High magnification of fibers revealed small surface irregularities. Transverse structures (arrow) were commonly observed in immersion-fixed tissue. S E M 12,700 x . Fig. 8. Luminal surface ofperfusion-fixed saphenous veins. The flattened nuclei (N)and borders IBJ of the stretched endothelial cells can be observed. The extreme thinness of the stretched endothelial cells made it possible to observe the presence of large, longitudinally oriented fibers IF). These were joined together by thin, obliquely oriented fibers (arrow).The obliquely oriented fibers were delicate and more complexly branched than the oblique fibers seen in immersion-fixed tissue. SEM, 1,300 X . 589 590 R.S. CRISSMAN, J.N. ROSS, JR.,AND T.DAVIS INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN Fig. 9. Cross section of denuded luminal surface of non-distended saphenous vein. Electron-lucent structures (F), which were the same diameter a s the fibers found by SEM, were located on the adluminal surface of circularly oriented smooth muscle cells (SM). These fibers, when stained with uranyl acetate and lead citrate, displayed the amorphous appearance of elastic fibers. However, no microfibrils were found a t their surface. The dark black line represented the gold on the surface of this embedded SEM specimen. TEM; 11,500 X . Fig. 10. Cross section of denuded luminal surface of non-distended saphenous vein. Fibers (F)the same size and location were positively stained with Verhoeff s iron hematoxylin for elastin. Note the lack of gold coat on the luminal surface. T E M 8,500 X , Fig. 11. Denuded luminal surface of non-distended saphenous vein. The fibers (F) were also positively stained with orcein stain for elastic tissue. The gold coat was intact, and fibers were the same size as previously observed. TEM 7,000 x. Fig. 12. Cross section ofdistended saphenous vein. Elastic fibers (F) stained positively with orcein were observed between the endothelium (El and the smooth muscle cells (SM). Two fibers appeared to be sectioned a t a point of fusion. The rnicrofibrillar component of the elastic fibers can he seen (arrow). TEM; 23,000 x . 591 592 R.S. CRISSMAN, J.N. ROSS, JR.,AND T.DAVIS INTERNAL ELASTIC LAMINA OF SAPHENOUS VEIN 593 Fig. 13. Cross section of luminal surface of distended saphenous vein. Stretched fibers (F)were classifiable as either large or small according to their diameter. Elastic fibers positively stained with orcein were observed between the endothelium (El and smooth muscle cells (SM). TEM; 17,000 x . Fig. 14. Longitudinal section of luminal surface of distended saphenous vein. Elastic fibers (F)from near the luminal surface were observed to pass deep to the smooth muscle cells (SM) lying on the luminal surface. Uranyl acetate and lead citrate. TEM; 9,000 x .