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Scanning electron microscopy of an elastic fiber network which forms the internal elastic lamina in canine saphenous vein.

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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/80/1984-0581$02.60@I 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,
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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:40@413.
Wolinsky, H., and S. Glagov (1967)A lamellar unit of aortic
medial structure and function in mammals. Circ. Res.,
ss: 9 9111.
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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 .
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