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Structure of mammalian portal veinPostnatal establishment of two mutually perpendicular medial muscle zones in the rat.

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Structure of Mammalian Portal Vein: Postnatal
Establishment of Two Mutually Perpendicular
Medial Muscle Zones in the R a t '
Department of Pathology, University of Chicago,
Pritzker School of Medicine,
Chicago, Illinois
In all mammalian species examined thus far, the media of the
adult mammalian portal vein consists of two mutually perpendicular smooth
muscle zones separated by a fibrous layer. The cells of the muscle zone nearest
the intima are arranged circumferentially and resemble smooth muscle of other
vessels; the muscle cells of the thicker, vascularized outer zone are longitudinal
and contain mitochondria and pinocytotic vesicles in great abundance, suggesting relatively high metabolic activity. The adult configuration is not present at
birth and only develops during the first three postnatal weeks in the rat. Partitioning of the media commences at about three days and contrasting orientation
and composition of the cells of the two muscle zones is not established until
seven to ten days after birth. Vasa vasorum appear in the outer muscle layer at
about two weeks and nerve fibers appear even later. Post-natal establishment of
a special, double layered neuromuscular structure may be related to the adaptation of the portal circulation to the effective closure of the ductus venosus.
Unlike other veins of comparable diameter in the adult rat, the media of the
extrahepatic segment of the portal vein
consists of two mutually perpendicular
zones of smooth muscle cells separated by
a relatively acellular fibrous layer containing nerve fibers (Ts'ao et al., '70). The inner muscular zone is devoid of vasa
vasorum and its cells are oriented circumferentially. The outer zone is thicker than
the inner and richly vascularized; its component cells are longitudinally oriented and
show evidence of relatively enhanced metabolic activity. Portal veins of six additional
species have now been examined; the same
double layered medial architecture is evident regardless of species or vessel diameter (unpublished observations). In relatively large animals, the intermediate
fibrous layer and the outer muscle zone is
strikingly vascular and innervated. The
special position of the portal vein, as a
chamber interposed between afferent and
efferent branching beds may be related to
its special medial organization. The two
mutually perpendicular medial muscular
layers, reminiscent of intestinal wall, may
ANAT. REC., 171: 457-470.
provide the basis for a propulsive, sphincteric or reservoir function related to
changes in afferent potal flow or hepatic
resistance. If this is so, it is reasonable to
expect that the special portal medial architecture may be established or accentuated
after birth, when the portal circulation
must adapt to the functional closure of the
ductus venosus. The purpose of this paper
is to show that in the rat portal vein, the
double layered medial configuration does
indeed develop after birth. At birth, the
media consists of uniform globular cells.
By the third postnatal day, these cells separate into two distinct medial zones separated by a layer of connective tissue fibers.
By seven days, smooth muscle cells of the
inner muscle zone are distributed circumferentially while those of the outer layer
are longitudinal. The intemediate fibrous
layer is well established by seven to ten
days. Vascularization of the outer longitudinal muscles is not evident until three
weeks later.
Received Feb. 11, '71. Accepted July 6, '71.
1 Supported by grant 05654 of the National Institutes of Health of the United States Public Health
The animals used in this study were
derived from two litters of Sprague-Dawley
rats. One litter contained 15 young, the
other, eight. Two littermates of comparable weight were used within 30 minutes
of birth and two others were sacrificed at
each of the following time intervals after
birth: 3, 5, 7, 10, 14, 18, 21, 28, and 42
days. For very young rats, i.e., newborn
and 3, 5, 7, and 10 days old, the hepatic
hilus was excised and immersed in cold,
phosphate-buffered 5% glutaraldehyde at
pH 7.3. For animals 14 to 42 days old, the
portal vein was flushed with fixative in
situ before excision while the animal was
anesthetized by means of ether inhalation.
Specimens of portal vein were prepared by
transecting the fixed hepatic hilus and embedding portal vein segments so as to
obtain transverse sections of vein wall.
Tissue processing methods for this work are
described elsewhere (Ts’ao et al., ’70).
Ultrathin sections were stained with lead
citrate and uranyl acetate and examined in
an RCA EMU-3 electron microscope. Topography was studied by light microscopic
examination of sections stained with 1%
toluidine blue. Step or serial sections were
used to determine special orientation of
The overall appearance of the rat portal
vein wall during postnatal development is
shown in figure 1. At birth, endothelial
and medial cells are morphologically similar. The media consists of a uniform layer,
three to four cells wide; sectioned adventitial cells appear long and flat with the
long dimension parallel to the intimal surface. By three days, medial thickness increases to three to seven cells, and the
cells are segregated into two discernible
zones. By the seventh day, medial cells of
the inner zone are arranged circumferentially while those of the outer zone are
clearly longitudinal. T e n days after birth,
the two-layered structure of the media is
clearly established along with a relatively
acellular intervening fibrous layer. As the
media differentiates into two distinct, mutually perpendicular smooth muscle zones,
the component cells undergo striking ultra-
structural changes. These are presented in
Differentiation and orientation of medial
smooth muscle
At birth, medial cells of the rat portal
vein were round or globular, and were
similar in most ultrastructural details to
endothelial cells. Cytoplasmic processes of
medial cells were prominent and interdigitated with those of adjacent media cells
and overlying endothelial cells (fig. 2).
There was no evidence of syncytial bridging
between cells, nor were there any demonstrable desmosomes. No definite basement
membranes were present and small groups
of collagen fibers were present focally between cells. Myofilaments were sparse and
tended to occur in small fascicles with
occasional dense bodies in peripheral portions of the cells. Clusters of glycogen
particles were numerous, and rough endoplasmic reticulum and Golgi formations
were prominent. Nuclei of medial cells at
this stage were large and showed occasional
indentations. Mitotic figures could be seen
on many sections.
Segregation of medial cells. By the
third day of extrauterine life, medial cells
were still mainly rounded, but formed two
zones separated by a narrow cleft containing connective tissue fibers. The inner
subendothelial muscle zone was one to
three cells wide; the outer zone was two
to four cells wide and the component cells
were definitely larger than those of the
inner zone (fig. 3 ) . As medial thickness
increased with age, demarcation of the two
medial smooth muscle zones became more
distinct. Increasing medial thickness was
associated with an increase in the number
of medial cells and an increase in the
width of the intervening fibrous layer.
Mitoses were present in both muscle zones,
but were more easily found in the outer.
Once the muscle zones were established,
the width of the outer zone increased more
rapidly with age than the inner due to a
continuing relative increase in both size
and number of the cells of the outer zone.
There were no cells which bridged the
intermediate fibrous area. By the seventh
day, cells of the inner layer were oriented
circumferentially and those of the outer
layer were longitudinal. There was con-
siderable variation from section to section
in both the thickness and compactness of
each of the cellular zones. In general,
both zones became increasingly compact
with age and the outer was usually more
compact than the inner. Cytoplasmic protrusions, prominent in the newborn, became less prominent with age. Distinct
basement membranes were discernible at
seven days. Intercellular collagen fibers
increased in number and were oriented
in the same direction as the cells, i.e., circumferentially in the inner muscle zone
and longitudinally in the outer. The nearly
definitive structure of the rat portal vein
wall by ten days is shown in figure 4. Establishment of distinct medial smooth muscle
zones coincided with the appearance of
distinguishing features between medial and
endothelial celIs and between the cells
which comprised each of the two muscle
Cytoplasmic structures. MyofilamentsBy the fifth postnatal day, myoflaments
were most abundant in cells of the outer
longitudinal zone (fig. 5), but by two weeks
no difference between the zones with regard to abundance of myofilaments was
apparent. In any given cell, abundance of
myofilaments was associated with relatively
decreased endoplasmic reticulum. Mitochondria - Striking and persistent differences between muscle cells of inner and
outer zones occurred in the relative numbers and distribution of mitochondria. In
cells of the adult inner media, mitochondria were comparable in both number and
distribution to those of other vein walls,
i.e., a few perinuclear mitochondria were
present (Ts'ao et al., '70). By contrast,
mitochondria in smooth muscle cells of the
outer longitudinal zone were much more
numerous and tended to be more widely
dispersed within the cell. This difference
was obvious at seven days and became increasingly evident with time. By 18 days,
the number of mitochondria per cell in the
external medial zone reached the adult
level (fig. 6). Pinocytotic vesicles - Before
medial cells separated into two oriented
zones, pinocytotic vesicles were comparable
in number in all of the cells. The number
of pinocytotic vesicles per cell increased
with age, but increased more rapidly in
cells of the external longitudinal zone. By
18 days, cells of the inner medial zone
contained pinocytotic vesicles comparable
in number to those usually seen in smooth
muscle cells; meanwhile, the number of
pinocytotic vesicles per cell in the external
zone had increased markedly and reached
a maximum (fig. 6).
Vasa vasorum. The inner muscle zone,
devoid of demonstrable vasa in the adult,
remained avascular throughout the period
studied, i.e., up to 42 days. The outer longitudinal zone, richly vascular in the adult,
was avascular at birth. Capillaries were
demonstrable in the outer zone in three
week old rats, but not sooner. Vessels were
abundant in the adventitia from birth and
became conspicuously aligned along the
adventitial aspect of the outermost medial
cell zone at 14 days. By 21 days, vascular
channels were unmistakable within the
external zone and became increasingly
abundant with age during the period under
study (fig. 7). Capillary channels occupied an increasing proportion of the external smooth muscle zone but did not occur
near the innermost layer of cells of the
outer zone.
Intermediate medial fibrous layer.
When this layer was first apparent at three
days, it contained collagen fibers and less
abundant fragments of newly forming
elastin. Connective tissue fibers in this
layer increased with age but there was a
gradual increase in the proportion of elastin (figs. 4, 7). In general, collagen fibers
in the intervening fibrous layer were oriented circumferentially, i.e., in the same
direction as the cells of the inner medial
muscle zone. By two weeks, bands of elastin were formed and oriented in the same
direction. The unmyelinated nerve fibers
found in the intermediate fibrous layer of
portal veins in adult rats, were not detected
in the vessels of rats up to six weeks of age.
Intima and adventitia
At birth, when endothelial cells were
morphologically similar to underlying medial cells, there were no intervening intimal
structures or connective tissue fibers. Cytoplasmic extensions from the medial side
of endothelial cells interdigitated with similar projections from subjacent medial cells
(fig. 2). Basal dense bodies similar to
those described in arterial endothelium
(Stehbens, '66; Ts'ao and Glagov, '701,
The findings presented above indicate
abundant numbers of ribosomes and promi- that the special structural features characnent endoplasmic reticulum were present. teristic of the extrahepatic segment of
Basement membranes were absent. Nu- mammalian portal veins are not present at
clear indentations were strikingly more birth but develop rapidly during early postprominent in endothelial cells than in natal life. At birth, intimal and medial
medial cells. With increasing age, endo- cells form a wall of poorly differentiated
thelial cells became flattened; cytoplasmic interdigiting rounded cells distinct from
projections and nuclear indentations de- the adventitia. Within a week, the media
creased in prominence. By ten days, endo- is distinct from the endothelium and has
thelial cells were as attenuated as those been partitioned into two smooth muscle
seen in adult portal veins; ribosomes and zones with an intervening fibrous layer.
Golgi zones remained very prominent until In the ensuing two weeks, cells of the inner
smooth muscle zone adopt a circumferenthree weeks. Pinocytotic vesicles, infretial orientation, while those of the external
quent in endothelial cells at birth; graduzone become longitudinal. The collagen
ally increased and were quite abundant at and elastin fibers of the intervening medial
three weeks. At three days, a gap contain- fibrous layer are eventually oriented ciring small clumps of newly formed elastin cumferentially while those of the adventiwas noted between endothelial cells and tia are longitudinal. Differences between
the innermost layer of medial cells. At this the medial smooth muscle zones with retime, the interdigitating cytoplasmic pro- gard to cell structure become manifest
jections of these adjacent layers were mark- during the period of segregation into two
edly decreased in prominence (fig. 8A). By zones. Inner zone cells become indistintwo weeks, many of the elastin fragments guishable from usual vascular smooth mushad coalesced to form plates. (fig. 8B). The cle, while those of the outer zone acquire
subendothelial space thereafter remained many more mitochondria and pinocytotic
sharply demarcated but much narrower vesicles than is usual. In general, myofithan the intermediate fibrous layer and did brils accumulate more rapidly in the outer
not develop a continuous internal elastin medial muscle zone. These differences,
along with the development of a striking
In the adventitia, elastin and collagen abundance of blood vessels in the outer
fibers as well as vascular channels and zone suggest that this portion of the media
unmyelinted nerves were present at birth. comes to function at a relatively higher
Though there was at first no sharp demar- metabolic level. Furthermore, this pattern
cation between adventitia and media, ad- of architecture has been seen in adult portal veins of all mammalian species examventitial cells were distinctly flattened as ined thus far.
compared to the spherical medial cells.
Flow through the portal vein probably
Step sections revealed that adventitial changes markedly soon after birth as a
fibroblasts were generally disposed as scat- result of the suppression of ductus venosus
tered and separate thin plates parallel to circulation. It is reasonable to presume
the intimal surface of the vessel and not that the postnatal establishment of an inelongated preferentially in either trans- nervated and vascularized media with two
verse or longitudinal directions. This ori- mutually perpendicular smooth muscle
entation was similar to that of mature zones is related to this transformation,
endothelial cells. There was an increase reflecting an adaptation to altered and spein all adventitial elements with age, partic- cial hemodynamic conditions. In the aorta,
ularly connective tissue fibers (fig. 7). Ad- the media is organized into successive, relaventitial collagen was increasingly oriented tively uniform distinct lamellar units, each
longitudinally. Myelinated nerve fibers, consisting of an elastin plate with a subjaeasily found in the adventitia of mature rat cent layer of circumferentially oriented
portal veins, were not observed in speci- smooth muscle cells and collagen and elasmens from animals up to six weeks of age. tin fibers (Wolinsky and Glagov, '67a).
The number of these lamellar units in
adults mammals is very nearly proportional
to aortic diameter or estimated medial tangential tension. The depth to which medial
vasa vasorum can be demonstrated in adult
mammalian aortas depends on the thickness of the media. Lamellar units outside
the inner 29 are always vascularized (Wolinsky and Glagov, '67b). Lamellar units
which accrue on the adventitial side after
birth apparently incorporate pre-existing
adventitial vessels. In the portal vein of the
rat, medial vasa are not present at birth
but appear in the outer layers of the growing outer muscular zone in association with
aligned capillaries at the medial-adventitial
border. It would appear that in the portal
vein, as in the aorta, medial vasa are
required only when the vessel has reached
some critical thickness. The relationship
between medial cell layers and medial
stress in the portal vein has not been studied in detail. However, regardless of
species, vessel diameter or the number of
smooth muscle cells in the portal vein
media, a characteristic two zoned structure prevails in mammals. In this respect,
it is reminiscent of intestinal architecture.
Whether the portal vein has an associated
propulsive, sphincteric or reservoir function analogous to that of the gut or any
other hollow viscus with innervated, organized and oriented smooth muscle zones,
remains to be proved; the anatomical evidence is quite suggestive.
Studies of the morphogenesis of vessels
and their components have been confined
to arteries and aortas (Karrer, '60, '61;
Pease and Paule, '60; Keech, '60; Haust.,
'65; Haust et al., '65; Cliff, '67; Jarmolych
et al., '68; KAdir et al., '69). Our data
support the notion that medial cells, indistinguishable from smooth muscle, probably elaborate medial fibrous proteins.
Increase in both collagen and elastin occurred within each medial smooth muscle
zone and in the intervening fibrous zone.
Prior to the establishment of the medial
zones at three days of age, medial cells
were globular and of uniform ultrastructural appearance. Presumably, both the
cells of the smooth muscle zones and the
fibroblasts of the fibrous layer were derived
from these original medial cells. In general, elastin production was relatively
46 1
greater than collagen production in the
postnatal period. This was particularly
true in the intermediate fibrous layer,
where fibroblasts were demonstrable. Difierential synthesis of these elements in the
human aorta during growth has been ascribed to the changes of medial tension
associated with rapid growth and rapidly
increasing flow after birth (Feldman and
Glagov, '71). A similar situation may prevail in the portal vein as flow increases
after birth (Lind, '63). The gradual replacement of endoplasmic reticulum by
myofibrils with age in smooth muscle cells
of the media suggests that fibrous protein
synthesis of each cell decreases steadily
after birth. The rapid proliferation of
medial cells after birth, however, probably
maintains total mEdial fibrous protein synthesis a t a relatively high level.
The potentialities of fetal medial cells
are further emphasized by the marked differences in ultrastructure which occurred
as the cells were segregated into internal
and external muscle zones. The greater
thickness and vascularity of the external
zone probably reflect its relatively greater
role in the coordinated function of the
media. The large numbers of mitochondria
and pinocytotic vesicles in its smooth
muscle cells indicate a high level of energy
transfer and therefore lend additional support to this concept.
The parallelism in orientation between
endothelial and adventitial cells is another
noteworthy feature of vascular morphogenesis. Endothelial cells eventually formed
an uninterrupted flattened intimal lining,
but were not preferentially elongated in
either circumferential or longitudinal directions. Even in the aorta, the longitudinal
dimensions of endothelial cells are only
slightly greater than the circumferential
(Poole et al., '58). Adventitial cells, though
not mutually attached and usually relatively distant from one another, were also
flattened and arranged as plates on cylinders concentric with the intimal surface
and without preferential elongation in
either circumferential or longitudinal directions. Thus, in relatively large vessels,
endothelial and adventitial cells, appear
to be distributed along the interfaces on
the inside and outside of the relatively
rigid muscular medial zones, making little
Karrer, H. E. 1960 Electron microscope study
of developing chick embryo aorta. J. Ultrastruct.
Res., 4: 420454.
Karrer, H. E., and J. Cox 1961 A n electron
microscopic study of the aorta in young and
aging mice. J. Ultrastruct. Res., 5: 1-27.
Cliff, W. J. 1967 The aortic tunica media in
Keech, M. K. 1960 Electron microscopic study
growing rat with the electron microscope. Lab.
of the normal rat aorta. J. Biophys. Biochem.
Invest., 17: 599-615.
Cytol., 7: 533-538.
Fahrenback, W. H.,L. B. Sandberg and E. G.
Cleary 1966 Ultrastructural studies of early Lind, J. 1963 Changes in the liver circulation
at birth. Ann. N. Y. Acad. Sci., 111: 110-120.
elastogenesis. Anat. Rec., 155: 563-575.
Pease, D. C., and W. J. Paule 1960 Electron
Feldman, S. A., and S. Glagov 1971 Transmicroscopy of elastic arteries. The thoracic
medial collagen and elastin gradients in human
aorta of rat. J. Ultrastruct. Res., 3: 469-483.
aortas. Atherosclerosis, 13: 385-394.
Poole, J. C. F., A. G. Sanders and H. W. Florey
Fyfe, F. W.,T. Gillman and I. B. Oneson 1968
1958 The regeneration of aortic endothelium.
A combined quantitative chemical, light and
J. Path. Bact., 75: 133-143.
electron microscopic study of aortic develop- Ross, R., and P. Borstein 1969 The elastic fiber.
ment i n normal and nitrile-treated mice. Ann.
I. The separation and partial characterization
N. Y.Acad. Sci., 147: 591-627.
of macromolecular components. J. Cell Biol.,
Haust, M. D. 1965 Fine fibrils of extracellular
40: 366-381.
space (microfibrils). Amer. J. Path., 47: Stehbens, W. H. 1966 The basal attachment
1113-1 137.
of endothelial cells. J. Ultrastruct. Res., 15:
Haust, M. D., R. H. More, S. A. Bencosme and
J. U. Balis 1965 Elastogenesis in human
Ts’ao, C. H., and S. Glagov 1970 Basal endoaorta. A n electron microscopic study. Expt.
thelial attachments. Tenacity a t cytoplasmic
Molec. Path., 4: 508-524.
dense zones i n the rabbit aorta. Lab. Invest.,
Jarmolych, J., A. S . Daoud, J. Landau, K. E.
23: 510-516.
Fritz and E. McElvene 1968 Aortic media
Ts’ao, C. H., S . Glagov and B. F. Kelsey 1970
implants. Cell proliferation and production of
Special structural features of the rat portal
mucopoly-saccharide, collagen and elastic tisvein. Anat. Rec., 266: 529-539.
sue. Expt. Molec. Path., 9: 171-188.
Wolinsky, H., and S. Glagov 1967a A lamellar
unit of aortic medial structure and function
Kid&, A., B. Veress and H. Jellinek 1969 Rein mammals. Circulation Res., 20: 99-111.
tionship of elastic fiber production with smooth
1967b Nature of species differences in
muscle cells and pulsation effect i n large vessels. Acta Morph. Acad. Sci. Hung., 17:
the medial distribution of aortic vasa vasorum
i n mammals. Circulation Res., 20: 409-421.
direct contribution to the development of
tension in the wall.
Wall of rat portal veins during early postnatal differentiation. At
birth (fig. l A ) , there is a fairly uniform cell population. At three days
(fig. l B ) , inner (S) and outer ( T ) smooth muscle zones are evident,
but there is little evidence for either circumferential or longitudinal
orientation of the component cells. Ten days after birth (fig. l C ) , the
inner circumferential ( S ) , and outer longitudinal ( T ) smooth muscle
zones and the intermediate fibrous zone ( F ) are well established.
Magnifications: figure l A , x 380; figure lB, x 415; figure lC, X 240.
Electron micrograph of the portal vein of a newborn rat. Endothelial
cells ( E ) have numerous cytoplasmic projections ( X ) which extend
into the lumen ( L ) and downward into the media and often interdigitate loosely with similar projections from the underlying globular
medial cells ( M ) . Distances from endothelial to medial cells and
between adjacent medial cells are similar and there is no apparent
special subendothelial demarcation between intima and media. There
are no endothelial or smooth muscle basement membranes. Narrow
groups of niyofilainents (arrows) are present in some medial cells
but are confined to the periphery. Cells also contain glycogen ( G )
and ribosomes ( R ) . Extracellular filaments in thc media, include
small clusters of collagen fibers ( C ) . x 5000.
C.-H. Ts’ao, S. Glagov and B. F. Kelsey
3 Portal vein of three day old rat. Medial cells are clearly segregated
into two smooth muscle zones separated by a fibrous zone (F). Cells
of the outer muscle zone CT) are larger than those of the inner
zone (S), but there is as yet no definite predominent orientation of
cells. As myofilaments increase in number with age, endoplasmic
reticulum and ribosomes decrease. These changes are most apparent
in the outer zone. Endothelial cells (E) still resemble inner zone
medial cells. Adventitial cells ( A ) are flattened. x 2900.
At ten days, cells of the inner (S) and outer ( T ) medial zones of the
rat portal vein are mutually perpendicular, the cells of the thicker
outer zone are longitudinal; those of the inner zone are circumferential. Connective tissue fibers in both the intermediate fibrous layer ( F )
and the adventitia ( A ) are much more abundant than at three days.
X 4300.
C.-H. Ts’ao, S. Glagov and B. F. Kelsey
5 Medial smooth muscle of portal vein five days after birth. Myofibrils
( m f ) and dense bodies ( d ) occupy much of cytoplasm of most cells.
Endoplasmic reticulum ( e r ) is still prominent. Distinct basement
membranes are infrequent. x 14,500, insert x 33,000.
Smooth muscle cells of the outer longitudinal medial zone at 18 days.
Mitochondria (arrows) are numerous and scattered throughout the
cytoplasm of the cells. Marginal pinocytotic vesicles are abundant
and basement membranes, though incomplete, are evident. Insert: An
enlargement of the area marked on the figure l o show the pinocytotic
vesicles ( P ) and the basement membranes (B). x 11,000; insert
X 21,500.
C.-H.Ts’ao, S. Glagov and B. F. Kelsey
Distribution of vasa vasorum i n portal vein of a 28 day old rat.
Capillaries (Va) i n the adventitia (A) are aligned along the outer
limit of the external longitudinal medial zone ( T ) . Other vessels (Vm)
are present within the external muscle zone. No vessels were found
i n the inner muscle zone ( S ) or the intermediate fibrous layer ( F ) .
>: 2900.
Endothelial-medial interface of rat portal vein. When first apparent
at three days (fig. S A ) , the subendothelial space ( S S ) contains fine
microfilaments and amorphous material (arrow) but very little collagen or elastin. Endothelial cells ( E ) are on the left inner medial
cells ( S ) on the right. At 21 days (fig. 8B), a well defined zone containing the internal clastin lamina ( E l ) separates the endothelium
( E ) from the inner media ( S ) . Figure 8A, x 10,500; fig. 8B, x 34,000.
C.-H. Ts’ao, S . Glagov and B. F. Kelsey
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