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On the granule-containing cells in the aortic wall of the young chick.

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On the Granule-containing Cells in the Aortic Wall
of the Young Chick
Department of A n a t o m y , T h e Tohoku University School of Medicine,
Sendai, J a p a n
The granule-containing cells in the wall of the arch of the aorta
proximal to its union with the ductus arteriosus in young chicks were examined
by electron microscopy. These cells contain many electron-opaque vesicles, about
1500 A in a n average diameter, and show basically similar cytological characteristics to the granule-containing cells described in autonomic ganglia, around the
abdominal aorta, and in the carotid body of mammals. Occasionally the cells are
in close apposition to smooth muscle cells, fibroblasts or endothelial cells. Other
surfaces of the granule-containing cells are partly covered by satellite cells. Synapses are rarely found on the granule-containing cells in the tunica media of
the aorta.
A few bundles of elongated cells which enclose several nerve fibers in the
cytoplasm penetrate perpendicularly into the tunica media of the aorta. In these
bundles are also a few granule-containing cells. Three types of nerve endings
terminate on the granule-containing cells; that is, endings with small clear synaptic vesicles, with small cored vesicles, and with large cored vesicles. In addition, presumptive afferent nerve endings are found in the bundles. They show
a variable diameter along their tortuous courses and contain numerous mitochondria.
The existence of specialized epithelioid
cells within the wall of the arch of the
aorta hats been reported by several authors
under various names, such as paraganglion, aortic body, encapsulated nerve ending (Palme, '34; Noaidez, '35; Muratori,
'62). Nonidez recognized them to be similar in structure to the parenchymal cells
of the camtid body and regarded the clustered areas of these cells as a sensory
nerve ending. Recently, using a fluorescent
microscopic technique, Bennett ('71 ) revealed the presence of specially fluorescent
cells within the wall of the arch of the
aorta of the chick and concluded that the)=
cells corresponded to the epithelioid cells
described previously. However, no reports
on their ultrastruoture have so far been
In the present paper, an ultrastructural
observation of these epithelioid cells was
Tissues from eight male white leghorn
chicks, two days to five weeks old, were
ANAT. REC., 178: 253-266.
examined. Under ether anesthesia, parts
of the arch of the aorta proximal to its
union with the ductus arteriosus were
excised, immersed for 1.5 hours in 3%
glutaraldehyde buffered at pH 7.4 with
sodium cacodylate, and postfixed in 1%
osmium tetroxide buffered at pH 7.4 with
sodium cacodylate for two hours. The fixed
blocks were finally tre'ated for 30 minutes
with 1% uranyl acetate solution. After
dehydration in a graded series of ethanol,
the blocks were embedded in Epon 812
(Luft, '61). Ultrathin sections were stained
with uranyl acetate and lead citrate (Reynolds, '63), and examined with a Hitachi
HS-7 electron microscope.
Light microscopy
The aortic wall in young chicks is mainly
composed of an endothelium and a tunica
media. The latter consists of cell layers
alternating with layers of elastic laminae
and gradually merges into a loosely built
Received April 4, '73. Accepted Aug. 13, '73.
adventitia. The epithelioid cells in question
are about 15 in diameter and characterized by the clear cytoplasm and an oval
nucleus which is poor in chromatin. They
are scattered singly or in groups, frequently interrupting the stratiform structure ,of the media (fig. I, cf. with fig. 2).
Some of them are seen to reach the subendothelial layer. At several points of the
aortic wall, bundles of elongated cells, 50
in width, penetrate perpendicularly into
the outer half of the media of the aorta.
Fig. 1 A light micrograph of a toluidine bluestained transverse section through the arch of
the aorta of the young chick. The epithelioid cells
(arrows) interrupt the stratiform structure of the
media. A bundle ( B ) of elongated cells penetrates
perpendicularly into the outer half of the media.
L, lumen of the arch of the aorta. X 540.
Electron microscopy
The epithelioid cells appear as granulecontaining cells easily discernible from
other cellular elements of the media, such
as smooth muscle cells and fibroblasts
(fig. 2). They have an irregular outline
and an oval nucleus with a single nucleolus. The cytoplasm contains numerous
rounded vesicles, 900 to 3000 A in diamelter (1500 A average), with an electron
opaque core (fig. 3 ) . The cores are spherical in most vesicles but sometimes may
show a crescent or dumbbell shape. They
are separated from the vesicular membrane by a narrow light halo. These granulated vesiclels are usually widely distributed
throughout the cytoplasm and sometimes
crowded at one pole of the cell.
In addition to the granulated vesicles,
the cells contain numerous free ribosomes
and a few cisternae of the granular endoplasmic reticulum. Spheroid and elongated
mitochondria are dispersed in the cytoplasm. Centrioles and cilia are located near
the nucleus. Filaments and microtubules
are also contained throughout the cytoplasm. Colgi complexeis are seen around
the nucleus (fig. 5 ) . In the Golgi areas
small dense granulated vesicles seem to
represent an early stage in the formation
of the granulated vesicles noted above.
Mitotic granule-containing cells are rarely
A narrow gap of about 150 A in width
occurs between apposed granule-containing cells. No specializations of cell membranes are found at these places. On other
surfaces, the granule-containing cells are
surrounded by either satellite cell cyboplasm or are separated from the intercellular space by a basal lamina continuous
onto the outer surface of the satellite cell
(fig, 3 ) . Such exposed areas of the cell
surfaces become greater in the cells situated nearer to the tunica intima. Beneath
the intima, the granule-containing cells
are almost entirely denuded of satellite
cells (fig. 4).
In some places where the internal elastic
lamina is interrupted, the granule-containing cells occur in direct contact with the
endothelial cells over the distance of 0.55 (fig. 4). A basal lamina separates the
granule-containing cells from the endothelial cells. Occasionally the intercellular
space between the endothelial cell and the
granule-containing cell is reduced to a gap
of about 50 A (fig. 4, inset). In such places
of direct apposition, the cytoplasm of the
Fig. 2 Electron micrograph showing the stratified structure of the media which consists of cell
layers alternating with intercellular layers. The granule-containing cells ( G ) are seen in the cell
layer. EL, elastic lamina; F, fibroblast; SM, immature smooth muscle cell. X 6,000.
endothelial cells becomes very thin, 7001500 A. No structural specializations of
the apposed cell membranes are found including attachment devices or pinocytotic
Some of the granule-containing cells are
closely apposed to adjacent smooth muscle cells or fibroblasts. At the closest apposition, the intercellular gap may be as narrow as 50 A and lack the basal lamina.
Mitotic satellite cells are rarely found.
A few nerve fibers and axon terminals
with clear synaptic vesicles protrude into
the satellite cells, forming mesaxons (fig.
5). In addition, small round profiles of
nerve fibers with only several neurotubules
are found to be in contact with the granule-containing cells. However, typical synaptic contacts between these axons and
the granule-containing cells are hardly
seen. It is likely that these granule-containing cells lack a functional innervation.
A large amount of elastic and collagen
fibers occupy the intercellular spaces of
the media. The elastic fibers tend to form
continuous laminae. No vasa vasorum are
seen in the media.
As revealed by light microscopy, bundles
of elongated cells penetrate perpendicularly into the outer zone of the media of
the aorta. These elongated cells are 5-15
in diameter. They have a slender nucleus
with dispersed clumps of chromatin and a
single nucleolus. The perikaryal cytoplasm
contains numerous free ribosomes and
cisternae of the granular endoplasmic
reticulum (fig. 6 ) . Centrioles are located
near the nucleus. Microtubules and filaments are rich in the cytoplasm but other
organelles, such as mitochondria, agranular endoplasmic reticulum and Golgi complexes, are not developed well. Outer surfaces of the cell are lined with thin basal
laminae. The elongated cells are rather
closely aggregated with intervening narrow
intercellular spaces which conltain abundant collagen fibers. At the periphery of
the bundle, the cells are scatteTed separately and intermingled with otheT cellular
elements of the media.
The elongated cells usually ensheath a
few axons by forming mesaxons (fig. 6 ) .
Some of these axons terminate with a narrow cleft of about 150 A, on the granulecontaining cells which are sparsely distributed within the bundles. No membrane
specializations are seen on these apposed
surfaces. Accmding to the population of
the synaptic vesicles, these endings can
be divided into three types. The commonest endings contain a crowd of clear synaptic vesicles, about 500 A in diameter,
and a few cored vesicles, about 1000 A in
diameter (fig. 7). Other nerve endings contain numerous small cored vesicles, about
500 A in diameter, mixed with a few clear
vesicles of similar size and some large
cored vesicles (fig. 8). The third type of
ending is characterized by dense accumulation of large cored vesicles, 900 A in
diameter (fig. 8). The electron opaque
cores of the vesicles are homogen(eous in
density and are separated by a light halo
from the vesicular membrane. The density
of the cores is a little lower than that of the
vesicles of the granule-containing cells.
Some of the endings are partly covered by
the cytoplasm of the elongated cells; the
other wide area of surface being sleparated
from the intercellular space only by a
basal lamina which passes from the elongated cells onto the ending. A direct contact between the third type of ending and
the granule-conltaadning cell in the bundle
is occasionally observed.
In addition to these nervous components
described above, there are found peculiar
axon profiles (fig. 9 ) . Thetse show a variable diameter along their tortuous courses
and contain numerous mitochondria besides neurofdaments d neurotubules.
They are completely enclosed by Schwann
cells and never appose to the granule-containing cells nor are exposed to the intercellular space.
The granule-containing cells observed in
the present study show fundamentally
similar cytoplasmic characteristics to the
granule-oontaining cells reported in various
parts of the vertebrate body in association
with the autonomic nervous system; such
as in the autonomic ganglia (Elfvin, '68;
Siegrist et al., '68; Matthews and Raisman,
'69; Williams and Palay, '69; Watanabe,
'71), around the abdominal aorta (Coupland and Weakley, '70; Hervonen, "7l),
and in the carotid body (Lever et al., '59;
Biscoe and Stehbens, '66; Kondo, '71). As
discussed recently by Bennett ( ' T l ) , all of
these granule-containing cells may be
variations of a basically single element
which probably corresponds to the "Paraganglion" proposed by Kohn ('03).
The present fine struotural findings suggest a secretory function for the granulecontaining cells. Bennett ('71) noted that
the granule-containing cells in the aortic
wall of the chick showed positive reaction
to the Falck-Hillarp's technique (Falck and
Owman, '65) for demonstration of biogenic
amineis. If the granule-containing cells
secrete any bioactive substances including
amines, these secretory substances may
have either local or blood-borne distant
effect. In consideration of this problem, it
should be noted that no capillaries have
belen found around the granule-containing
cells in the aortic wall and that the granulecontaining cells are frequently located in
a close topographical apposition to smooth
muscle cells and fibroblasts. Such a close
interrelation may suggest that the granulecontaining cells have a direct effect on
theee cells.
The elongated cells constituting the bundles can be identified as Schwann cells by
the following features : They ensheath one
or a few profiles of axons, the nucleus is
relatively dark and characterized by dispersed clumps of chromatin, and the cytoplasm contains numerous filaments and
microtubules and is relatively scarce of
cell organelles. The bundles may be regarded as the extrinsic nerves of the aorta.
The granule-containing cells found in
the bundles receive a rich innervation. In
birds, as in mammals, the existence of
adrenergic and cholinergic components
in the aueonomic nervous system is suggested physiologically and pharmacologically (Burnstock, '69). Since the nerve endings with a crowd of small clear and a
few large cored synaptic vesicles are generally considered to be cholinergic efferent,
and those with small cored and clear
synaptic vesicleis to be adrenergic efferent
(De Robertis et al., '63; Richardson, '64;
Van Orden et al., '66; Farrell, '68; Tranzer
and Thoenen, '68; Watanabe, '71), the
present study strongly suggests that the
granule-containing cells in the bundles
relceive both adrenergic and cholinergic
innervation. Bennett ('71 ) demonstrated
the preseance of adrenergic nerves in the
chick aorta by fluorescenit microscopy.
The nerve endings containing a dense
accumulation of large cored vesicles found
in the present study probably correspond
to the adrenergic nerve endings described
by Burnstock and Iwayama ('71). This
study has failed to clarify, however,
whether the endings with a dense accumulation of large cored vesicles and those with
small cored and clear vesicles belong to
different axons from separate neurons.
The peculiar axon profiles with a crowd
of mitochondria found in the bundles
closely resemble in structure the nerve profiles in the wall of the carotid sinus (Rees,
'67). It has often been described that the
sensory nerve endings contain a dense
accumulation of mitochondria (Cauna and
Ross, '60; Munger, '65; Yamamoto, '66).
Moreover, the portion of the aorta examined in the present study is supplied by
the depressor nerve (Nonidez, '35). So,
these a x m profiles possibly represent the
baroreceptive nerve endings in this area.
Bock et al. ('70) and K m d o ('71) pointed
out that the coexistence of both granulecontaining cells and presumptive sensory
nerve endings might be the structural
basis for the chemoseceptive mechanism
of the carotid body. It is possible that the
peculiar axons described here have a
chemoreceptive function in the chick aorta.
I wish to thank Prof. Toshi Yuki Yamamot0 for his encouragement and suggestion throughout this study.
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3 A nest of the granulecontaining cells ( G ) and the satellite cells (S).
The rounded vesicles of the granulecontaining cell vary in diameter
and contain cores of homogeneous electron density. However, some
of them may show a fine granular appearance. The greater part of
the surface of the granulecontaining cell is separated from the intercellular space only by a basal lamina (arrows). co, collagen fibers; el,
elastic fiber. x 11,000.
Hisatake Kondo
4 The granule-containing cells ( G ) are in direct contact with the endothelial cells ( E ) . The inset represents a higher magnification of the
contact area. The gap between the endothelial cell and the granulecontaining cell is only 50 A. No membrane specializations are found
i n this area. EL, elastic lamina; L, lumen of the arch of the aorta;
S, satellite cell. x 10,000. Inset, X 100,000.
Two nerve fibers ( n ) with several neurotubules are enveloped by a
satellite cell ( S ) forming a mesaxon. Go, Golgi complex of the
granule-containing cell. X 25,000.
Hisatake Kondo
A bundle of elongated cells (Lo) penetrating the media of the aorta.
These cells are closely aggregated and their cytoplasm is rich in
microtubules and filaments and ensheaths several nerve fibers ( n ) .
x 20,000.
A nerve ending (ChE) in direct contact with a granule-containing
cell ( G ) in the bundle. The ending contains a crowd of clear synaptic
vesicles and a few large cored vesicles, together with mitochondria.
The synaptic membranes show n o density increases. Lo, elongated
cells. x 30,000.
Hisatake Kondo
Nerve endings containing many small cored vesicles (AdE) and a
crowd of large cored vesicles (gE). The former shows direct contact
with the granule-containing cell ( G ) in the bundle. Note the differences in size and density of the cores of vesicles found in these t w o
types of endings and in the granulecontaining cell. X 35,000.
Peculiar axon profiles ( N ) found in the bundle of elongated cells.
They have a variable diameter along their tortuous course and contain numerous mitochondria. X 10,000.
Hisatake Kondo
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young, containing, wall, granules, chick, aortic, cells
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