Cell Tissue Res (1992) 270:165-172 Cell &Tissue

Research �9 Springer-Verlag 1992

The cytoarchitecture of the medial layer in rat thoracic aorta: a scanning electron-microscopic study Takashi Fujiwara 1 and Yasuo Uehara a

1 Laboratory Animal Center, School of Medicine, Ehime University, Shigenobu, Ehime, 791-02, Japan 2 Department of Anatomy, Kumamoto University, Medical School, Kumamoto, 860, Japan

Received December 12, 1991 / Accepted June 17, 1992

Summary. The cytoarchitecture of the medial layer of rat thoracic aorta was examined by scanning electron microscopy after removal of the connective tissue. The outermost lamella showed a lattice-like structure of mus- cle bundles of closely apposed smooth muscle cells (SMCs), whereas the inner lamellae consisted of more- or-less continuous muscle sheets of vaguely defined sub- groups of parallel SMCs. Longitudinal rows of ridges ran along the adventitial surface of these muscle bundles and sheets. The SMCs of the outermost lamella, were 5.1 gm wide, and varied in shape, whereas those of the inner lamellae, were 52.7 gm long, 2.6 gm wide and 4.1 gm thick, and were elongated, spindle-shaped cells with serrated outlines. These latter SMCs extended obliquely, and partially overlapped each other. The sur- face of the SMCs in the outermost lamella exhibited a rugged texture, with nodular protrusions and oblique and longitudinal laminar folds, while the inner lamellar cells showed longitudinal laminar folds and finger-like processes on both sides of the ridges, pointing in oppo- site directions to the ridges. The angle of deviation from the transverse axis of the vessel, of the muscle bundles and subgroups in the outermost lamella, was 33.6 ~ in the second and third lamellae, 22.5 ~ and in the inner- most lamellae, 12.8 ~ The mean angle of the muscle bun- dle and subgroup arrangement, with respect to the long axis of the vessel, however, was basically 90 ~ in all lamel- lae.

Key words: Thoracic aorta - Smooth muscle cell - Cy- toarchitecture - Morphometry - Rat (Wistar)

The aortic media consists of a series of concentric lamel- lar units comprising the basic structural elements of the aortic wall (Wolinsky and Glagov 1967). The shape and

Correspondence to: T. Fujiwara

arrangement of the smooth muscle cells (SMCs) in this layer, have been the subject of numerous transmission electron-microscopic studies in a variety of species, e.g., rat (Keech 1960; Pease and Paule 1960; Paule 1963; Cliff 1970; Gerrity and Cliff/975; Osborne-Pellegrin 1978; Wasano and Yamamoto 1983; Berry and Sosa-Melgare- jo 1989; Sosa-Melgarejo and Berry 1989), rabbit (Bier- ring and Kobayasi 1963; Wolinsky and Glagov 1964; Clark and Glagov 1979), mouse (Karrer 1961), pig (Clark and Glagov 1979), bird (Karrer 1960; Moss and Benditt 1970; Berry et al. 1974), and human (Dingemans et al. 1981), and the data have been comprehensively reviewed by Rhodin (1980) and Gabella (1983). The re- sults of such studies have proved to be contradictory. For example, cross-sectioned SMCs have been described as possessing branching processes (Cliff/967, 1970) or multiform laminar projections (Dingemans et al. 1981), and in longitudinal section, as having a serrated outline in the human aorta (Dingemans et al. 1981) or a smooth, regular and relatively straight, outline in rabbits and pigs (Clark and Glagov 1979). Again, SMCs have been re- ported to span (Pease and Paule 1960; Bierring and Ko- bayasi 1963), or not to span (Clark and Glagov 1985), the space between neighboring elastic lamellae, and to lie with their longitudinal axis parallel (Clark and Gla- gov 1985), or not parallel, to the surface of the vessel (Keech 1960; Pease and Paule 1960; Cliff 1970), and to be arranged in a circular (Wolinsky and Glagov 1964; Hansen et al. 1980; Arner and Uvelius 1982; Gabella 1983), oblique (Keech 1960; Bierring and Kobayasi 1963; Wolinsky and Glagov 1964; Cliff 1967), spiral (Pease and Paule 1960), or helical orientation (Wolinsky and Glagov 1964; Osborne-Pellegrin 1978; Gabella 1983).

The present study examines the medial layer cytoar- chitecture of the rat thoracic aorta, and a morphometric analysis has been made of the medial SMCs by scanning electron microscopy, after the selective removal of the adventitial and medial connective tissue by HC1 hydroly- sis (Fujiwara and Uehara 1982; Uehara and Fujiwara 1984; Fujiwara and Iijima/990; Uehara et al. 1990).

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Materials and methods

Twenty male adult Wistar rats (270-440 g) were housed with free access to food and water in the Laboratory Animal Center of Ehime University School of Medicine. They were sacrificed using an overdose of ether. The thoracic aorta was fixed in situ by open- ing up the thoracic cavity and flooding it with phosphate buffered (pH 7.3) 3% glutaraldehyde, at room temperature, for more than 4 h. A 3 cm length of aorta (extending approximately from the 3rd to 12th thoracic vertebra) was excised, and cut into small pieces (5-7 mm in length), washed in distilled water, and then post-fixed with 2% OsO4, at 4 ~ C, for 2 h. After a brief rinse in distilled water, specimens were placed in 8N HC1 for 25-30 min, at 60 ~ C (to remove the connective tissue), washed in distilled water, dehy- drated in a graded series of ethanols, immersed in isoamyl acetate, and critical-point dried with CO2. They were sputtercoated with platinum and examined in a Hitachi S-500A scanning electron mi- croscope.

Scanning electron micrographs were taken from the adventitial surface of the vessel and enlarged to a final magnification of x 3000-x 6000 in order to measure the width perpendicular to the long axis of medial SMCs. In order to measure cell-length and thickness in the radial direction (Hua and Cragg 1980), micro- graphs (magnification: x 3000- x 7000) of the lateral side were tak- en by tilting and rotating the specimen stage.

The angle of alignment of muscle bundles and muscle sub- groups with respect to the long axis of the vessel, and the angle of their deviation from the transverse axis, were both measured on micrographs (magnification: x 150-x 250), and measurements of both angles were determined in relation to the center line of the vessel drawn parallel to the long axis of the vessel (Fig. 1). Angles of all muscle bundles and muscle subgroups situated on the center line were measured.

Results

the muscle lamellae had, in some cases, produced a par- tial or total exfoliation of the outer muscle lamellae, exposing the inner muscle lamellae. Thus, almost all the muscle lamellae of the aortic medial layer could be exam- ined from the adventitial side. The medial layer was seen to consist of 7-9 concentric SMC lamellae, with the out- ermost and inner lamellae demonstrating differences in their overall structure and organization of their cellular components.

Outermost lamella

The outermost lamella was seen to consist of irregularly oriented SMC bundles (Fig. 2a). Most muscle bundles appeared to connect with, overlaped, or crossed over each other, forming a lattice-like structure with the per- forations showing variations in shape and size (Fig. 2 b). Some muscle bundles, however, ended freely, and ap- peared unattached to any other bundle. The muscle bun- dles varied in length, from less than 20 gm to more than 150 gm, and in width, from about 3 gm to more than 100 gm. Generally, the inner bundles were broader.

Although the outline of individual muscle cells was not usually discernible, because of the close apposition of adjacent cells with few intercellular clefts, some could be observed, and were seen to vary in shape (Fig. 2c). The adventitial surface of SMCs exhibited a rough tex- ture with numerous nodular protrusions (less than 3 gm in diameter), and laminar folds which were obliquely, or longitudinally, oriented to the cell's long axis.

Removal of the adventitial and medial connective tissue with HC1, produced no apparent adverse effects on me- dial SMCs and allowed direct observation of the adven- titial aspect of the medial layer. The removal of connec- tive tissue from between the SMCs and from between

B

A < a 2 ~

Fig. 1. Diagram showing measurements taken of muscle orienta- tion. The rectangle represents a segment of the aorta from the adventitial aspect. A Transverse axis of the vessel, B longitudinal axis of the vessel, al and a2 angle of deviation from transverse axis, bl and b2 angle of deviation from the longitudinal axis of the vessel

Inner lamellae

The inner muscle lamellae consisted of broader, more- or-less continuous SMC sheets, which, in the second la- mella, were sometimes continuous with the muscle bun- dles of the outermost lamella, and, in some places, with the muscle sheets of the third lamella, which, in turn, were continuous with those of the fourth larnella (Fig- s. 3 a, b). Thus, all the lamellae were interconnected to adjacent lamellae, from the outermost to the innermost lamella, rather in the manner of "pas t ry" layers than in the manner of " o n i o n " layers (Dingemans et al, 1981 ; Clark and Glagov 1985).

In and between these muscle sheets were perforations of various diameters. SMCs of the inner muscle lamellae were practically transversely oriented (Fig. 3 b), except for the quite rare occurrence of longitudinally running muscle cells in the innermost lamella. Vaguely-defined subgroups, composed of parallel arrays of muscle cells, were discernible, and their disposition varied in the dif- ferent muscle lamellae and in different areas of the same muscle sheet (Fig. 3 c).

Within a muscle sheet, the individual SMC was an elongate, spindle-shaped structure, and was flattened in the axial direction of the vessel. The adventitial cell sur- face showed an extremely rugged texture with numerous longitudinal laminar folds, approximately 0.3 gm in thickness and less than 15 gm in length. There were also

Fig. 2a-e. SEM image of the outermost lamella of the thoracic aortic media, a The lamella has a lattice-like structure. IL inner lamella, OL outermost lamella, A adventitia. • 95. Bar: 200 gm. b The longitudinal axis of the aorta is from left to right. Note irregularly oriented SMC bundles in the lattice network. Connec- tions between the bundles, and between bundles and the underlying

lamellae are clearly observable in some places. Arrows indicate ridges and gutters more or less longitudinally oriented with respect to the long axis of the vessel, x 240. Bar: 50 gm. e SMC bundles at a higher magnification. The bundles consist of closely apposed SMCs. The adventitial surface exhibits a rough texture with nodu- lar protrusions (NP) and laminar folds (L/;). x 1500. Bar: 10 gm

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Fig. 3a-d. Inner lamellae showing broader, more-or-tess continu- ous sheet-like structures, a Second and third lamellae. Smooth mus- cle subgroups are somewhat irregularly arranged. Ridges and gut- ters (arrow) wind longitudinally with respect to the long axis of the vessel, x 80 gm. Bar: 200 I~m. b Inner lamellae beyond the 4th lamella. SMCs are basically transversely oriented. Arrow shows longitudinal ridges and gutters, x 70. Bar: 200 gm. e SMC sheet. Vaguely-defined subgroups are shown by the dotted lines in upper

right of the figure. The orientation of subgroups varies in different areas of the same muscle sheet, x 270. Bar: 50 gm. d SMCs at a high magnification. The adventitial surface of the muscle cells shows a highly rugged texture with numerous longitudinal laminar folds (LF). Finger-like processes (FP) project from one end of some laminar folds. The neighboring SMCs are connected by many cellu- lar processes (arrow). x 3000. Bar: 5 gm

finger-like processes, abou t 0.3 lam in thickness and less t han 1 gm in length, project ing f rom one end of some l amina r folds (Fig. 3 d). The lateral profile of the muscle cell, was an elongated, sp indle-shaped body with a ser-

rated out l ine (Fig. 4). Adjacen t muscle cells were sepa- rated by relatively wide intercel lular clefts (less than 2 gm), t raversed by n u m e r o u s fine cellular processes in- ter.connecting ne ighbor ing SMCs.

Fig. 4. A lateral view of SMCs. The SMCs are elongated spindle- shaped with serrated outlines and overlap each other. All longitudi- nal laminar folds and finger-like processes observed here on the surface of the SMCs are pointing in the same direction, exhibiting a " tha tched roof" appearance. x 1900. Bar: 10 ~m

Fig. 5. Parallel rows of alternating ridges and gutters run horizontal- ly in the middle part of the fig- ure. The ridges consist of cell protuberances aligned in register. Each protuberance is composed of an aggregation of nodular pro- trusions. The longitudinal laminar folds and the finger-like processes are pointing in the opposite direc- tion to the rows of ridges; those in the upper one third of the fig- ure point to upwards and those in the lower one third to down- wards, x 2900. Bar: 5 ~m

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Fig. 6. An oblique view of ridges and gutters. The longitudinal la- minar folds and the finger-like processes are pointing in the op- posite direction to the ridges. The laminar folds and the finger-like processes on the left side of the figure point to the left and those on the right side point to the right, x 3600. Bar: 5 gm

Longitudinal ridges and cell orientation

The adventitial surface of SMC bundles of the outermost lamella and SMC sheets of the inner lamellae, were, in some areas, covered by rows of alternating ridges and gutters running longitudinally to the long axis of the aorta (Figs. 2b; 3a, b). The ridges were under 400 gm in length, 1-4 I-tm in thickness, and 0 . 5 4 gm in height. The ridge-to-ridge distance varied between 2 12 gm (Fig. 5). The ridges consisted of cell protuberances pro- jecting toward the adventitial surface, the protuberances of adjacent cells being aligned so as to form a ridge on the surface of the lamella. Each protuberance was an aggregation of smaller nodular protrusions less than 3 gm wide. The longitudinal laminar folds, and the finger-like processes of the SMCs, were directed away from the rows of ridges (Fig. 6).

The SMCs were not exactly parallel, but rather ex- tended obliquely to the surface of each muscle lamellal Thus, when viewed laterally, the end of the cell nearest to a ridge faced the inner (luminal) side of the muscle lamella, whereas the cell-end farthest from the ridge ter- minated near the adluminal surface (Fig. 4). The SMCs overlapped each other for about a quarter to one third of their length, exhibiting a " thatched roof" appearance as a whole. Though SMC sheets were generally only one cell-layer thick, at the areas of overlapping, the sheets were two cell-layers thick.

Morphometry

In the outermost lamella, the component SMCs were twice as wide as those of the innermost lamellae (Ta- ble 1). In the innermost lamellae, the value of cell-thick- ness was greater than that of the cell width, thus con- firming the visual evidence of a flattening of the cells.

The mean angle of deviation from the transverse ori- entation of the muscle bundles of the outermost lamella, was 33.6 ~ (range 0~176 and this decreased progressive- ly in the muscle subgroups of concentric muscle sheets, moving in a luminal direction, to a mean of 12.8 ~ (range 0~ ~ in the innermost lamellae (Table 2). Although the angle of deviation from the transverse orientation

Table 1. Morphometric measurements of medial smooth muscle cells from thoracic aorta of the rat. Values are mean_ S.E.

Lamella* Cell length Cell width Cell thickness (gm) (gm) (gin)

ist - 5.1 • - (n = 33)

4th onwards 52.7+1.7 2.6+_0.1 4.1 _+0.1 (n = 38) (n = 35) (n = 28)

* Lamellae are counted from the adventitial surface of the media towards the vessel lumen

Table 2. Morphometric measurements of smooth muscle bundle and subgroup orientation in the medial lamellae. Values are mean • S.E.

Lamella* Angle of deviation Angle with respect from transverse to the long axis orientation of the vessel

1st 33.6 _ 1.8 (n = 170) 87.4_+ 3.0 (n = 170) 2nd-3rd 22.5 • 1.3 (n = 140) 90.0 • 2.3 (n = 140) 4th onwards 12.8 + 1.0 (n = 120) 88.1 + 1.5 (n = 120)

* Lamellae are counted from the adventitial surface of the media towards the vessel lumen

differed from one lamella to the next, the muscle bundles and muscle subgroups showed closely similar mean angle values of approximately 90 ~ to the long axis of the vessel.

Discussion

The thoracic aorta has long been considered to be a passive, elastic tube, periodically distended by pulsatile pressure (Hamilton and Dow 1939), thus discounting a possible functional role for its smooth muscle cells. Contractions of aortic SMCs, however, have been re- corded following stimulation by various neural and hor- monal agents (Altura and Altura 1974; Deth and van Breemen 1977), and direct stimulation by the sympathet- ic nerves (Gerovfi et al. 1973). The morphology of this

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vessel, however, still remains a subject of much discus- sion.

The present study of the cytoarchitecture of the me- dial layer of rat thoracic aorta, demonstrates that it con- sists of an outer sheath of a lattice-like lamella composed of discrete subgroups of SMC bundles, and inner con- centric layers of sheet-like lamellae composed of vaguely defined subgroups of commonly aligned SMCs. A struc- ture, therefore, markedly different from other arterial vessels. Vaguely defined subgroups packed side by side into a muscle sheet, have previously been reported in a light-microscopic study (Clark and Glagov 1985).

The lattice-like structure of the peripheral medial la- mella, appears to be a characteristic feature of rat thor- acic aorta. The few papers that have so far appeared on the medial structure of arterial vessels, describe longi- tudinally oriented SMC bundles on the adventitial sur- face in monkey mesenteric vessels (Fujiwara and Uehara 1982), and dog brain vessels (Shiraishi et al. 1986), but make no reference to a lattice-like structure. Longitudin- ally oriented SMCs on the adventitial surface, have also been reported for aortic media (Schmid et al. 1982; Wa- sano and Yamamoto 1983; Clark and Glagov 1985), and for the media of the renal artery (Osborne-Pellegrin 1978), and coronary artery (Yohro and Burnstock 1973).

Since longitudinally arranged SMCs occur most fre- quently at vessel branching sites, they may have devel- oped in these locations in response to the increased stress that such sites are subjected to; serving to strengthen the arterial wall (Yohro and Burnstock 1973; Osborne- Pellegrin 1978) and contribute to the regulation of blood flow (Yohro and Burnstock 1973). SMCs found at the non-branching sites may also support the arterial wall against longitudinal stress parallel to the muscles (Ball e ta l . 1963; Shiraishi et al. 1986). In this context, the lattice-like muscle bundle network of the peripheral la- mella, may act to resist stress parallel to each muscle bundle. In addition, it may also be exposed to a tensile stress from the muscle sheets of the inner lamellae, with which it is interconnected, distributing it throughout the lattice of muscle bundles. Such a role may, however, be a minor one, since the muscle bundles seem to be too thin and loosely connected, to endure the stress re- sulting from a high systolic blood pressure. The outer lamellar lattice may thus have some other, but as yet unknown, physiological role.

The longitudinal ridges and gutters on the adventitial surface of the lamellae are prominent structures, similar to those found on the luminal surface of the innermost medial muscle cells of the mesenteric arteries (Fujiwara and Uehara 1982), and outer medial surface of the mes- enteric arteries constricted with norepinephrine (Uehara and Fujiwara 1984). They differ, however, from the lon- gitudinal ridges and gutters of the rat thoracic aorta in that the ridges and gutters of the thoracic aorta are localized to certain areas of the muscle lamella, whereas those of the mesenteric arteries were found all over the vessel.

The longitudinal folds, and the numerous knob-like and finger-like processes of SMCs, are the cause of the irregular outline noted in transversely sectioned (Cliff

1967; Stein etal . 1969; Cliff 1970; Rhodin 1980; Dingemans et al. 1981), and longitudinally sectioned, smooth muscle cells (Dingemans et al. 1981), noted in some transmission electron microscopy studies. These irregular profiles enlarge the surface area to volume ratio of SMCs (Gerrity and Cliff 1975), providing a greater surface area for attachment of connective tissue ele- ments, and enables the aortic SMCs to endure the high systolic pressure, as has been suggested in the cerebral artery of hypertensive rat (Fujiwara et al. 1990).

Neighboring SMCs are connected by gap junctions (communicating junctions) (Cliff 1967, 1970; Moss and Benditt 1970; Dingemans et al. 1981; Berry and Sosa- Melgarejo 1989; Sosa-Melgarejo and Berry 1989) and simple appositions (mechanical contacts) (Henderson 1975; Sosa-Melgarejo and Berry 1989). The many cyto- plasmic processes found in the intercellular clefts bridg- ing neighboring SMCs, may be involved in the forma- tion of gap junctions and simple appositions.

There are few available data on the size of the aortic SMC, except for a study by Seifert (1962). The present data show morphometrically the difference in the width of the cells between the outermost and the inner lamel- lae, and show that the SMCs of the inner lamellae are flattened axially. The 90 ~ orientation to the long axis of the vessel, means that the arrangement of smooth muscles in rat thoracic aorta is neither helical nor spiral, and suggests that the forces produced by each muscle bundle and muscle sheet might be integrated into forces directed transversely with respect to the long axis of the vessel.

Acknowledgements. The authors wish to thank Dr. T. Tenkova for her critical reading of the manuscript, Miss Y. Toda, Mr. D. Shimizu and K. Okugawa for their help, and Miss E. Felt for improving the English of the manuscript.

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