the cytoarchitecture of the wall and the innervation pattern of the microvessels in the rat mammary...

THE AMERICAN JOURNAL OF ANATOMY 170:39-54 (1984)

The Cytoarchitecture of the Wall and the Innervation Pattern of the Microvessels in the Rat Mammary Gland: A Scanning Electron Microscopic Observation

TAKASHI FUJIWARA AND YASUO UEHARA Department ofAnatomy, School of Medicine, Ehime University, Shigenobu, Ehime, 791-02 Japan

ABSTRACT This report describes the morphology of the smooth muscle cells, pericytes, and the perivascular autonomic nerve plexus of blood vessels in the rat mammary gland as visualized by scanning electron microscopy after removal of connective-tissue components. From the differences in cellular morphology, eight vascular segments were identified: 1) terminal arterioles (10-30 pm in outer diameter), with a compact layer of spindle-shaped and circularly oriented smooth muscle cells; 2) precapillary arterioles (6-12 pm), with a less compact layer of branched smooth muscle cells having circular processes; 3) arterial capillaries (4-7 pm), with %pidery” pericytes having mostly circularly oriented processes; 41 true capillaries (3-5 pm), with widely scattered pericytes having longitudinal and several circular processes; 5) venous capillaries (5-8 pm), with spidery pericytes having ramifying processes; 6) postcapillary venules (10-40 pm), with clustered spidery pericytes; 7) collecting venules (30-60 pm), with a discontinuous layer of circularly oriented and elongated stellate or branched spindle-shaped cells which may represent primitive smooth muscle cells; and 8) muscular venules (over 60 pm), with a discontinuous layer of ribbon-like smooth muscle cells having a series of small lateral projections. No focal precapillary sphincters were found.

The nerve plexus appears to innervate terminal arterioles densely and precapillary arterioles less densely. Fine nerve fibers are only occasionally associated with arterial capillaries. Venous microvessels in the rat mammary gland seemingly lack innervation.

Vascular smooth muscle cells and pericytes which are often collectively referred to as periendothelial cells (Rhodin, 1968; Beacham et al., 1976) have been directly visualized with scanning electron microscopy (SEMI (Uehara and Suyama, 1978; Murakami et al., 1979; Shimada, 1981; Fujiwara and Uehara, 1982; Mazanet and Franzini-Armstrong, 1982; Miller et al., 1982; Fujiwara et al., 1983; Holley and Fahim, 1983). The use of procedures recently developed to selectively remove connective-tissue components and the basal lamina (Evan et al., 1976; Uehara and Suyama, 1978; Mazanet and Franzini-Arm- strong, 1982; Miller et al., 1982) provides detailed information as to the over-all shape, arrangement, and dimensions of the periendothelial cells a t higher resolution than has hitherto been possible with classical silver

impregnation techniques (Zimmermann, 1923; Plenk, 1927) and by modern histochemical methods (Wolter, 1962; Kuwabara and Cogan, 1963; Williamson et al., 1980). This information has direct relevance to regula- tory mechanisms of the peripheral blood circulation.

Nevertheless, these previous SEM studies have been limited to the observation of randomly exposed portions of the vessels, and none have been successful in following the morphological changes in the cytoarchitecture of microvessels along the entire vessel

Address reprint requests to T. Fujiwara, Department of Anat- omy, School of Medicine, Ehime University, Shigenobu, Ehime, 791-02 Japan.

Received September 29, 1983. Accepted January 10,1984.

o 1984 ALAN R. Lrss. rNc

40 T. FUJIWARA AND Y. UEHARA

length from the arterial to the venous side. In those studies, the vessels tended to be disrupted during specimen preparation, and moreover they were partly covered by over- lying parenchymal structures. In the present investigation, however, the microvessels distributed in the interacinal spaces of the mammary gland of the rat were fully exposed as an almost continuous three-dimensional network. Loose connective tissue and adipose cells filling the interacinal space were removed with little damage to the vascular structures. The autonomic nerve plexuses innervating the vessels were also well preserved.

The present observations may contribute toward a better understanding of the functional specialization of various segments of the microvessels in the mammary gland and may also facilitate an accurate identification of individual segments a t the ultrastructural level. Functional aspects and ultrastructural features of various microvascular segments have been reviewed in recent years (Rhodin, 1973; Stromberg and Wiederhielm, 1973; Wolff, 1977).

MATERIALS AND METHODS

The mammary glands of nonlactating fe- male Wistar rats (200-300 gm) were cut into small pieces in Eagle's minimum essential medium (MEM, pH 7.0; Eagle, 1959). The procedures used to remove perivascular connective-tissue components were previously described (Nagato et al., 1980). In brief, the tissue blocks were digested in MEM containing collagenase (Worthington, type 11; 2.3 mg/ ml) and hyaluronidase (Amano; 6 TRU/ml) for 30-60 min at 37"C, fixed with 3% glutaraldehyde in 0.1 M phosphate buffer for 2 hr, washed in distilled water, and hydrolyzed in 8N HC1 for 30-50 min at 60°C. They were dehydrated through a graded series of ethan- o l ~ , immersed in isoamyl acetate and critical- point dried. The specimens were coated with platinum and examined with a Hitachi S- 500A scanning electron microscope.

Several microvessels were selectively pho- tographed to estimate the relative surface area not covered by periendothelial cells to the total surface area of the microvessels (see Nagato et al., 1980). To minimize the perspec- tive error introduced by the curvature of the blood vessel, several steps were taken. First, the microvessel was tilted so as to be perpendicular to the direction of observation. Next, photographs were taken of the central part

of the blood vessel surface only, excluding both edges (more than 10% of the diameter) where the surface sloped steeply. Tracings of the profiles of periendothelial cells were made from photographic enlargements a t a magnification of at least 3,000. The areas of periendothelial cells were measured with a Leitz Texture Analyzing System and ex- pressed as a percentage of the total area of the microvessels.

RESULTS

The microvessels are readily visible in the interacinal space of the glands when connective-tissue components are adequately removed by enzymatic digestion and HC1 hydrolysis. The vessels branch and anastomose repeatedly to form a randomly arranged, three-dimensional network. Stereo- pair micrographs of such preparations allow the vessels to be followed almost continu- ously from the arteriolar to the venular side via an extensive capillary network (Fig. 1).

The periendothelial cells and the vascular autonomic nerve plexuses are fully disclosed along the vessel length, except for small areas where a layer of adventitial fibroblasts or veil cells (Rhodin, 1968) has not been removed. The shape and arrangement of the periendothelial cells vary markedly in different vascular segments, although there always exists a gradual intersegmental transition.

We classified the vessels somewhat arbi- trarily into eight segments mainly on the basis of the morphological features of the periendothelial cells: 1) terminal arterioles, 2) precapillary arterioles, 3) arterial capillaries, 4) true capillaries, 5 ) venous capillaries, 6) postcapillary venules, 7) collecting venules, and 8) muscular venules.

Terminal arterioles Terminal arterioles are readily identified

by a single layer of elongated spindle-shaped periendothelial cells which obviously represent smooth muscle cells (Fig. 2). Individual smooth muscle cells consist of a central bulge 5-6 pm in diameter and unbranched tapered ends. They range from 50 to 60 pm in length as measured by tilting the specimen stage at maximum angle around the long axis of the vessel. The smooth muscle cells are oriented circularly around the vessels, being offset with respect to one another so that the thick central portion of one cell is generally juxta- posed to the tapered end of the adjacent one.

MICROVESSEL WALL CYTOARCHITECTURE 41

Fig. 1. Stereo-SEM micrographs showing a microvascular network exposed in the interacinal space of the rat mammary gland. The pathway of the vessels can be

followed from the arteriolar (A) to the venular (V) side via a capillary bed. G) Terminal portions of the gland; F) fat cell. X200.

Helical orientation of the cells has not been observed. The adjacent cells are closely ap- posed to each other with a gap narrower than 1 pm, and they form an almost continuous layer. Slender side processes 0.2 pm to 0.5 pm thick are often found bridging adjacent cells.

The vessels with these features range from 10 to 30 pm in outer diameter. Since the smooth muscle cells are rather constant in their length irrespective of the vessel size, they encircle small vessels thinner than about 15 pm in one turn or more and form an incomplete hoop on larger vessels.

At their proximal ends, where the diameter is 30 pm and more, the vessels tend to have a larger number of smooth muscle cell layers.

Precapillary arterioles Farther distally, smooth muscle cells grad-

ually acquire longitudinal bifurcations on

one or both sides of the bulged cell body (Fig. 3). The vessels defined as precapillary arterioles here are characterized by much more highly branched periendothelial cells which resemble an octopus clinging to a pipe (Fig. 4). These periendothelial cells possibly represent smooth muscle cells, because our previous serial-section reconstruction study showed similar branched cells which had many of the ultrastructural features of typi- cal smooth muscle cells (Komuro et al., 1982). The gaps between neighboring cells start to increase, reaching a distance of 2-3 pm. On both sides of the central bulge, which is about 4 pm in diameter, each cell possesses 6 to 10 processes 0.5-1 pm thick which further ramify into smaller secondary processes. All the processes are arranged almost parallel to each other and are oriented circularly, although there are a few small longitudinal side projections. The cells are about 20 to 30 pm in over-all length, being shorter than the


Fig. 2. A terminal arteriole about 20 pm in outer diameter with a compact layer of spindle-shaped smooth muscle cells. Each muscle cell has a central bulge and unbranched tapered ends. A few slender side projections (PI connect the adjacent cells. The vascular autonomic

nerve plexus (N) closely attaches onto the outer aspect of the muscle layer. An irregular small expansion of the plexus fits into the shallow cleft between adjacent smooth muscle cells (arrow). ~4,200.

spindle-shaped smooth muscle cells in the terminal arterioles. The vessels with these features range from 6 pm to 12 pm in diameter and frequently give rise to capillary off- shoots (Fig. 6).

Condensation of muscle cells suggestive of a precapillary sphincter of a focal type have not been found at any branching points of the terminal or precapillary arterioles (Fig. 6).

Arterial capillaries Farther distally, the periendothelial cells

become more highly branched with ramifying processes, assuming a spider-like shape (Fig. 5) . They are no longer recognizable as smooth muscle cells and are regarded here as pericytes. These cells are scattered along

the length of the vessel with a center-to-center distance between the adjacent cell bodies of about 10 pm. The cell processes, 0.5 to 1 pm in diameter, are generally oriented in a circular fashion and often give rise to thinner processes less than 0.5 pm in diameter. These processes are frequently interconnected to form a continuous loose cellular network around the endothelial tube. More than 40% of the adventitial surface area of the vessel is not covered by the pericytes. The arterial capillaries range from 4 to 7 pm in outer diameter.

True capillaries True capillaries are identified as the thin-

nest vessels in the whole vasculature, ranging from 3 to 4 pm in diameter except for the

Fig. 3. The smooth muscle cells with longitudinally split ends found in the transitional region between the terminal arteriole and the precapillary arteriole. x 6,000.

Fig. 4. A precapillary arteriole about 6 pm in diameter characterized by highly branched smooth muscle cells. The main cellular processes are circularly oriented

with a few small side projections oriented longitudinally. N) A small nerve fiber associated with the vessel. X6,OOO.

Fig. 5. An arterial capillary about 7 pm in diameter, having a few scattered spidery pericytes with mostly circularly oriented processes. A relatively large surface area ofthe vessel is not covered by the pericytes. ~5,000.


Fig. 6. A portion of the precapillary arteriole (PA) branching into an arterial capillary (AC) with scattered spidery pericytes. Autonomic nerves (N) from the precapillary arteriole continue along the arterial capillary. ~2,400. points. x 1,500.

Fig. 7. A network of true capillaries studded with scattered pericytes which tend to occw at branching

bulging portions (5 pm) containing endothelial nuclei. Capillaries anastomose repeatedly in an extensive three-dimensional network (Fig. 7). Pericytes are studded along the vessel at intervals of 15 to 30 pm and tend to occur a t the branching points of the capillaries. Each capillary pericyte has a round or spherical cell body about 5 pm in diameter with longitudinal primary processes less than 1 pm thick on both of its sides and several thinner, circularly oriented secondary processes (Fig. 8). These pericytes resemble those of muscle capillaries (Uehara et al., 1978; Mazanet and Franzini-Arm-

strong, 1982) but have shorter longitudinal processes and fewer circularly oriented processes. The endothelial tube is largely exposed; more than 60% of the surface area lacks pericytic investment.

Venous capillaries Several capillaries join to form a thicker

vessel, ranging from 5 to 8 pm in diameter, designated here as a venous capillary (Fig. 9). The vessels are associated with spidery pericytes which resemble those of the arterial capillaries (Fig. 10). However, they have more ramifying processes which are con-


Fig. 8. A true capillary pericyte with two primary longitudinal processes and several secondary circular processes. Large parts of the adventitial surface of the vessel lack pericytic investment. ~4,400.

Fig. 9. Several true capillaries (TC) join a venous capillary (VC) about 5 to 7 I m in diameter. The periendothelial cells are more densely distributed along the latter vessel than over the true capillary. x 1,300.

nected and/or overlap each other to form a loose network around the endothelial tube. The processes tend to be oriented rather randomly with respect to the vessel axis. About 45% of surface area of the endothelium is exposed.

Postcapillary venules The venous capillaries join successively

into thicker vessels ranging from 10 to 40 pm in diameter which are regarded here as postcapillary venules (Fig. 11). These vessels also receive true capillaries as direct tributaries along their length. The postcapillary venules

are characterized by highly clustered spidery pericytes. Individual pericytes are similar in shape to those of the venous capillaries described above, but have more ramifying processes which are oriented randomly with respect to the vessel axis (Fig. 12). The primary processes radiating from the cell body often form expanded ends which give rise to several thinner secondary processes. These processes overlap and/or attach to each other in an intricate cellular lacework. Small areas of the endothelium can be seen through the meshes. Farther proximally, the venules have a more or less loose cellular lacework


Fig. 10. A venous capillary with pericytes with rami- Fig. 11. Confluence of venous (VC) and true (TC) cap- fying processes which are randomly oriented with re- illaries with a postcapillary venule (PV). Note the differ- spect to the vascular axis. The processes contact andior ences in the vascular diameter and the density of overlap each other. A true capillary (TC) joins the venous periendothelial cells among the three segments. x 1,200. capillary. ~3,000.

with an increasing intercellular space. Con- comitantly, the pericytes acquire a polygonal cell body with fewer processes, which appear to be flattened against the endothelial tube (Fig. 13).

Small round cells about 5 pm in diameter which probably represent blood cells are occasionally observed on the outer aspect of the vessels, apparently being trapped by the pericytic processes (Figs. 12,131.

Collecting uenules The vessels following the postcapillary

venules are associated with stellate periendothelial cells which tend to be elongated in the direction perpendicular to the vascular axis (Fig. 14). The cells are much more loosely distributed along the length of the vessel than those in the postcapillary venules (Fig. 15). Therefore, fairly large areas of the endothelium (about 30%) are exposed. The cell bodies are about 15 wm long and about 2.5 pm in diameter, and their processes are less than 1.5 pm thick. The over-all length of the cell may exceed 50 pm. These periendothelial

cells may correspond to the primitive smooth muscle cells as discussed by Rhodin (1968). The vessels with these features range from 30 to 60 pm in diameter and are regarded here as the collecting venules. In the most proximal portion of this segment, the periendothelial cells are spindle-shaped and branched. They are more crowded and are almost circularly oriented (Fig. 16). The entire length can be followed for as long as 100 pm. What appear to be blood cells can be also seen on the surface of these vessels.

Muscular uenules The vessels thicker than 60 pm in diameter

are endowed with irregular ribbon-like smooth muscle cells less than 5 pm wide and about 100 pm long, which form a discontinuous layer with intercellular gaps less than 4 pm wide (Fig. 17). These smooth muscle cells have numerous thin lateral projections less than 0.5 pm wide which connect the neighboring cells. Thus, they differ strik- ingly in their shape from the arteriolar smooth muscle cells described earlier. The


Fig. 12. Two postcapillary venules with a dense lacework of spidery pericytes. Arrows indicate the expansions of their primary processes which give rise to finer secondary processes. B) A spherical body possibly represents an emigrating blood cell. ~2 ,000 .

Fig. 13. A large postcapillary venule about 25 pm in diameter. The wall cytoarchitecture is similar to that shown in Figure 12, but the pericytes have a polygonal cell body. The pericytes are flattened against the endothelium. B) What appears to be an emigrating blood cell trapped by pericytic processes. x 1,800.

Fig. 14. The distal portion of the venular side of the microvessels, showing the continuous arrangement of true capillaries (TC), venous capillaries WC), postcapillary venule (PV), and collecting venule (CV). The collecting venule is endowed with scattered periendothelial

cells, which tend to be circularly oriented. ~ 4 6 0 . Fig. 15. A collecting venule about 55 pm in diameter,

characterized by sparsely distributed stellate periendothelial cells which tend to be circularly oriented. The endothelial surface is largely exposed. X 1,200.


Fig. 16. A transitional area between the collecting endowed with irregular ribbon-like smooth muscle cells. venule and the muscular venules. The periendothelial The smooth muscle cells are oriented circularly in a cells are branched, spindle-shaped, and are arranged in discontinuous fashion. Note thin lateral projections a circular fashion. x 1,600. which connect the neighboring cells. x 1,900.

Fig. 17. A muscular venule about 60 pm in diameter

smooth muscle cells are oriented circularly, but not helically as expected in previous studies (Rhodin, 1968).

Vascular autonomic nerve plexus The vascular autonomic nerve plexus is

closely associated with the adventitial surface of the microvessels. This plexus consists of thin nerve fiber bundles or single fibers ranging from 0.2-1.5 pm in diameter, and the fusiform or triangular bulges of the associated Schwann cell bodies range from 3 to 5 pm in diameter (Fig. 18). The varicosities of the nerve fibers, as demonstrated in previous fluorescence histochemical studies, are not evident in our preparations. However, the thin portions, less than about 0.3 pm, often exhibit irregular expansions (Fig. 2) or

varicosities (Fig. 18) which fit into shallow depressions on the surface of the smooth muscle cells or into the clefts between adjacent cells. The innervation pattern varies in different vascular segments. The terminal arterioles and the proximal portion of the precapillary arteriole are densely innervated (Figs. 2, 18). The innervation density gradually decreases distally; only one or two nerves can be seen in association with distal portions of precapillary arterioles Fig. 4) and arterial capillaries (Figs. 6, 19). True capillaries, venous capillaries, postcapillary venules, and collecting venules seemingly lack innervation. The plexus was not demonstrated in muscular venules in the rat mammary gland. This segment may be innervated in other tissues, however, since our previous


Fig. 18. The autonomic plexus associated with a terminal arteriole, consisting of a Schwann cell body (S) and nerve fibers (N) which range from less than 0.2 to 1.5 pn in diameter. The thinnest portions have varicose

structures (arrows). ~5,200.

Fig. 19. A nerve fiber (Nf less than 1 pm in diameter closely associated with an arterial capillary. ~4 ,800 .

SEM study showed a loose plexus on the tion of periendothelial cells associated with muscular venules in rat skeletal muscle microvessels in the rat mammary gland. Al- (Uehara et al., 1981). though the procedure involves rather drastic

acid treatment, periendothelial cells are preserved without obvious artifactual altera- tions. In fact, the arteriolar smooth muscle cells thus exposed are almost identical in

DISCUSSION

The selective removal of connective-tissue components permits direct SEM visualiza-


TABLE 1. Cytological features and innervation pattern of various microvascular segments in the rat mammary gland

Innervation Vascular Periendothelial cells segment Diameter Cell shape, type, and orientation Arrangement pattern

Terminal 10-30 pm Spindle-shaped smooth muscle Compact Dense arteriole cells, circularly oriented

Precapillary 6-12 pm Branched smooth muscle cells Less compact Less dense arteriole with circular processes to sparse

Arterial 4-7 pm Spidery pericytes with mostly Scattered Sparse capillary circular processes

True 3-5 wm Pericytes with longitudinal Widely None capillary and circular processes scattered

Venous 5-8 brn Spidery pericytes with Scattered None capillary randomly oriented processes

Postcapillary 1 0 4 0 pm Spidery pericytes with highly Densely None venule ramified overlapped processes clustered

Collecting 30-60 pm Elongated or branched spindle- Scattered None venule shaped primitive smooth muscle

cells, circularly oriented

cells with many side processes, oriented circularly

Muscular > 60 pm Ribbon-like smooth muscle Discontinuous None venule

their shape and dimensions to the three-dimensional models of comparable cells recon- structed from ultrathin serial sections of glutaraldehyde-osmium fixed and Epon- embedded preparations (Komuro et al., 1982).

The present study reveals marked morphological differences in periendothelial cells throughout the length of the vessel from terminal arteriole to muscular venule. The results confirm and extend Zimmermann’s description (1923; see also Benninghoff, 1930; Majno, 1965) which has been questioned due to the capricious nature of the silver technique used. Although there is always a gradual intersegmental transition, the differences in the periendothelial cell morphology per- mit an accurate identification of various microvascular segments at the ultrastructural level (Table 1).

Our classification of the venular segments coincides well with that proposed by Rhodin (1968) and subsequently supported by several investigators (Baez, 1977; Anderson and Anderson, 1981). However, the classification of the arteriolar segments still remains de- batable. The terminal arteriole designates the final arterial ramification with a continuous muscle layer (Chambers and Zweifach, 1944). Although eliminated from the Nomina Histologica (1977) and from the list of Ander- son and Anderson (1981), the term is used by a number of authors (Baez, 1977; Beacham et al., 1976; Rhodin, 1973). The vessel endowed with branched muscle cells and directly following the terminal arteriole is

referred to here as the precapillary arteriole. The term was used by‘ Zweifach (1961) to designate the final arteriolar segment and was adopted recently by several authors (Furness and Marshall, 1974; Stingl, 1976; Anderson and Anderson, 1981). Zweifach (1961) also termed this segment the “metarteriole.” However, the metarteriole more generally refers to a thoroughfare or prefer- ential channel (Chambers and Zweifach, 1944; Beacham et al., 1976). No such channel has been found in our preparations. The oc- currence of the channel appears to depend on the tissues and organs in which the microvessels are contained (Zweifach, 1961; Wiede- man, 1963). The metarteriole is characterized by a discontinuous muscle layer spaced at irregular intervals (Chambers and Zweifach, 1944; Zweifach, 1961; Beacham et al., 1976). The adjacent smooth muscle cell processes in our precapillary arterioles are actually sepa- rated from each other by a wider gap than those in the terminal arterioles. However, this gap reaches 2 pm to 3 pm at the most, and a gap of this range does not seem to be wide enough to be visibly discontinuous by light microscopy.

In the precapillary arteriole, smooth muscle cells are branched, assuming a shape like an octopus clinging to a pipe. This unique configuration can be seen in Zimmermann’s drawing (1923), but has not been recognized in subsequent histological and SEM studies. The existence of a precapillary sphincter structure in relation to arterioles is disputed.


Some authors have considered that the sphincter is a focal structure consisting of condensation of smooth muscle cells encir- cling the orifice of capillaries and smaller arterioles (Chambers and Zweifach, 1944; Ni- coll and Webb, 1955; Beacham et al., 19761, whereas some thought that it represents the final arteriolar smooth muscle cells (Wiede- man, 1973; Wiedeman et al., 1976). In the present study, neither condensed smooth muscle cells have been found at any branching points of the arterioles (see also Holley and Fahim, 1983), nor could the final smooth muscle cells be identified due to the gradual transformation of smooth muscle cells into pericytes.

The spindle-shaped smooth muscle cells of the terminal arterioles, forming a compact layer, are circularly oriented and often wrap the vessel with more than one turn. The branched smooth muscle cells of the precapillary arterioles are also circularly oriented but wrap the vessel incompletely. Although claimed in early histological studies (Hayek, 1952; Rhodin, 1967; Phelps and Luft, 1969; Hua and Cragg, 1980), the helical orientation of muscle cells has not been found in the present study (for a more detailed discussion see Fujiwara and Uehara, 1982). The circular orientation of the muscle cells is appropriate to vasoconstriction upon their contraction; the smooth muscle cells of the terminal arteriole appear to be more powerful in narrow- ing the vascular lumen than those of the precapillary arteriole, as expected from morphological differences. It is likely that capillary blood flow is principally regulated by the terminal arterioles and to a lesser extent by the precapillary arterioles as a whole, rather than by the focal type of sphincter; such a possibility has been proposed by a number of previous investigators (Rhodin, 1973; Stingl, 1976; Gorczynski et al., 1978; Anderson and Anderson, 1981).

Attempts have been made to classify capillaries into three subdivisions; the arterial capillary, the true capillary, and the venous capillary (Majno, 1965; Rhodin, 1968; Ander- son and Anderson, 1981). However, little is known about the differences of their wall structures. The present study shows, for the first time, that these subdivisions differ in the shape, arrangement, and distribution of their associated pericytes as summarized in Table 1.

Among various functions proposed (for re- views see Majno, 1965; Rhodin, 1968; Wolff,

1977; Gabbiani and Majno, 19801, a possible contractile capability of capillary pericytes in regulating blood flow has been empha- sized in recent works (Kuwabara and Cogan, 1963; Forbes et al., 1975, 1977a,b; Altura, 1978; Tilton et al., 1979a.b). The presence of contractile elements in pericytes (Le Beux and Willemot, 1978; Wallow and Burnside, 1980) and their apparent gradual transformation into smooth muscle cells (Zimmer- mann, 1923; present study) would support the view that pericytes are the precursors of smooth muscle cells (Rhodin, 1968, 1973). Mazanet and Franzini-Armstrong (19821, in their SEM study of pericytes of skeletal muscle capillaries, suggested that the longitudinal processes of the cell would buckle the vessels upon contraction, whereas contraction of their circular side branches would result in reduction of the vascular lumen. The same can be applied to the pericytes of the true capillaries in our preparations; whereas the pericytes of the arterial capillaries would constrict rather than buckle the vessel, since their processes are mostly circularly oriented. The effect of the constriction of the venous capillary pericytes is not easily predicted because of the random orientation of their processes.

The percentage of the total vascular surface area not covered by pericytes differs in the three capillary subdivisions: more than 60% in the true capillaries, about 45% in the venous capillaries, and about 40% in the arterial capillaries. This may indicate that the exchange of substances through the endothelium is greatest in the true capillary and less extensive in the venous and the arterial capillaries.

The postcapillary venules are characterized by densely packed pericytes which form an intricate cellular lacework. This feature may represent the thick connective-tissue sheath, or a t least a part of it, as described by Zweifach (1934, 1961). A similar configuration has previously been depicted by Zim- mermann (1923) in certain small venules. However, it has not been disclosed at the ultrastructural level, although overlappings of the pericytes which gave the impression of several cell layers were noted by Rhodin (1968) in the postcapillary venule. The signif- icance of these features is not known at present. Majno (1965) and Cotran (1965) showed that intravenously administered foreign substances which might escape through the highly permeable endothelium were trapped


by the pericytes. Movat and Fernando (1964) also showed that the pericytes increased in number and often became multilayered upon antigenic stimulation. Blood cell emigration (Marchesi and Florey, 1960; Florey and Grant, 1961; present observation) and plasma exudation (Majno and Palade, 1961; Majno et al., 1961) take place through the endothelium. Rhodin (1968) suggested that the pericytes are involved in a protective mechanism against an excessive loss of blood elements. We are undertaking further studies to clarify the actual morphological changes of the pericytes in various experimental and patholog- ical conditions.

The collecting venules ranging from 30 to 60 pm in diameter are endowed with elongated stellate or branched, spindle-shaped periendothelial cells which are sparsely distributed and mostly circularly oriented. These cells may represent; an intermediate cell type between pericytes and smooth muscle cells, i.e., the primitive muscle cells of Rhodin (1968). What appear to be emigrating blood cells are also found in this segment. The collecting venules as well as postcapillary venules may be common sites of cellular emigration.

The collecting venules gradually change into muscular venules with a diameter of more than 60 pm. The venular smooth muscle cells differ from the arteriolar ones. Un- like the spindle-shaped arteriolar smooth muscle cells, they are irregularly ribbon-like in shape, are slightly flattened against the endothelium, and are arranged in a discontinuous layer. The neighboring venular smooth muscle cells are bridged by numerous small side projections as reported previously for the smooth muscle cells of mesenteric veins (Fujiwara et al., 1983). Fewer bridges can be seen in the terminal arteriole. Whether these contacts represent mechanical attachment sites or electrically coupled gap-junctions remains to be clarified. The sparse distribution of primitive smooth muscle cells in the collecting venule and the apparently weak contractile capability of the smooth muscle cells in the muscular venules may well be correlated to the properties of these segments as volume capacitance vessels (Stromberg and Wiederhielm, 1973).

The present study provides information on the morphology of the vascular autonomic nerve plexus and its decreasing density along the length of the microvessels to its disap- pearance at the level of the true capillary.

The density of the plexus varies considerably in different vascular segments (Table 1). The findings generally coincide with observations by the fluorescence histochemical method for adrenergic nerves (Furness and Marshall, 1974; Burnstock et al., 1980). The differences in the innervation density may be correlated with the physiological findings which indicate different responses of various microvascular portions to nerve stimulation (Furness and Marshall, 1974; Altura, 1978). The present SEM procedure, however, could neither distinguish between different histochemical categories of autonomic nerves nor reveal the functional relationship of individual nerve fibers to pericytes, particularly to the pericytes in the arterial capillary.

The architecture of microvessels differs markedly in different animal species, tissues, and organs (Wiedeman, 1963). Hence, further systematic comparative surveys are needed as a basis for the classification and identification of microvascular segments.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Peter Baluk for his critical reading of the manuscript and valuable suggestions.

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the cytoarchitecture of the wall and the innervation pattern of the microvessels in the rat mammary...

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