- Email: [email protected]
Scanning electron microscopy of pericytes in rat red muscle
MICROVASCULARRESEARCH23, 361--369 (1982)
Scanning Electron Microscopy of Pericytes in Rat Red Muscle ROSEMARY MAZANET AND CLARA FRANZINI-ARMSTRONG
Departments of Anatomy and Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received July 8, 1981 We describe the overall shape of pericytes on the surface of small blood vessels in a rat red muscle, as seen by scanning electron microscopy. Once connective tissue components and the basal lamina are removed, pericytes can be easily recognized. The cells have two or more major processes parallel to the long axis of the blood vessel. Numerous short secondary branches arise at right angles to the major process and partially encircle the blood vessel. The longest processes average 77 ~xm and at least one of them is present over 82% of the length of the microcirculatory bed. Bundles of microfilaments are contained within the pericytic processes. In light of these findings, it is likely that pericytes are contractile and that they play a role in microvascular control.
INTRODUCTION Pericytes are vascular companion cells located at the periphery of small blood vessels. They were first described in the nictitating membrane of the frog by Rouget (1873), and were interpreted by him and others as having Contractile properties (Krogh, 1929). Using the Golgi-Kopsch silver impregnation technique and light microscopy, Zimmermann (1923) provided complete and extraordinary drawings of the complexity of pericyte shape. He reported on the extent to which processes originating from the cell body cover the vessel walls in various organs of many vertebrates. Somewhat similar, though less Complete, low-resolution images were obtained by other methods of rendering the whole cell visible in the light microscope (Wolter, 1962; Williamson et al., 1980). However, the extensive pericytic processes are not normally visible in living tissue. Hence, some early studies of circulatory control in the capillary bed by direct light microscopic observation of transparent tissues (Saunders et al., 1940; Chambers and Zweifach, 1944) failed to assign a direct role to pericytes. Similarly, since information on the overall shape of the cell is lost in random thin sections for EM, responsibility for changes in small vessels under the influence of histamine has been assigned to endothelial cells rather than to pericytes (Majno et al., 1969). Indeed, it has been often assumed that either precapillary sphincters, or the endothelial cells themselves are responsible for regulatory effects at the level of the microcirculation (see reviews by Majno, 1965; Zweifach, 1973;Rosell, 1980). Our new approach to the study of pericyte morphology has the major advantage of providing as complete-a view of the cell as that obtained from the Golgi 361 0026-2862/82/030361-09502.00/0 Copyright©~1982by AcademicPress, Inc. All rights of reproductionin anyform reserved. Printed in U.S.A.
362
MAZANET AND FRANZINI-ARMSTRONG
staining, but at a much higher level of resolution. In providing a direct confirmation that Zimmermann's drawings were quite accurate and not merely a result of artifact, we again bring into evidence the fact that pericytes have unusually extensive branches and are well designed for interaction with endothelial cells. Results of serial sectioning (Matter et al., 1969; Matsusaka, 1975; and, particularly, the complete reconstructions of myocardial pericytes by Forbes et al., 1977a) are extended to show that there are two predominant orientations of the processes: longitudinal and circumferential. The former provides coverage of long lengths of the vessel, the latter encircles a good part of its circumference. In view of these findings, the possible contractile role of pericytes, as emphasized in the recent work of Altura (1978), Forbes et al. (1977a), and Tilton et al. (1979a,b), becomes more significant. MATERIALS AND METHODS For SEM, adult rats were fixed by vascular perfusion with 2% glutaraldehyde in 0.15 M Na+-phosphate buffer, pH 7.2-7.4. The sternomastoid muscle was removed and the red bundle (Dulhunty, 1977) separated. The muscles were teased into small bundles. Components of the connective tissue were eliminated by enzymatic digestion in 0.2% trypsin in 0.15 M phosphate buffer for 8-12 hr at 38°C, and maceration in 4% unbuffered OsO4 at 40°C for 8-12 hr. The muscles were then critical-point dried, coated with gold-palladium, and viewed in an AMR 1000A SEM. Outlines of all cells initially hidden by basal lamina are clearly visible in "cleaned" tissue (Shotton et al., 1978; Mazanet et al., 1979, 1980). Unfortunately, the technique suffers from severe limitations, particularly in the case of muscles with large amounts of collagen. The major difficulty is in obtaining a reproducible digestion. In overdigested tissue, we may lose entire cells or portions of branches. When digestion is not complete, pericytes are still hidden under the basal lamina. Other muscles were perfused in 2% glutaraldehyde in 0.15 M cacodylate buffer containing 2% tannic acid. Postfixation in 2% osmium in cacodylate, staining en bloc with 1% uranyl acetate, and embedding in Epon or Spurr followed. RESULTS The red bundle of the sternomastoid is richly vascularized. In cross sections of the muscle, two to five small vessel profiles are located at the periphery of each fiber. Both capillaries (diameter up to 8 ~m) and postcapillary venules (diameter, 8-20 ~m) lie in close proximity to the surface of the muscle fiber, and are often located in grooves. They can be distinguished from each other only on the basis of diameter, not of disposition. The majority of vascular profiles in a random cross section of the muscle belong to capillaries. The endothelial cells are partially covered by pericyte profiles of variable size. The largest contain nuclei, others have mitochondria and bundles of filaments in variable amounts. The pericyte is enveloped by a basal lamina on all sides, except for the smaller profiles which penetrate the endothelial cells' basal lamina and come to close proximity with their plasma membrane (Fig. 1). Grooves accommodate some of the deeper branches. We do not find any readily char-
PERICYTES IN RAT RED MUSCLE
363
acterizable type of junction at the site of proximity between pericyte side branches and endothelial cells. The distance between the two membranes remains large (10-20 nm) except at focal points, and there are no visible densities on the cytoplasmic surfaces of either side. Following tannic acid treatment, however, we observe small dense strands apparently joining the two membranes across the intervening space (Fig. 2). The blood vessels that we examined by SEM have an outside diameter of less than 14 Ixm, and are not covered by a layer of smooth muscle (Figs. 3, 4), i.e., do not resemble the surface of arterioles as seen by SEM (Uehara and Suyama, 1978). They can be identified as either capillaries or postcapillary venules. A more precise distinction is not possible because diameter cannot be measured precisely from this type of surface view, and because we cannot assess what, if any, changes in diameter have occurred during preparative procedures. In addition, we do not find any shape differences in pericytes that might distinguish the vessels. Following the cleaning procedure, only cells closely associated with the muscle fiber (satellite cells), the nerve fiber (Schwann cells), and the blood vessel (pericytes) remain. All cells belonging to the interstitial tissue are lost. The surface of the endothelial cell is smooth at the level of resolution of these images. Figure 3 shows two longitudinal capillaries situated between muscle fibers (m)
FI6. 1. Cross section showing a major pericyte process (mp) and side branches. One of the latter penetrates beneath the basal lamina of the endothelial cell (e). The area outlined by the black box is shown in Fig. 2. x 7200. FIc. 2. Three pericyte profiles (p) are filled with microfilaments. Slight densities (arrows) are noticeable in the space where the pericyte closely apposes the endothelial cell (e). x 26,500.
364
MAZANET AND FRANZINI-ARMSTRONG
that are still covered by a veil of basal lamina. A pericyte cell body lies in the nook of a transverse connecting branch: this location has been described in the literature (Krogh, 1929). Other cell bodies lie along the vessel. Long processes originate from the cell body and we can follow them along the surface of the blood vessel for distances up to 60 p~m. Two components can be distinguished: a main process arranged in a predominantly longitudinal fashion, and side (or secondary) branches which arise at right angles from it and are circumferentially arranged. These partially encircle the capillary much like hoops (Fig. 4). A visible gap separates the main branch from the endothelial cell surface, while the tapered tips of the smaller branches seem to touch the vessel's surface.
Fl~. 3. SEM micrograph of a pericyte cell body (b) nestled in a transverse capillary connection (tc). Long pericyte extensions run over the capillary surface. A veil of basal lamina remains on the muscle fiber (m). x 1000. FI~. 4. Detail of a major process, showing numerous short secondary side branches (arrowheads) arising from a major longitudinal process (1). At right, a long circumferential branch (arrows) surrounds at least half of the circumference of the capillary (c). x 2700.
PERICYTES IN RAT RED MUSCLE
365
Hence, the large branches are likely to be separated from the endothelial cell surface by a layer of basal lamina, whereas the smaller branches are the processes that cross the basal lamina and form junctions with the endothelial cells (see above). The shape of processes and their disposition are variable. Some are very long with regularly spaced, short side branches (Fig. 4). The majority of branches surround at most a third of the circumference. It must be noted, however, that these shorter side branches seem to occur more frequently where the vessel is accompanied longitudinally by two or more parallel main processes. Other processes have less uniformly distributed side branches, but notably some of them encircle the entire visible portion of the blood vessel (Fig. 4, arrows). Circumferential branches may also arise directly from cell bodies and surround the vessels to a variable extent. Some processes run parallel to the vessel's long axis, but suddenly shift to a different circumferential position. In this case, the two portions of the long branch are joined by a short circumferential side branch (Fig. 3). Several main processes may run parallel to each other over the same segment of the vessel, but their number cannot be determined from SEM images, since only half of the surface can be photographed. In order to assure ourselves that these long branches are common, we determined the average length of pericyte branches by a simple morphometric analysis (Abercrombie, 1946) on thin cross sections. This is feasible due to the preferred orientation of the structures. Randomness was insured by taking photographs of the entire section, where not covered by grid bars. All profiles of small blood vessels which were cut transversely, or nearly so, were scored for the presence or absence of nuclear and nonnuclear pericyte profiles (see Fig. 5). Table 1 (a to d) gives the actual counts. Pericyte nuclear length was measured from three longitudinally cut nuclei; Table 1 (e) gives the average of the three. The results are shown in Table 1. From this we calculate the following (see Fig. 5):
D
~i~ C ,
D
FIG. 5. Diagram of two hypothetical adjacent pericytes along a capillary. Letters A - D mark distances between obvious landmarks (see text).
366
MAZANET AND FRANZINI-ARMSTRONG TABLE 1 COUNTS OF NUCLEAR AND NONNUCLEAR PERICYTE PROFILES a b c d e
Total capillary profiles N o pericyte N o n n u c l e a r pericyte Nuclear pericyte Pericyte nuclear length
243 44 188 11 9 ~m
(a) average center-to-center distance between adjacent pericyte nuclei (A), a
e~ = 199 fxm; (b) average distance covered by the longest branches of two adjacent pericytes (B minus C), ¢
e~ = 154 ~m; (c) average length of longest pericyte branches (D), c
e~-~ = 77 txm; (d) percentage of capillary length covered by at least one pericyte branch and/ or cell body, c+d - -
a
100%
=
82%.
Thus, the majority of branches must be quite long and most of the capillary surface in this red muscle is covered by at least one branch. In thin sections, the small secondary processes contain bundles of microfilaments oriented circumferentially to the blood vessel (Fig. 2). Larger profiles are cross sections of the main branches and also contain microfilaments. Many of these run roughly parallel to the long axis of the blood vessel. DISCUSSION One of the most widely accepted theories on the control of the microcirculation is that blood flow through individual segments is controlled by precapillary sphincters, which are composed of smooth muscle cells, and located at sites where capillaries arise from metarterioles (Zweifach, 1973). This theory disregards not only the possible role of pericytes in the local control of circulation, but also the fact that at least in some tissues there is a graded morphological continuum of perivascular cells from smooth muscle around arterioles, to pericytes around capillaries and venules (Zimmermann, 1923). On the other hand, the interesting possibility that pericytes may have a direct role in local control of microcirculation in some tissues gains credibility from numerous lines of evidence. First of all, pericyte processes, although small, can
PERICYTES IN RAT RED MUSCLE
367
be quite extensive (Zimmermann, 1923; Wolter, 1962; Forbes et al., 1977a; Williamson et al., 1980; this report), a fact that has been disregarded in some of the literature (see Rhodin for recent review, 1980). Secondly, there is an intimate association and possible attachment between pericytes and endothelial cells (Rhodin, 1968, 1974; Weibel, 1974; Matsusaka, 1975; Forbes et al., 1977a; Ryan et al., 1979; Wallow and Burnside, 1980; this report). Thirdly, there are small sympathetic nerve endings in proximity of pericytes (Majno et aI., 1969; Forbes et al., 1977b; Tilton et al., 1979a). Finally, pericytes contain ordered bundles of thin filaments (Forbes et al., 1977a), which are shown to contain actin (LeBeux and Willemot, 1978; Wallow and Burnside, 1980). In the richly vascularized red bundle of the rat sternomastoid, about 82% of the length of each small blood vessel is covered by at least one pericytic profile. The main arrangement is longitudinal with frequent circumferential branches and "choking points." This arrangement provides thorough pericyte coverage, a n d an appropriate disposition for constricting the blood vessel. A contraction of the longitudinal main branches could result in forces obliging the capillary to buckle along its length. Contraction of the side arms, particularly where they are more extensive, would result in wrinkling of the underlying endothelial cells and possibly in reduction of the vessel lumen. Interestingly, both effects have been noted in other tissues following either stimulation of the sympathetic nervous sy~stem (Saunders et al., 1940), or application of vasoactive agents (Chambers and Zweifach, 1944; Majno et al., 1969). Tilton et al. (1979b) have shown that, in particular, the wrinkling of the endothelial cell surface upon application of vasoconstrictor agents is limited to areas immediately below pericyte processes. Clearly, however, the importance and the function of such a regulatory role may vary greatly from one microcirculatory bed to another, and even along the same bed. There are large known variations in the extent to which pericytes cover blood vessels in different tissues, in homologous tissues from different species, and along different portions of the same circulatory bed (Zimmermann, 1923; Wolter, 1962; Weibel, 1974; Tilton et al., 1979a). Pericytes in different tissues may vary in their innervation (Forbes et al., 1977b; Tilton et al., 1979a) and in their responsiveness to drugs (Tilton et al., 1979b). Additionally, pericytes along different areas of the same circulatory bed have different immunoreactivities to myosin antibodies (Drenckhahn et al., 1980). Skeletal muscle is one of the tissues where pericyte coverage of small blood vessels is most extensive, and, interestingly, it is also a tissue where the existence of precapillary sphincters has not been proven (see Zweifach, 1973). Hence, a direct control at the level of postarteriolar small vessels is most likely to exist in this tissue. ACKNOWLEDGMENTS Supported by MDA (H. M. Watts Research Center) and NIH (HL 15835-08) to the Pennsylvania Muscle Institute. The work presented in this paper will be included in a dissertation to be submitted to the Graduate Faculty of the University of Pennsylvania by R.M. in partial fulfillment of the requirements for the Ph.D.
368
MAZANET AND FRANZINI-ARMSTRONG
REFERENCES ABERCROMmE, M. (1946). Estimation of nuclear population from microtome sections. Anat. Rec. London 94, 239-247. ALTURA, B. M. (1978). Pharmacology of venular smooth muscle: New insights. Mierovasc. Res. 16, 91-117. CHAMBERS, R., AND ZWEIEACH,B. W. (1944). Topography and function of the mesenteric capillary circulation. Amer. J. Anat. 75, 173-205. DRENCKHAHN, D., GROSCHEL-STEWART, U., AND STUMPH, B. (1980). Different affinities of antigizzard myosin and anti-uterus myosin to endothelial cells and pericytes of the rat lymph node and brain. J. Muscle Res. Cell Motil. 1, 485-486. DOLnVNTY, A. F. (1977). K ÷ -contractures and membrane potential in mammalian skeletal muscle. Nature (London) 266, 75-78. FORBES, M. S., RENNELS, M. C., ANDNELSON, E. (1977a). Ultrastructure of pericytes in mouse heart. Amer. J. Anat. 149, 47-70. FORBES,M. S., RENNELS,M. C., ANDNELSON, E. (1977b). Innervation of myocardial microcirculation: Terminal autonomic axons associated with capillaries and postcapillary venules in mouse heart. Amer. J. Anat. 149, 71-91. KROGrt, A. (1929). "The Anatomy and Physiology of Capillaries." Yale Univ. Press, New Haven. LEBEUx, Y. J., AND WILLEMOT, J. (1978). Actin and myosin-like_filaments in rat brain pericytes. Anat. Rec. 190, 811-826. MAJNO, G. (1965). The ultrastructure of the vascular membrane. In "Handbook of Physiology: Circulation" (W. F. Hamilton and P. Dow, eds.), Vol. III, Sect., 2, pp. 2293-2375. Amer. Physiol. Soc., Washington, D.C. MAJNO, G., SHEA, S. M., AND LEVENTaAL, M. (1969). Endothelial contraction induced by histamine type mediators: An electron microscopic study. J. Cell Biol. 46, 647-672. MATSUSAKA,T. (1975). Tridimensional views of the relationship of pericytes to endothelial cells of capillaries in the human choroid and retina. J. Electron Microsc. 24, 13-18. MATTER,A., ORO, L., AND ROUILLER,C. (1969). Die dreidimensionale Rekonstuktion des Kapillarperiziten im Muskel. Verh. Anat. Ges. 63, 125-130. MAZANET, R., REESE, B. F., FRANZlm-ARMSTRONG,C., AND REESE, T. S. (1979). SEM of satellite cells and their response to muscle fiber injury. J. Cell Biol. 83, 383a. MAZANET, R., AND FRANZIM-ARMSTRONG,C. (1980). SEM of vascular pericytes in rat red muscle. J. Cell Biol. 87, 259a. RHODIN, J. A. G. (1968). Ultrastructure of mammalian venous capillaries, venules, and small collecting veins. J. Ultrastruct. Res. 25, 452-500. RHODIN, J. A. G. (1974). "Histology: A Text and Atlas," pp. 331-370. Oxford Univ. Press, New York. RHODIN, J. A. G. (1980). Architecture of the vessel wall. In "Handbook of Physiology: The Cardiovascular System" (D. F. Bohr, A. P. Somlyo, and H. V. Sparks, eds.), Vol. II, Sect., 2, pp. 1-31. Amer. Physiol. Soc., Washington, D.C. ROSELL, S. (1980). Neuronal control of microvessels. Annu. Rev. Physiol. 42, 359-371. ROOGET, C. (1873). Memoire sur le development, la structure et les proprietes physiologiques des capillaires sanguins et lymphatiques. Arch. Physiol. Norm. Pathol. 5, 603-663. RYAN, U. S., RYAN, J. W., AND WHITAKER,C. (1979). How do kinins affect vascular tone? Advan. Exp. Med. Biol. 102A, 237-291. SAUNDERS,A. G., EBERT, R. H., AND FLOREY,H. W. (1940). The mechanism of capillary contraction. Q. J. Exp. Physiol. 30, 281-287. SHOTTON, D. M., HEUSER, J. E., REESE, B. F., AND REESE, T. S. (1977). Postsynaptic membrane folds of the frog neuromuscular junction visualized by scanning electron microscopy. Neuroscience 4, 427-435. TILTON, R. G., Kmo, C., AND WILLIAMSON,J. R. (1979a). Pericyte-endothelial relationships in cardiac and skeletal muscle capillaries. Microvasc. Res. 18, 325-335. TILTON, R. G., KILO, C., WILHAMSON,J. R., AND MURCH, D. W. (1979b). Differences in pericyte contractile function in rat cardiac and skeletal muscle microvasculatures. Microvasc. Res. 18, 336-352. UEnARA, Y., AND SUYAMA,K. (1978). Visualization of the advential aspect of the vascular smooth muscle cells under the scanning electron microscope. J. Electron Microsc. 27, 157-159.
PERICYTES IN RAT RED MUSCLE
369
WALLOW,I. H., AND BURNSIDE,B. (1980). Actin filaments in retinal pericytes and endothelial cells. Invest. Ophthalmol. Vis. Sci. 19, 1433-1441. WEIBEL, E. R. (1974). On pericytes, particularly their existence on lung capillaries. Microvasc. Res. 8, 218-235. WILLIAMSON, J. R., TILTON, R. G., KiLo, C., AND YU, S. (1980). Immunofluorescent imaging of capillaries and pericytes in human skeletal muscle and retina. Microvasc. Res. 20, 233-241. WOLTER,J. R. (1962). The pericytes of the human retina. Amer. J. Ophthalmol. 53, 981-988. ZIMMERMANN, K. W. (1923). Der feinere bau der blutcapillaren. Z. Anat. Entwicklungsgesch. 68, 29-109. ZWEIFACH, B. W. (1973). Microcirculation. Annu. Rev. Physiol. 35, 117-150.
Scanning Electron Microscopy of Pericytes in Rat Red Muscle ROSEMARY MAZANET AND CLARA FRANZINI-ARMSTRONG
Departments of Anatomy and Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received July 8, 1981 We describe the overall shape of pericytes on the surface of small blood vessels in a rat red muscle, as seen by scanning electron microscopy. Once connective tissue components and the basal lamina are removed, pericytes can be easily recognized. The cells have two or more major processes parallel to the long axis of the blood vessel. Numerous short secondary branches arise at right angles to the major process and partially encircle the blood vessel. The longest processes average 77 ~xm and at least one of them is present over 82% of the length of the microcirculatory bed. Bundles of microfilaments are contained within the pericytic processes. In light of these findings, it is likely that pericytes are contractile and that they play a role in microvascular control.
INTRODUCTION Pericytes are vascular companion cells located at the periphery of small blood vessels. They were first described in the nictitating membrane of the frog by Rouget (1873), and were interpreted by him and others as having Contractile properties (Krogh, 1929). Using the Golgi-Kopsch silver impregnation technique and light microscopy, Zimmermann (1923) provided complete and extraordinary drawings of the complexity of pericyte shape. He reported on the extent to which processes originating from the cell body cover the vessel walls in various organs of many vertebrates. Somewhat similar, though less Complete, low-resolution images were obtained by other methods of rendering the whole cell visible in the light microscope (Wolter, 1962; Williamson et al., 1980). However, the extensive pericytic processes are not normally visible in living tissue. Hence, some early studies of circulatory control in the capillary bed by direct light microscopic observation of transparent tissues (Saunders et al., 1940; Chambers and Zweifach, 1944) failed to assign a direct role to pericytes. Similarly, since information on the overall shape of the cell is lost in random thin sections for EM, responsibility for changes in small vessels under the influence of histamine has been assigned to endothelial cells rather than to pericytes (Majno et al., 1969). Indeed, it has been often assumed that either precapillary sphincters, or the endothelial cells themselves are responsible for regulatory effects at the level of the microcirculation (see reviews by Majno, 1965; Zweifach, 1973;Rosell, 1980). Our new approach to the study of pericyte morphology has the major advantage of providing as complete-a view of the cell as that obtained from the Golgi 361 0026-2862/82/030361-09502.00/0 Copyright©~1982by AcademicPress, Inc. All rights of reproductionin anyform reserved. Printed in U.S.A.
362
MAZANET AND FRANZINI-ARMSTRONG
staining, but at a much higher level of resolution. In providing a direct confirmation that Zimmermann's drawings were quite accurate and not merely a result of artifact, we again bring into evidence the fact that pericytes have unusually extensive branches and are well designed for interaction with endothelial cells. Results of serial sectioning (Matter et al., 1969; Matsusaka, 1975; and, particularly, the complete reconstructions of myocardial pericytes by Forbes et al., 1977a) are extended to show that there are two predominant orientations of the processes: longitudinal and circumferential. The former provides coverage of long lengths of the vessel, the latter encircles a good part of its circumference. In view of these findings, the possible contractile role of pericytes, as emphasized in the recent work of Altura (1978), Forbes et al. (1977a), and Tilton et al. (1979a,b), becomes more significant. MATERIALS AND METHODS For SEM, adult rats were fixed by vascular perfusion with 2% glutaraldehyde in 0.15 M Na+-phosphate buffer, pH 7.2-7.4. The sternomastoid muscle was removed and the red bundle (Dulhunty, 1977) separated. The muscles were teased into small bundles. Components of the connective tissue were eliminated by enzymatic digestion in 0.2% trypsin in 0.15 M phosphate buffer for 8-12 hr at 38°C, and maceration in 4% unbuffered OsO4 at 40°C for 8-12 hr. The muscles were then critical-point dried, coated with gold-palladium, and viewed in an AMR 1000A SEM. Outlines of all cells initially hidden by basal lamina are clearly visible in "cleaned" tissue (Shotton et al., 1978; Mazanet et al., 1979, 1980). Unfortunately, the technique suffers from severe limitations, particularly in the case of muscles with large amounts of collagen. The major difficulty is in obtaining a reproducible digestion. In overdigested tissue, we may lose entire cells or portions of branches. When digestion is not complete, pericytes are still hidden under the basal lamina. Other muscles were perfused in 2% glutaraldehyde in 0.15 M cacodylate buffer containing 2% tannic acid. Postfixation in 2% osmium in cacodylate, staining en bloc with 1% uranyl acetate, and embedding in Epon or Spurr followed. RESULTS The red bundle of the sternomastoid is richly vascularized. In cross sections of the muscle, two to five small vessel profiles are located at the periphery of each fiber. Both capillaries (diameter up to 8 ~m) and postcapillary venules (diameter, 8-20 ~m) lie in close proximity to the surface of the muscle fiber, and are often located in grooves. They can be distinguished from each other only on the basis of diameter, not of disposition. The majority of vascular profiles in a random cross section of the muscle belong to capillaries. The endothelial cells are partially covered by pericyte profiles of variable size. The largest contain nuclei, others have mitochondria and bundles of filaments in variable amounts. The pericyte is enveloped by a basal lamina on all sides, except for the smaller profiles which penetrate the endothelial cells' basal lamina and come to close proximity with their plasma membrane (Fig. 1). Grooves accommodate some of the deeper branches. We do not find any readily char-
PERICYTES IN RAT RED MUSCLE
363
acterizable type of junction at the site of proximity between pericyte side branches and endothelial cells. The distance between the two membranes remains large (10-20 nm) except at focal points, and there are no visible densities on the cytoplasmic surfaces of either side. Following tannic acid treatment, however, we observe small dense strands apparently joining the two membranes across the intervening space (Fig. 2). The blood vessels that we examined by SEM have an outside diameter of less than 14 Ixm, and are not covered by a layer of smooth muscle (Figs. 3, 4), i.e., do not resemble the surface of arterioles as seen by SEM (Uehara and Suyama, 1978). They can be identified as either capillaries or postcapillary venules. A more precise distinction is not possible because diameter cannot be measured precisely from this type of surface view, and because we cannot assess what, if any, changes in diameter have occurred during preparative procedures. In addition, we do not find any shape differences in pericytes that might distinguish the vessels. Following the cleaning procedure, only cells closely associated with the muscle fiber (satellite cells), the nerve fiber (Schwann cells), and the blood vessel (pericytes) remain. All cells belonging to the interstitial tissue are lost. The surface of the endothelial cell is smooth at the level of resolution of these images. Figure 3 shows two longitudinal capillaries situated between muscle fibers (m)
FI6. 1. Cross section showing a major pericyte process (mp) and side branches. One of the latter penetrates beneath the basal lamina of the endothelial cell (e). The area outlined by the black box is shown in Fig. 2. x 7200. FIc. 2. Three pericyte profiles (p) are filled with microfilaments. Slight densities (arrows) are noticeable in the space where the pericyte closely apposes the endothelial cell (e). x 26,500.
364
MAZANET AND FRANZINI-ARMSTRONG
that are still covered by a veil of basal lamina. A pericyte cell body lies in the nook of a transverse connecting branch: this location has been described in the literature (Krogh, 1929). Other cell bodies lie along the vessel. Long processes originate from the cell body and we can follow them along the surface of the blood vessel for distances up to 60 p~m. Two components can be distinguished: a main process arranged in a predominantly longitudinal fashion, and side (or secondary) branches which arise at right angles from it and are circumferentially arranged. These partially encircle the capillary much like hoops (Fig. 4). A visible gap separates the main branch from the endothelial cell surface, while the tapered tips of the smaller branches seem to touch the vessel's surface.
Fl~. 3. SEM micrograph of a pericyte cell body (b) nestled in a transverse capillary connection (tc). Long pericyte extensions run over the capillary surface. A veil of basal lamina remains on the muscle fiber (m). x 1000. FI~. 4. Detail of a major process, showing numerous short secondary side branches (arrowheads) arising from a major longitudinal process (1). At right, a long circumferential branch (arrows) surrounds at least half of the circumference of the capillary (c). x 2700.
PERICYTES IN RAT RED MUSCLE
365
Hence, the large branches are likely to be separated from the endothelial cell surface by a layer of basal lamina, whereas the smaller branches are the processes that cross the basal lamina and form junctions with the endothelial cells (see above). The shape of processes and their disposition are variable. Some are very long with regularly spaced, short side branches (Fig. 4). The majority of branches surround at most a third of the circumference. It must be noted, however, that these shorter side branches seem to occur more frequently where the vessel is accompanied longitudinally by two or more parallel main processes. Other processes have less uniformly distributed side branches, but notably some of them encircle the entire visible portion of the blood vessel (Fig. 4, arrows). Circumferential branches may also arise directly from cell bodies and surround the vessels to a variable extent. Some processes run parallel to the vessel's long axis, but suddenly shift to a different circumferential position. In this case, the two portions of the long branch are joined by a short circumferential side branch (Fig. 3). Several main processes may run parallel to each other over the same segment of the vessel, but their number cannot be determined from SEM images, since only half of the surface can be photographed. In order to assure ourselves that these long branches are common, we determined the average length of pericyte branches by a simple morphometric analysis (Abercrombie, 1946) on thin cross sections. This is feasible due to the preferred orientation of the structures. Randomness was insured by taking photographs of the entire section, where not covered by grid bars. All profiles of small blood vessels which were cut transversely, or nearly so, were scored for the presence or absence of nuclear and nonnuclear pericyte profiles (see Fig. 5). Table 1 (a to d) gives the actual counts. Pericyte nuclear length was measured from three longitudinally cut nuclei; Table 1 (e) gives the average of the three. The results are shown in Table 1. From this we calculate the following (see Fig. 5):
D
~i~ C ,
D
FIG. 5. Diagram of two hypothetical adjacent pericytes along a capillary. Letters A - D mark distances between obvious landmarks (see text).
366
MAZANET AND FRANZINI-ARMSTRONG TABLE 1 COUNTS OF NUCLEAR AND NONNUCLEAR PERICYTE PROFILES a b c d e
Total capillary profiles N o pericyte N o n n u c l e a r pericyte Nuclear pericyte Pericyte nuclear length
243 44 188 11 9 ~m
(a) average center-to-center distance between adjacent pericyte nuclei (A), a
e~ = 199 fxm; (b) average distance covered by the longest branches of two adjacent pericytes (B minus C), ¢
e~ = 154 ~m; (c) average length of longest pericyte branches (D), c
e~-~ = 77 txm; (d) percentage of capillary length covered by at least one pericyte branch and/ or cell body, c+d - -
a
100%
=
82%.
Thus, the majority of branches must be quite long and most of the capillary surface in this red muscle is covered by at least one branch. In thin sections, the small secondary processes contain bundles of microfilaments oriented circumferentially to the blood vessel (Fig. 2). Larger profiles are cross sections of the main branches and also contain microfilaments. Many of these run roughly parallel to the long axis of the blood vessel. DISCUSSION One of the most widely accepted theories on the control of the microcirculation is that blood flow through individual segments is controlled by precapillary sphincters, which are composed of smooth muscle cells, and located at sites where capillaries arise from metarterioles (Zweifach, 1973). This theory disregards not only the possible role of pericytes in the local control of circulation, but also the fact that at least in some tissues there is a graded morphological continuum of perivascular cells from smooth muscle around arterioles, to pericytes around capillaries and venules (Zimmermann, 1923). On the other hand, the interesting possibility that pericytes may have a direct role in local control of microcirculation in some tissues gains credibility from numerous lines of evidence. First of all, pericyte processes, although small, can
PERICYTES IN RAT RED MUSCLE
367
be quite extensive (Zimmermann, 1923; Wolter, 1962; Forbes et al., 1977a; Williamson et al., 1980; this report), a fact that has been disregarded in some of the literature (see Rhodin for recent review, 1980). Secondly, there is an intimate association and possible attachment between pericytes and endothelial cells (Rhodin, 1968, 1974; Weibel, 1974; Matsusaka, 1975; Forbes et al., 1977a; Ryan et al., 1979; Wallow and Burnside, 1980; this report). Thirdly, there are small sympathetic nerve endings in proximity of pericytes (Majno et aI., 1969; Forbes et al., 1977b; Tilton et al., 1979a). Finally, pericytes contain ordered bundles of thin filaments (Forbes et al., 1977a), which are shown to contain actin (LeBeux and Willemot, 1978; Wallow and Burnside, 1980). In the richly vascularized red bundle of the rat sternomastoid, about 82% of the length of each small blood vessel is covered by at least one pericytic profile. The main arrangement is longitudinal with frequent circumferential branches and "choking points." This arrangement provides thorough pericyte coverage, a n d an appropriate disposition for constricting the blood vessel. A contraction of the longitudinal main branches could result in forces obliging the capillary to buckle along its length. Contraction of the side arms, particularly where they are more extensive, would result in wrinkling of the underlying endothelial cells and possibly in reduction of the vessel lumen. Interestingly, both effects have been noted in other tissues following either stimulation of the sympathetic nervous sy~stem (Saunders et al., 1940), or application of vasoactive agents (Chambers and Zweifach, 1944; Majno et al., 1969). Tilton et al. (1979b) have shown that, in particular, the wrinkling of the endothelial cell surface upon application of vasoconstrictor agents is limited to areas immediately below pericyte processes. Clearly, however, the importance and the function of such a regulatory role may vary greatly from one microcirculatory bed to another, and even along the same bed. There are large known variations in the extent to which pericytes cover blood vessels in different tissues, in homologous tissues from different species, and along different portions of the same circulatory bed (Zimmermann, 1923; Wolter, 1962; Weibel, 1974; Tilton et al., 1979a). Pericytes in different tissues may vary in their innervation (Forbes et al., 1977b; Tilton et al., 1979a) and in their responsiveness to drugs (Tilton et al., 1979b). Additionally, pericytes along different areas of the same circulatory bed have different immunoreactivities to myosin antibodies (Drenckhahn et al., 1980). Skeletal muscle is one of the tissues where pericyte coverage of small blood vessels is most extensive, and, interestingly, it is also a tissue where the existence of precapillary sphincters has not been proven (see Zweifach, 1973). Hence, a direct control at the level of postarteriolar small vessels is most likely to exist in this tissue. ACKNOWLEDGMENTS Supported by MDA (H. M. Watts Research Center) and NIH (HL 15835-08) to the Pennsylvania Muscle Institute. The work presented in this paper will be included in a dissertation to be submitted to the Graduate Faculty of the University of Pennsylvania by R.M. in partial fulfillment of the requirements for the Ph.D.
368
MAZANET AND FRANZINI-ARMSTRONG
REFERENCES ABERCROMmE, M. (1946). Estimation of nuclear population from microtome sections. Anat. Rec. London 94, 239-247. ALTURA, B. M. (1978). Pharmacology of venular smooth muscle: New insights. Mierovasc. Res. 16, 91-117. CHAMBERS, R., AND ZWEIEACH,B. W. (1944). Topography and function of the mesenteric capillary circulation. Amer. J. Anat. 75, 173-205. DRENCKHAHN, D., GROSCHEL-STEWART, U., AND STUMPH, B. (1980). Different affinities of antigizzard myosin and anti-uterus myosin to endothelial cells and pericytes of the rat lymph node and brain. J. Muscle Res. Cell Motil. 1, 485-486. DOLnVNTY, A. F. (1977). K ÷ -contractures and membrane potential in mammalian skeletal muscle. Nature (London) 266, 75-78. FORBES, M. S., RENNELS, M. C., ANDNELSON, E. (1977a). Ultrastructure of pericytes in mouse heart. Amer. J. Anat. 149, 47-70. FORBES,M. S., RENNELS,M. C., ANDNELSON, E. (1977b). Innervation of myocardial microcirculation: Terminal autonomic axons associated with capillaries and postcapillary venules in mouse heart. Amer. J. Anat. 149, 71-91. KROGrt, A. (1929). "The Anatomy and Physiology of Capillaries." Yale Univ. Press, New Haven. LEBEUx, Y. J., AND WILLEMOT, J. (1978). Actin and myosin-like_filaments in rat brain pericytes. Anat. Rec. 190, 811-826. MAJNO, G. (1965). The ultrastructure of the vascular membrane. In "Handbook of Physiology: Circulation" (W. F. Hamilton and P. Dow, eds.), Vol. III, Sect., 2, pp. 2293-2375. Amer. Physiol. Soc., Washington, D.C. MAJNO, G., SHEA, S. M., AND LEVENTaAL, M. (1969). Endothelial contraction induced by histamine type mediators: An electron microscopic study. J. Cell Biol. 46, 647-672. MATSUSAKA,T. (1975). Tridimensional views of the relationship of pericytes to endothelial cells of capillaries in the human choroid and retina. J. Electron Microsc. 24, 13-18. MATTER,A., ORO, L., AND ROUILLER,C. (1969). Die dreidimensionale Rekonstuktion des Kapillarperiziten im Muskel. Verh. Anat. Ges. 63, 125-130. MAZANET, R., REESE, B. F., FRANZlm-ARMSTRONG,C., AND REESE, T. S. (1979). SEM of satellite cells and their response to muscle fiber injury. J. Cell Biol. 83, 383a. MAZANET, R., AND FRANZIM-ARMSTRONG,C. (1980). SEM of vascular pericytes in rat red muscle. J. Cell Biol. 87, 259a. RHODIN, J. A. G. (1968). Ultrastructure of mammalian venous capillaries, venules, and small collecting veins. J. Ultrastruct. Res. 25, 452-500. RHODIN, J. A. G. (1974). "Histology: A Text and Atlas," pp. 331-370. Oxford Univ. Press, New York. RHODIN, J. A. G. (1980). Architecture of the vessel wall. In "Handbook of Physiology: The Cardiovascular System" (D. F. Bohr, A. P. Somlyo, and H. V. Sparks, eds.), Vol. II, Sect., 2, pp. 1-31. Amer. Physiol. Soc., Washington, D.C. ROSELL, S. (1980). Neuronal control of microvessels. Annu. Rev. Physiol. 42, 359-371. ROOGET, C. (1873). Memoire sur le development, la structure et les proprietes physiologiques des capillaires sanguins et lymphatiques. Arch. Physiol. Norm. Pathol. 5, 603-663. RYAN, U. S., RYAN, J. W., AND WHITAKER,C. (1979). How do kinins affect vascular tone? Advan. Exp. Med. Biol. 102A, 237-291. SAUNDERS,A. G., EBERT, R. H., AND FLOREY,H. W. (1940). The mechanism of capillary contraction. Q. J. Exp. Physiol. 30, 281-287. SHOTTON, D. M., HEUSER, J. E., REESE, B. F., AND REESE, T. S. (1977). Postsynaptic membrane folds of the frog neuromuscular junction visualized by scanning electron microscopy. Neuroscience 4, 427-435. TILTON, R. G., Kmo, C., AND WILLIAMSON,J. R. (1979a). Pericyte-endothelial relationships in cardiac and skeletal muscle capillaries. Microvasc. Res. 18, 325-335. TILTON, R. G., KILO, C., WILHAMSON,J. R., AND MURCH, D. W. (1979b). Differences in pericyte contractile function in rat cardiac and skeletal muscle microvasculatures. Microvasc. Res. 18, 336-352. UEnARA, Y., AND SUYAMA,K. (1978). Visualization of the advential aspect of the vascular smooth muscle cells under the scanning electron microscope. J. Electron Microsc. 27, 157-159.
PERICYTES IN RAT RED MUSCLE
369
WALLOW,I. H., AND BURNSIDE,B. (1980). Actin filaments in retinal pericytes and endothelial cells. Invest. Ophthalmol. Vis. Sci. 19, 1433-1441. WEIBEL, E. R. (1974). On pericytes, particularly their existence on lung capillaries. Microvasc. Res. 8, 218-235. WILLIAMSON, J. R., TILTON, R. G., KiLo, C., AND YU, S. (1980). Immunofluorescent imaging of capillaries and pericytes in human skeletal muscle and retina. Microvasc. Res. 20, 233-241. WOLTER,J. R. (1962). The pericytes of the human retina. Amer. J. Ophthalmol. 53, 981-988. ZIMMERMANN, K. W. (1923). Der feinere bau der blutcapillaren. Z. Anat. Entwicklungsgesch. 68, 29-109. ZWEIFACH, B. W. (1973). Microcirculation. Annu. Rev. Physiol. 35, 117-150.