Cell Tiss. Res. 163, 169-174 (1975) - 9 by Springer-Verlag 1975
Fenestrated Blood Capillaries and Lymphatic Capillaries in Rat Skeletal Muscle Helge Korneliussen* Anatomical Institute, University of Oslo, Oslo, Norway
Summary. Capillary fenestrae occur in one of about 60 cross-sectioned blood capillaries in normal adult rat skeletal muscles. The fenestrae occur singly or in groups. Fenestrated capillaries are found close to muscle fibers as well as in the perimysial and perineurial connective tissue. Small numbers of lymphatic capillaries are also present, mostly in the perimysial connective tissue. Key words: Muscles - Blood supply - Ultrastructure.
Capillaries -
Lymphatic system
Introduction It is generally held that the blood capillaries of skeletal muscles possess a continuous complete basal lamina, lack endothelial fenestrations, and possess an incomplete pericapillary cellular investment (e.g. Bennett et al., 1959; Karnovsky, 1967; Bruns and Palade, 1968; Stingl, 1971). However, Hammersen (1966) also described infrequent capillaries of the fenestrated variety in the connective tissue of rat skeletal muscles. Lymphatic capillaries are evidently very infrequent in skeletal muscle, and are confined to the connective tissue rich portions (Yoffey and Courtice, 1970). Studies of a large number of tissue samples from rat muscles over several years have confirmed the occurrence of fenestrated capillaries, some of them situated close to muscle fibers, in addition to lymphatic capillaries, in this tissue.
Material and Methods Nine adult albino rats of both sexes were used. Small samples of muscle tissue were rapidly obtained from the diaphragm or hindlimb muscles, mostly m. soleus, after decapitation or anesthesia with ether or a barbiturate. The blocks were immediately fixed by immersion overnight in 2% paraformaldeSend offprint requests to." Dr. Helge Korneliussen, Anatomical Institute, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway. *
The author is indebted to Miss Bjorg Riber and Miss Berit Branil for technical assistance.
170
H. Korneliussen
hyde +2.5% glutaraldehyde in 0.1 or 0.15 M Na-cacodylate, pH 7.4, at 4 ~ C. After dividing the samples into suitable smaller blocks and after rinses for 89hour in the buffer, the blocks were postfixed in 1% OsO4 in the same buffer for 1-2 hours, dehydrated in acetone and embedded in TAAB Embedding Resin. Part of the material was stained en bloc with 2% uranyl acetate in 0.3 M iso-acetate buffer, pH 6.5, before dehydration. Additional material was fixed with OsO4 or aldehydes in buffers of various molarities, with various buffer rinses, at various pH's, and at various temperatures, as described in an earlier publication (Korneliussen, 1972). Ultrathin sections were stained with lead citrate and examined with a Siemens Elmiskop I.
Results
Most capillaries in rat skeletal muscles (see Fig. 2, lower left corner) match descriptions given by Bruns and Palade (1968) and Stingl (1971). A few thin-walled vessels (Fig. 1) are evidently lymphatic capillaries (CasleySmith and Florey, 1961 ; Leak and Burke, 1968). These are considerably larger (diameter over about 8 pm) than blood capillaries. Their lumina do not contain profiles of sectioned erythrocytes. The endothelial lining of these lymphatic capillaries is thin and irregular, often with microvilli-like projections into the lumen. No fenestrae or discontinuities have been found. The endothelial cells lack a basal lamina. Anchoring filaments (see Leak and Burke, 1968) extend outwards from the outer leaflet of the external plasma membrane at patches where the cytoplasmic side of the membrane is especially dense. Pericytic cell processes are very sparse. Lymphatic capillaries occur for the most part in the perimysial connective tissue, but also very rarely close to muscle fibers. Fenestrated blood capillaries are seen more often than lymphatic capillaries. In transverse sections through muscles, one out of every 60 cross-sectioned capillaries possesses fenestrae (Figs. 2-5) in all nine animals. Material from many additional rats has confirmed the universal occurrence of fenestrated capillaries in rat skeletal muscles. No differences were noted with regard to the occurrence of fenestrated capillaries between the sexes, between different muscles, or between samples obtained with different preparative procedures. Fenestrated capillaries occur in the perimysial connective tissue and in the connective tissue that accompanies intramuscular nerves, as reported by Hammersen (1966), but even more often close to muscle fibers (Figs. 2, 3, 5). Fenestrae occur only in attenuated portions of endothelial cells, where the thickness is comparable to the diameter of cytoplasmic vesicles (Figs. 2-5). The diameters of the fenestrae are 600-800 A, and the fenestrae are always closed by a diaphragm which is thickest (about 60 A) at the centre of the pore. The fenestrae may occur singly (Figs. 3, 4) or in groups (Figs. 2, 5), and up to 20 fenestrae have often been found in one cross-sectioned capillary. The fenestrae face towards (Figs. 2, 5) or away from (Fig. 3) muscle fibers with equal frequency. Sometimes (Fig. 5) the region outside
Fig. 1. Lymphatic capillary from rat soleus muscle. 1, lumen. • 18000 Fig. 2. Fenestrae (black triangles) in capillary interposed between two soleus muscle fibers (m) of rat. Another capillary in lower left corner possesses a continuous endothelium, e, erythrocyte, p, pericyte. x 18000
Muscle Microvessels
171
172
H. Korneliussen
Muscle Microvessels
173
the fenestrated portion of a capillary is occupied by a finely granular substance which appears similar to the blood plasma and which may obscure the basal lamina.
Discussion
For some time there has been a tendency to differentiate between morphological types of capillaries (e.g. Bennett et al., 1959). In particular the presence or absence of fenestrae has been regarded as a major distinguishing feature. The demonstration of fenestrae even in muscle capillaries, classically considered nonfenestrated (Bennett et al., 1959; Bruns and Palade, 1968; Stingl, 1971), tends to weaken the hitherto strict distinction drawn between fenestrated and nonfenestrated capillaries. This fits with the dynamic view on the formation of fenestrae expressed by Palade and Bruns (1968) and Maul (1971), involving fusion of cytoplasmic vesicles with both luminal and abluminal plasma membrane in attenuated regions of endothelial cells. Cytoplasmic vesicles are numerous in muscle capillaries, whereas attenuated portions of endothelial cells where the thickness is similar to the diameter of vesicles and where fenestrae are observed, occur rarely. Thus, as a speculation, the capability for forming thin cytoplasmic sheets may determine the number of fenestrae in capillaries. In addition, as a further implication of this dynamic view, the fenestrae may appear in special cases of the transendothelial channels reported by Simionescu et al. (1975) to form by fusion of chains of vesicles. They found that some of the vesicle channel stomata possess diaphragms. The relatively rare occurrence of fenestrae in muscle capillaries may indicate that only some endothelial cells in this tissue are capable of forming fenestrae at the time of fixation, either due to the fixation procedure or due to factors inherent locally in the tissue. The incidence of fenestrated capillaries noted in the present material, i.e. one per about 60 cross-sectioned capillaries, means that each endothelial cell theoretically can hold several fenestrated portions if endothelial cells are as large as thought (about 10 x 30 ~tm; see Bruns and Palade, 1968). Thus, whether only some or all endothelial cells possess fenestrated portions remains an open question. The extent of fenestration in muscle capillaries is certainly by far exceeded by that in "typical" fenestrated capillaries in renal glomeruli, intestinal villi, and endocrine glands. However, the fenestrated portions of muscle capillaries must also represent areas with high permeability for large molecules as well as for water and electrolytes (see Clementi and Palade, 1969; Simionescu et al., 1975). Therefore, in future studies of the permeability of muscle capillaries and of interstitial fluid dynamics in muscle, the fenestrated portions of the capillaries as well as the presence of lymphatic capillaries must be taken into consideration. Figs. 3 - 5 . Details of higher magnification from fenestrated portions of capillaries from rat soleus muscle. In Figs. 3 4 single fenestrae (black triangles) face away from muscle fibers (m), whereas multiple fenestrae (black triangles) in Fig. 5 face towards two muscle fibers (m). e, erythrocyte, l, lumen, p, pericyte. • 18000
174
H. Korneliussen
References Bennett, H.S., Luft, J.H., Hampton, J.C.: Morphological classification of vertebrate capillaries. Amer. J. Physiol. 196, 381-390 (1959) Bruns, R.R., Palade, G.E. : Studies on blood capillaries. I. General organization of blood capillaries in muscle. J. Cell Biol. 37, 244-276 (1968) Casley-Smith, J.R., Florey, H.W. : The structure of normal small lymphatics. Quart. J. exp. Physiol. 46, 101-106 (1961) Clementi, F., Palade, G.E. : Intestinal capillaries. I. Permeability to peroxidase and ferritin. J. Cell Biol. 41, 33-58 (1969) Hammersen, F. :Poren- und Fenster-Endothelien der Kapillaren in der Skeletmuskulatur der Ratte. Z. Zellforsch. 69, 296-310 (1966) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Korneliussen, H. : Elongated profiles of synaptic vesicles in motor endplates. Morphological effects of fixative variations. J. Neurocytol. 1,279-296 (1972) Leak, L.V., Burke, J.F. : Ultrastructural studies on the lymphatic anchoring filaments. J. Cell Biol. 36, 129-149 (1968) Maul, G.G. : Structure and formation of pores in fenestrated capillaries. J. Ultrastruct. Res. 36, 768-782 (1971) Palade, G.E., Bruns, R.R. : Structural modulations of plasmalemmal vesicles. J. Cell Biol. 37, 633~49 (1968) Simionescu, N., Simionescu, M., Palade, G.E. : Permeability of muscle capillaries to small hemipeptides. Evidence for the existence of patent transendothelial channels. J. Cell Biol. 64, 586 607 (1975) Stingl, J. : Zur Ultrastruktur des terminalen Gefhl3bettes der Skeletmuskulatur. Acta anat. (Basel) 80, 255-272 (1971) Yoffey, J.M., Courtice, F.C. : Lymphatics, lymph and the lymphomyeloid complex. London : Academic Press 1970
Received May 20, 1975 / Accepted July 26, 1975
Fenestrated Blood Capillaries and Lymphatic Capillaries in Rat Skeletal Muscle Helge Korneliussen* Anatomical Institute, University of Oslo, Oslo, Norway
Summary. Capillary fenestrae occur in one of about 60 cross-sectioned blood capillaries in normal adult rat skeletal muscles. The fenestrae occur singly or in groups. Fenestrated capillaries are found close to muscle fibers as well as in the perimysial and perineurial connective tissue. Small numbers of lymphatic capillaries are also present, mostly in the perimysial connective tissue. Key words: Muscles - Blood supply - Ultrastructure.
Capillaries -
Lymphatic system
Introduction It is generally held that the blood capillaries of skeletal muscles possess a continuous complete basal lamina, lack endothelial fenestrations, and possess an incomplete pericapillary cellular investment (e.g. Bennett et al., 1959; Karnovsky, 1967; Bruns and Palade, 1968; Stingl, 1971). However, Hammersen (1966) also described infrequent capillaries of the fenestrated variety in the connective tissue of rat skeletal muscles. Lymphatic capillaries are evidently very infrequent in skeletal muscle, and are confined to the connective tissue rich portions (Yoffey and Courtice, 1970). Studies of a large number of tissue samples from rat muscles over several years have confirmed the occurrence of fenestrated capillaries, some of them situated close to muscle fibers, in addition to lymphatic capillaries, in this tissue.
Material and Methods Nine adult albino rats of both sexes were used. Small samples of muscle tissue were rapidly obtained from the diaphragm or hindlimb muscles, mostly m. soleus, after decapitation or anesthesia with ether or a barbiturate. The blocks were immediately fixed by immersion overnight in 2% paraformaldeSend offprint requests to." Dr. Helge Korneliussen, Anatomical Institute, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway. *
The author is indebted to Miss Bjorg Riber and Miss Berit Branil for technical assistance.
170
H. Korneliussen
hyde +2.5% glutaraldehyde in 0.1 or 0.15 M Na-cacodylate, pH 7.4, at 4 ~ C. After dividing the samples into suitable smaller blocks and after rinses for 89hour in the buffer, the blocks were postfixed in 1% OsO4 in the same buffer for 1-2 hours, dehydrated in acetone and embedded in TAAB Embedding Resin. Part of the material was stained en bloc with 2% uranyl acetate in 0.3 M iso-acetate buffer, pH 6.5, before dehydration. Additional material was fixed with OsO4 or aldehydes in buffers of various molarities, with various buffer rinses, at various pH's, and at various temperatures, as described in an earlier publication (Korneliussen, 1972). Ultrathin sections were stained with lead citrate and examined with a Siemens Elmiskop I.
Results
Most capillaries in rat skeletal muscles (see Fig. 2, lower left corner) match descriptions given by Bruns and Palade (1968) and Stingl (1971). A few thin-walled vessels (Fig. 1) are evidently lymphatic capillaries (CasleySmith and Florey, 1961 ; Leak and Burke, 1968). These are considerably larger (diameter over about 8 pm) than blood capillaries. Their lumina do not contain profiles of sectioned erythrocytes. The endothelial lining of these lymphatic capillaries is thin and irregular, often with microvilli-like projections into the lumen. No fenestrae or discontinuities have been found. The endothelial cells lack a basal lamina. Anchoring filaments (see Leak and Burke, 1968) extend outwards from the outer leaflet of the external plasma membrane at patches where the cytoplasmic side of the membrane is especially dense. Pericytic cell processes are very sparse. Lymphatic capillaries occur for the most part in the perimysial connective tissue, but also very rarely close to muscle fibers. Fenestrated blood capillaries are seen more often than lymphatic capillaries. In transverse sections through muscles, one out of every 60 cross-sectioned capillaries possesses fenestrae (Figs. 2-5) in all nine animals. Material from many additional rats has confirmed the universal occurrence of fenestrated capillaries in rat skeletal muscles. No differences were noted with regard to the occurrence of fenestrated capillaries between the sexes, between different muscles, or between samples obtained with different preparative procedures. Fenestrated capillaries occur in the perimysial connective tissue and in the connective tissue that accompanies intramuscular nerves, as reported by Hammersen (1966), but even more often close to muscle fibers (Figs. 2, 3, 5). Fenestrae occur only in attenuated portions of endothelial cells, where the thickness is comparable to the diameter of cytoplasmic vesicles (Figs. 2-5). The diameters of the fenestrae are 600-800 A, and the fenestrae are always closed by a diaphragm which is thickest (about 60 A) at the centre of the pore. The fenestrae may occur singly (Figs. 3, 4) or in groups (Figs. 2, 5), and up to 20 fenestrae have often been found in one cross-sectioned capillary. The fenestrae face towards (Figs. 2, 5) or away from (Fig. 3) muscle fibers with equal frequency. Sometimes (Fig. 5) the region outside
Fig. 1. Lymphatic capillary from rat soleus muscle. 1, lumen. • 18000 Fig. 2. Fenestrae (black triangles) in capillary interposed between two soleus muscle fibers (m) of rat. Another capillary in lower left corner possesses a continuous endothelium, e, erythrocyte, p, pericyte. x 18000
Muscle Microvessels
171
172
H. Korneliussen
Muscle Microvessels
173
the fenestrated portion of a capillary is occupied by a finely granular substance which appears similar to the blood plasma and which may obscure the basal lamina.
Discussion
For some time there has been a tendency to differentiate between morphological types of capillaries (e.g. Bennett et al., 1959). In particular the presence or absence of fenestrae has been regarded as a major distinguishing feature. The demonstration of fenestrae even in muscle capillaries, classically considered nonfenestrated (Bennett et al., 1959; Bruns and Palade, 1968; Stingl, 1971), tends to weaken the hitherto strict distinction drawn between fenestrated and nonfenestrated capillaries. This fits with the dynamic view on the formation of fenestrae expressed by Palade and Bruns (1968) and Maul (1971), involving fusion of cytoplasmic vesicles with both luminal and abluminal plasma membrane in attenuated regions of endothelial cells. Cytoplasmic vesicles are numerous in muscle capillaries, whereas attenuated portions of endothelial cells where the thickness is similar to the diameter of vesicles and where fenestrae are observed, occur rarely. Thus, as a speculation, the capability for forming thin cytoplasmic sheets may determine the number of fenestrae in capillaries. In addition, as a further implication of this dynamic view, the fenestrae may appear in special cases of the transendothelial channels reported by Simionescu et al. (1975) to form by fusion of chains of vesicles. They found that some of the vesicle channel stomata possess diaphragms. The relatively rare occurrence of fenestrae in muscle capillaries may indicate that only some endothelial cells in this tissue are capable of forming fenestrae at the time of fixation, either due to the fixation procedure or due to factors inherent locally in the tissue. The incidence of fenestrated capillaries noted in the present material, i.e. one per about 60 cross-sectioned capillaries, means that each endothelial cell theoretically can hold several fenestrated portions if endothelial cells are as large as thought (about 10 x 30 ~tm; see Bruns and Palade, 1968). Thus, whether only some or all endothelial cells possess fenestrated portions remains an open question. The extent of fenestration in muscle capillaries is certainly by far exceeded by that in "typical" fenestrated capillaries in renal glomeruli, intestinal villi, and endocrine glands. However, the fenestrated portions of muscle capillaries must also represent areas with high permeability for large molecules as well as for water and electrolytes (see Clementi and Palade, 1969; Simionescu et al., 1975). Therefore, in future studies of the permeability of muscle capillaries and of interstitial fluid dynamics in muscle, the fenestrated portions of the capillaries as well as the presence of lymphatic capillaries must be taken into consideration. Figs. 3 - 5 . Details of higher magnification from fenestrated portions of capillaries from rat soleus muscle. In Figs. 3 4 single fenestrae (black triangles) face away from muscle fibers (m), whereas multiple fenestrae (black triangles) in Fig. 5 face towards two muscle fibers (m). e, erythrocyte, l, lumen, p, pericyte. • 18000
174
H. Korneliussen
References Bennett, H.S., Luft, J.H., Hampton, J.C.: Morphological classification of vertebrate capillaries. Amer. J. Physiol. 196, 381-390 (1959) Bruns, R.R., Palade, G.E. : Studies on blood capillaries. I. General organization of blood capillaries in muscle. J. Cell Biol. 37, 244-276 (1968) Casley-Smith, J.R., Florey, H.W. : The structure of normal small lymphatics. Quart. J. exp. Physiol. 46, 101-106 (1961) Clementi, F., Palade, G.E. : Intestinal capillaries. I. Permeability to peroxidase and ferritin. J. Cell Biol. 41, 33-58 (1969) Hammersen, F. :Poren- und Fenster-Endothelien der Kapillaren in der Skeletmuskulatur der Ratte. Z. Zellforsch. 69, 296-310 (1966) Karnovsky, M.J.: The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol. 35, 213-236 (1967) Korneliussen, H. : Elongated profiles of synaptic vesicles in motor endplates. Morphological effects of fixative variations. J. Neurocytol. 1,279-296 (1972) Leak, L.V., Burke, J.F. : Ultrastructural studies on the lymphatic anchoring filaments. J. Cell Biol. 36, 129-149 (1968) Maul, G.G. : Structure and formation of pores in fenestrated capillaries. J. Ultrastruct. Res. 36, 768-782 (1971) Palade, G.E., Bruns, R.R. : Structural modulations of plasmalemmal vesicles. J. Cell Biol. 37, 633~49 (1968) Simionescu, N., Simionescu, M., Palade, G.E. : Permeability of muscle capillaries to small hemipeptides. Evidence for the existence of patent transendothelial channels. J. Cell Biol. 64, 586 607 (1975) Stingl, J. : Zur Ultrastruktur des terminalen Gefhl3bettes der Skeletmuskulatur. Acta anat. (Basel) 80, 255-272 (1971) Yoffey, J.M., Courtice, F.C. : Lymphatics, lymph and the lymphomyeloid complex. London : Academic Press 1970
Received May 20, 1975 / Accepted July 26, 1975
