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The Intestinal Mucosal Lymphatic in Man
Vol. 51, No.6 Printed in U.S.A.
GAs·rnoENTER OL OGY
Copyright © 1966 by The Williams & Wilkins Co.
THE INTESTINAL MUCOSAL LYMPHATIC IN MAN A light and electron microscopic study WILLI AM
0.
DoBBINS,
III ,
M . D.
GastTOintestinal Research Laboratory, Veterans Administration Hospital, and Depm·tment of Medicine, Duke University Medical Center, Durham, Nort h Carolina
Normal intestinal mucosal lymphatic ult rastructure in animals is well defined ,1 - 6 as is lymphatic ultrastructure elsewhere in the body. 7 - 9 Two reports 10 • 11 mention briefly the ultrastructure of lacteals (lymphatics that transport chylomicrons) in man, but details of structure are not given. A study of normal intestinal mucosal lymphatics is necessary in order to recognize morphological changes that may occur in disease states such as intestinal lymphangiectasia. This paper reports the ultrastructure of intestinal mucosal lymphatics in man. A companion paper will report electron microscopic changes noted in intestinal lymphatics of a patient with lymphangiectasia. Materials and Methods
Each subj ect was fasted overnight prior to biopsy. Two subj ects, young adult volunteers without known disease, were biopsied before and 45 min after instillation of emulsified corn oil into the proximal duodenum. Three subj ects, adult patients without significant disease at the Durham Veterans Administration Hospital, were biopsied after an overnight fast. All biopsy specimens were obtained perorally at the duodenoj ejunal junction using the multipurpose biopsy tube."' Immediately after excision, the specimens were placed in ice cold 3.3% osmium tetroxide buffered with 0.067 M s-collidine, 0.1 M sodium bicarbonate," or 0.05 M cacodylate. Specimens from 1 subj ect were 13
Received April 29, 1966. Accepted July 17, 1966. Address requests for reprints to: Dr. W. 0. Dobbins, III, Veterans Adm inistration Hospita l, Durham, North Carolina 27705. This investigation was supported in part by Research Grant R01 AM095507-01 from the National Institutes of Arthritis and M etabolic Diseases, United States Public H ealth Service.
fixed in ice cold 1.25% glutaraldehyde buffered in 0.067 M cacodylate and postfixed in cacodylate buffered osmium.15 Five minutes after placing the specimens in fixative, the intact specimens were removed and cut into 1mm thick slices with a sharp razor blade and returned to the fixing solution for 1 to 1\12 hr. After fixation, the specimens were dehydrated and embedded in epoxy resin.'• Sections for light microscopy were cut at 1 to 1.5 J.J. and stained with methylene blue-azure II." Thin sections were stained with aqueous uranyl acetate'" and Reynold's lead solution,'" and photographs were taken with an RCA 3F electron microscope at original magnifications of 1300 to 7250 times. Results
Light microscopic studies showed that the biopsy specimens were histologically normal. Lymphatics within t he lamina propria were rarely observed in biopsy specimens from fasting subjects and only then after multiple "thick " sections had been examined. Lymphatics of the lamina propria were located at the center of villi and were tentatively identified by t heir thin walls (figs. 1 and 2). Centra l lymphatics were more easily ident ified in biopsy specimens obtained following a fat mea l (fig. 3). Submucosal lymph atics were identified with ease by their large diameter and thin walls (fig. 4). The occasional finding of a lymphocyte and the a bsence of red blood cells within lymphatic lumina further aided in their identification . Lymphatics within the lamin a propria and submucosa were identified in all seven biopsy specimens. Two t o three lymphatics in each specimen were examined ultrastructurally. Electron microscopy of the lymphatics tentatively identified at light microscopy 994
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FIG. 1 (upper left ) . Semidiagrammatic sect ion of intestine showing from left to right venous, art eri al, lymphatic, and nerve supply to intestinal Yilli. (From Verzar and McDougall."') FIG. 2 (npper right). Light mi crograph of 1.51-' section of intestinal villi obtained from a fasting subj eet . A central lacteal is outlined by arrows in the villus on the right. A central lacteal cannot be identified in the villus on the left (osm ium fixation, methylene blue-azure II stain, X 250). FIG. 3 (lower left). Light micrograph of 1.5-1-' section of villus obtained from a subject following ingestion of a fat meal. Central lacteal,
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revealed a morphological picture similar to that previously described for lymphatics.l-11 Cen~ral lymphatics of the lamina propria consisted almost exclusively of endot heli al cells ':ithout pericytes (fig. 5). A thin, often mcomplete, basal lamina consisting of fine fil aments about 40 A in diameter surrounded the endothelial cells. The endothelium and its basal lamina, when present . . ' were mt1mately related though not directly attached to surrounding groups of collagen fibers, smooth muscle cells, nerve elements, fibrocytes, and other cells of connective tissue (figs. 5 and 6) . Collagen fibers often extended from the basal lamina of adjacent smooth muscle to the endothelial basal lamina. The endothelium was flattened, in areas only 500 A thick, except for the area containing the nucleus (figs. 5 to 7). Portions of endothelium projected into the lumen and other portions projected abluminally into surrounding connective tissue (figs. 5 and 6). Cytoplasm of the endothelial cells contained the usual cell organelles. Mitochondria, usually rounded or elliptical though occasionally elongated in shape, were more numerous near nuclei but were scattered throughout the cytoplasmic matrix (figs. 5 to 7 and 9). A Golgi complex with an adjacent centriole could occasionally be seen near nuclei (figs. 7 and 12). Fine filam ents were sparsely scattered about the cytoplasmic matrix and were occasionally bunched together (fig. 8). Many vesicles and caveolae (pinocytotic vesicles) were scattered throughout the cytopl asmic matrix (figs. 8 and 9) . The cytoplasmic matrix was considerably less dense than that of other structures of the lamina propria (figs. 5 to 12). Endoplasmic reticulum, both granular and agranular, was scanty. Frequent ribosomes were found throughout the cytoplasmic matrix. Occasional lysooutlined by arrows, is distended and more apparent than lacteal seen in figure 2 (osmium fixation, methylene blue-azure II stain, X 250). Fro. 4 (lo wer right). Light micrograph of 1.51-' section of intestinal biopsy showing prominent and easily identified submucosal lymphatic (arrows) (glutaraldehyde fix ation, methylene blueazure II X 250).
FIG. 5. Electron micrograph showing cross section of central lacteal in biopsy obtained after a fat meal. The endothelium (E) is attenuated and has numerous luminal (LP) and abluminal projections (AP). The basal lamina (arrows) is discontinuous and partially invested by collagen fibers (Go). Cell junctions (J) vary greatly in type. Nerve fibers (N) are frequent. Chylomicrons are present only without the lumen. Base of intestinal absorptive cells (A) may be seen in right upper corner (s-collidine-buffered osmium, X 7000). FIG. 6 (left). Electron micrograph showing a portion of endothelium (E) of a central lacteal with adjacent smooth muscle (SM). Note close relation of abluminal projections (AP) to basal lamina of smooth muscle and paucity of endothelial basal lamina (arrow). Endothelial cytoplasmic organelles are easily seen at this magnification. Note pinocytotic vesicles (PV), mitochondria (M), ribosomes (R), endoplasmic reticulum (ER), lysosome (L) with dense lipid deposits, and cell junction (J) showing adhesion plate (s-collidinebuffered osmium, X 10,400). FIG. 7 (upper right). Electron micrograph showing portion of endothelial nucleus (N) with prominent nucleolus (Nu). Note small Golgi apparatus (G), centriole (arrow), and
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FIGs. 6 to 8. scattered fibrils (f). Mitochondria, M; endoplasmic reticulum, ER; lumen, L (s-collidinebuffered osmium, X 12,000) . FIG. 8 (lower right). Electron micrograph of lymphatic endothelium showing a cluster of fibrils (f) surrounded by numerous pinocytotic vesicles. Note large vesicles (V), cell junctions (J), and basal lamina (arrow). Smooth muscle, SM; lumen, L (cacodylate-buffered osmium, X 10,600) . 997
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FIG. 9.
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FIG. 10 (left) . Higher magnification of cell junction shown in figure 6. There appears to be dense material in the intercellular space (arrow) and increased density along the cell membranes (M), suggesting an adhesion plate between the endothelial cells (E) . Note fibrils (f) and pinocytotic vesicles or caveolae (C) (s-collidine-buffered osmium, X 72,500). FIG. 11 (right). Electron micrograph illustrating gap (arro w ) at junction of endothelial cells (E) . Lumen, L (s-collidine-buffered osmium, X 20,000). FIG. 9. Electron micrograph showing central lacteal of villus from a fasting subject. This figure illustrates features shown in figures 5 and 6 and shows in addition two endothelial nuclei (N), numerous mitochondria (M), numerous vesicles (V) , multivesicular bodies (B), and several lysosomes (L). Note prominent investment of endothelial cells by collagen fibers (Co) but only occasional presence of basal lamina. Smooth muscle, SM; fibro cyte, F (cacodylate-buffered osmium, X 5300).
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somes (heterogeneous dense bodies), multivesicular bodies, and lipid droplets were present, usually adjacent to nuclei (fig. 9). Intercellular junctions of endothelial cells varied from interlocking to edge-toedge approximations, usually without clearly defined adhesion plates (figs. 5, 6, and 8). Occasional dense regions along cell membranes with increased intercellular density were noted at cell junctions (fig. 10). There were gaps at some junctions (fig. 11). Chylomicrons were usually external to lymphatic endothelium and were not seen within open cell junctions. Occasionally, they were seen within endothelial vesicles and within lymphatic lumina. Submucosal lymphatics were similar to lymphatics of the lamina propria. However, submucosal lymphatics had a greater diameter, were usually distended, were more prominently invested by bundles of collagen fibers, were less intimately related to smooth muscle, had thicker endothelial walls, and did not contain prominent openings at cell junctions (fig. 12). Glutaraldehyde-fixed lymphatics were similar to osmium-fixed lymphatics. Capillaries were quite similar in ultrastructure to lymphatics (fig. 13). Capillaries, however, possessed a prominent basal lamina, had a fenestrated endothelium, and had pericytes. Capillary endothelial cell junctions usually had adhesion plates, though often there was only close proximity of endothelial cells similar to that seen in the usual lymphatic cell junction. Discussion
These observations have shown that the fine structure of intestinal mucosal lymphatics in man is similar to that of intestinal lymphatics in animals. 1 - 6 Lacteals of the lamina propria in man and animals had thin walls, a poorly developed discontinuous basal lamina, a close relationship to smooth muscle and connective tissue ele-
1001
ments, and cell organelles, as described. It was difficult to identify the central lacteal at light microscopy. This may have been a result of rapid emptying and collapse of the central lacteal at the time of biopsy excision. It has been observed in vivo that minimal stimuli cause the central lacteal to empty rapidly and become invisible.1 Central lacteals were dilated and more easily identified following a fat meal, as shown in this study, or following injection of serum into the muscular wall of the gut, as shown by Papp et al.l Though no direct connections were noted, collagen fibers and smooth muscle were closely apposed to lymphatic endothelial cells and appeared to perform a supporting function, as has been described in normal capillaries.21 Abluminal endothelial projections, in particular, could serve to anchor the lymphatic wall to surrounding connective tissue. Then expansions or contractions of smooth muscle would cause concomitant widening or narrowing of the vessel lumen. Papp et aP outlined the features that distinguish intestinal lymphatics from capillaries in the cat, and these same features apply in man. Lymphatic endothelium was quite similar to capillary endothelium, except that capillaries possess a well developed, continuous basal lamina, were fenestrated, and had well defined pericytes. Lymphatics of the lamina propria were centrally located, while capillaries were peripheral.2° Lymphatics tended to be irregular in shape, while capillaries were rounded. Capillaries usually had more clearly defined adhesion plates at endothelial cell junctions and did not have gaps at cell junctions. Submucosal lymphatics were largely similar to lymphatics of the lamina propria. However, submucosal lymphatics were greater in diameter and tended to remain distended, possibly because of their much greater investment by "supporting" collagen fibers. Submucosal lymphatics did
FIG. 12. Electron micrograph of submucosal lymphatic illustrating much greater investment by collagen fibers (Co), more prominent basal lamina (arrows), and overlapping and interlocking cell junctions (J). Lumen contains some precipitated material and occasional chylomicrons (Ch), though the biopsy was obtained from a fasting subject. Nucleus, N; centriole, C; Golgi apparatus, G (bicarbonate-buffered osmium, X 10,600).
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FIG. 13. Left, electron micrograph of capillary in lamina propria. Note well developed basal lamina (arrow) about the endothelial cells (E) with scattered collagen fibers externa l to the basal lamina. Pericytes (PC) are prominent and erythrocytes are present within the lumen (s-collidine-buffered osmium, X 6750). Right, higher magnification of capi llary endothelium showing fenestration (arrow). Note the dense material in th e intereellular space at cell junction (J) (X 20,000).
not have closely associated smooth muscle and did not possess prominent openings at cell junctions. The mode of entry of chylomicrons into the lymphatic lumen has not been sett1ed.1· 3 • 4 • 6 • 10 • 11 Rubin 11 and CasleySmith6 recently reviewed this subject. Rubin pointed out the paucity in which fat particles were seen entering lacteals by any route but noted that entry between gaps in endothelial cells was more common than "transport" across the endothelium.U In contrast, in the present study , chylomicrons were not seen within cell gaps, though they were occasionally seen within endothelial
vesicles. The usual absence of chylomicrons within the lymphatic lumen after fat ingestion may have been related to rapid emptying of the lacteal at the time of biopsy. Casley-Smith 6 has suggested that lack of adhesion plates and the presence of a poorly developed basal lamina facilitated separation of lymphatic endothelia to allow passage of particles. He further suggested that these open junctions acted as inlet valves and "force pumps" with muscular contraction. Occasional lipid droplets noted within lymphatic walls may well be metabolized in situ and are not necessarily evidence of endothelial transport. 22
December 1966
INTESTINAL MUCOSAL LYMPHATIC IN MAN
Summary and Conclusions
The light and electron microscopic structure of intestinal mucosal lymphatics in man has been described and briefly compared to the structure of intestinal capillaries. The mode of entry of chylomicrons into lymphatics and their presumably rapid exit from lymphatics were discussed. REFERENCES 1. P app, M ., P . Rohlich, I. Rusznyak, and I T oro. 1962. An electron microscopic study of the central lacteal in the intestinal villus of the cat. Z. Zellforsch . 57: 475-486. 2. D eane, H . W. 1964. Some electron microscopic observations on the lamina propria of the gut, with comments on the close association of macrophages, plasma cells, and eosinophils. Anat. Rec. 149 : 453-474. 3. P alay, S. L., and L. J. K arlin . 1959. An electron microscopic study of the intestinal vi llus. I. The fasting animal. J . Biophys. Biochem . Cytol. 5: 363-372. 4. Casley-Smith, J . R. 1962. The identification of chylomicra and lipoproteins in tissue lac tea ls. J. Cell Bioi. 15: 259-277. 5 . Weiss, J. M. 1955. The role of the Golgi complex in fat absorption as studied with the electron microscope with observations on the cytology of duodenal absorptive cells. J . Exp. Med. 10fi : 775-782. 6. Casley-Smith, J. R. 1964. An electron microscopic study of injured and abnormally perm eable lymphatics. Ann. N. Y. Acad. Sc-i. 116: 803- 830. 7. Casley-Smith, J. R., and H. W. Florey. 1961. The structure of normal small lymphatics. Quart. J. Exp . Physiol. 46: 101-106. 8. French, J. E., H . M . Florey, and B. Morris. 1960. The absorpt ion of particles by the lymphatics of th e diaphragm. Quart. J. Exp. Physiol. 45: 88-103. 9. Fraley, E. E., and L. Weiss. 1961. An electron microscopic study of the lymphatic vessels in the penile skin of the rat. Amer. J. Anat. 109: 85-101. 10. Ladman, A. J., H. A. Padykula, and E. W .
11.
12.
13 .
14.
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Strauss. 1963. A morphological study of fat transport in t he normal human jejunum . Amer. J . Anat.112: 389-419. Rubin, C . E . 1966. E lectron microscopic studies of triglyceride absorption in man. Gastroenterology 50: 65- 77. Brandborg, L. L., C. E. Rubin, and W. E. Quinton, 1959. A multipurpose instrument for suction bopsy of the esophagus, stomach, small bowel and colon. Gastroenterology 37: 1-16. Bennett, H . S., and J . H. Luft. 1959. s-Collidine as a basis for buffering fix atives. J . Biophys. Biochem. Cytol. 6: 113-114. Wood, R. L., and J. H. Luft. 1965. The influ ence of buffer systems on fixation with osmium t etroxide. J . Ultrastruct. R es. 12 : 22-
45. 15. Sabatini, D. D ., K. Bensch, and R. J. Barrnett. 1963. Cytochemistry and electron micros-
copy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J . Cell Bioi. 17 : 19-58. 16. Luft, J. H . 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9 : 409-414. 17. Richardson, K. C., L. Jarrett, and E. H. Fincke. 1960. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain T echn . 35: 313-323. 18. Watson, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J . Biophys. Biochem . Cytol. 4: 727730. 19. Reynolds, E. S. 1963. The use of lead citrate
at high pH as an electron-opaque stain in electron microscopy. J. Cell Bioi. 17 : 208-212 .
20. Verzar, R ., and E. J . McDougall. 1936. Absorption from the intestine, p. 9-14. Longmans, Green, and Company, New York . 21. Luft, J . H. 1964. Fine structure of the vascular wall, p. 3-4. In Evolution of th e atherosclerotic plaque. University of Chicago Press, Chicago. 22. Suter, E . R., and G. Majno. 1965. P assage of lipid across vascular endothelium in newborn rats. J. Cell Bioi. 27 : 163-177.
GAs·rnoENTER OL OGY
Copyright © 1966 by The Williams & Wilkins Co.
THE INTESTINAL MUCOSAL LYMPHATIC IN MAN A light and electron microscopic study WILLI AM
0.
DoBBINS,
III ,
M . D.
GastTOintestinal Research Laboratory, Veterans Administration Hospital, and Depm·tment of Medicine, Duke University Medical Center, Durham, Nort h Carolina
Normal intestinal mucosal lymphatic ult rastructure in animals is well defined ,1 - 6 as is lymphatic ultrastructure elsewhere in the body. 7 - 9 Two reports 10 • 11 mention briefly the ultrastructure of lacteals (lymphatics that transport chylomicrons) in man, but details of structure are not given. A study of normal intestinal mucosal lymphatics is necessary in order to recognize morphological changes that may occur in disease states such as intestinal lymphangiectasia. This paper reports the ultrastructure of intestinal mucosal lymphatics in man. A companion paper will report electron microscopic changes noted in intestinal lymphatics of a patient with lymphangiectasia. Materials and Methods
Each subj ect was fasted overnight prior to biopsy. Two subj ects, young adult volunteers without known disease, were biopsied before and 45 min after instillation of emulsified corn oil into the proximal duodenum. Three subj ects, adult patients without significant disease at the Durham Veterans Administration Hospital, were biopsied after an overnight fast. All biopsy specimens were obtained perorally at the duodenoj ejunal junction using the multipurpose biopsy tube."' Immediately after excision, the specimens were placed in ice cold 3.3% osmium tetroxide buffered with 0.067 M s-collidine, 0.1 M sodium bicarbonate," or 0.05 M cacodylate. Specimens from 1 subj ect were 13
Received April 29, 1966. Accepted July 17, 1966. Address requests for reprints to: Dr. W. 0. Dobbins, III, Veterans Adm inistration Hospita l, Durham, North Carolina 27705. This investigation was supported in part by Research Grant R01 AM095507-01 from the National Institutes of Arthritis and M etabolic Diseases, United States Public H ealth Service.
fixed in ice cold 1.25% glutaraldehyde buffered in 0.067 M cacodylate and postfixed in cacodylate buffered osmium.15 Five minutes after placing the specimens in fixative, the intact specimens were removed and cut into 1mm thick slices with a sharp razor blade and returned to the fixing solution for 1 to 1\12 hr. After fixation, the specimens were dehydrated and embedded in epoxy resin.'• Sections for light microscopy were cut at 1 to 1.5 J.J. and stained with methylene blue-azure II." Thin sections were stained with aqueous uranyl acetate'" and Reynold's lead solution,'" and photographs were taken with an RCA 3F electron microscope at original magnifications of 1300 to 7250 times. Results
Light microscopic studies showed that the biopsy specimens were histologically normal. Lymphatics within t he lamina propria were rarely observed in biopsy specimens from fasting subjects and only then after multiple "thick " sections had been examined. Lymphatics of the lamina propria were located at the center of villi and were tentatively identified by t heir thin walls (figs. 1 and 2). Centra l lymphatics were more easily ident ified in biopsy specimens obtained following a fat mea l (fig. 3). Submucosal lymph atics were identified with ease by their large diameter and thin walls (fig. 4). The occasional finding of a lymphocyte and the a bsence of red blood cells within lymphatic lumina further aided in their identification . Lymphatics within the lamin a propria and submucosa were identified in all seven biopsy specimens. Two t o three lymphatics in each specimen were examined ultrastructurally. Electron microscopy of the lymphatics tentatively identified at light microscopy 994
D ecember 1966
INTESTINAL MUCOSAL LYMPHATIC IN MAN
FIG. 1 (upper left ) . Semidiagrammatic sect ion of intestine showing from left to right venous, art eri al, lymphatic, and nerve supply to intestinal Yilli. (From Verzar and McDougall."') FIG. 2 (npper right). Light mi crograph of 1.51-' section of intestinal villi obtained from a fasting subj eet . A central lacteal is outlined by arrows in the villus on the right. A central lacteal cannot be identified in the villus on the left (osm ium fixation, methylene blue-azure II stain, X 250). FIG. 3 (lower left). Light micrograph of 1.5-1-' section of villus obtained from a subject following ingestion of a fat meal. Central lacteal,
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revealed a morphological picture similar to that previously described for lymphatics.l-11 Cen~ral lymphatics of the lamina propria consisted almost exclusively of endot heli al cells ':ithout pericytes (fig. 5). A thin, often mcomplete, basal lamina consisting of fine fil aments about 40 A in diameter surrounded the endothelial cells. The endothelium and its basal lamina, when present . . ' were mt1mately related though not directly attached to surrounding groups of collagen fibers, smooth muscle cells, nerve elements, fibrocytes, and other cells of connective tissue (figs. 5 and 6) . Collagen fibers often extended from the basal lamina of adjacent smooth muscle to the endothelial basal lamina. The endothelium was flattened, in areas only 500 A thick, except for the area containing the nucleus (figs. 5 to 7). Portions of endothelium projected into the lumen and other portions projected abluminally into surrounding connective tissue (figs. 5 and 6). Cytoplasm of the endothelial cells contained the usual cell organelles. Mitochondria, usually rounded or elliptical though occasionally elongated in shape, were more numerous near nuclei but were scattered throughout the cytoplasmic matrix (figs. 5 to 7 and 9). A Golgi complex with an adjacent centriole could occasionally be seen near nuclei (figs. 7 and 12). Fine filam ents were sparsely scattered about the cytoplasmic matrix and were occasionally bunched together (fig. 8). Many vesicles and caveolae (pinocytotic vesicles) were scattered throughout the cytopl asmic matrix (figs. 8 and 9) . The cytoplasmic matrix was considerably less dense than that of other structures of the lamina propria (figs. 5 to 12). Endoplasmic reticulum, both granular and agranular, was scanty. Frequent ribosomes were found throughout the cytoplasmic matrix. Occasional lysooutlined by arrows, is distended and more apparent than lacteal seen in figure 2 (osmium fixation, methylene blue-azure II stain, X 250). Fro. 4 (lo wer right). Light micrograph of 1.51-' section of intestinal biopsy showing prominent and easily identified submucosal lymphatic (arrows) (glutaraldehyde fix ation, methylene blueazure II X 250).
FIG. 5. Electron micrograph showing cross section of central lacteal in biopsy obtained after a fat meal. The endothelium (E) is attenuated and has numerous luminal (LP) and abluminal projections (AP). The basal lamina (arrows) is discontinuous and partially invested by collagen fibers (Go). Cell junctions (J) vary greatly in type. Nerve fibers (N) are frequent. Chylomicrons are present only without the lumen. Base of intestinal absorptive cells (A) may be seen in right upper corner (s-collidine-buffered osmium, X 7000). FIG. 6 (left). Electron micrograph showing a portion of endothelium (E) of a central lacteal with adjacent smooth muscle (SM). Note close relation of abluminal projections (AP) to basal lamina of smooth muscle and paucity of endothelial basal lamina (arrow). Endothelial cytoplasmic organelles are easily seen at this magnification. Note pinocytotic vesicles (PV), mitochondria (M), ribosomes (R), endoplasmic reticulum (ER), lysosome (L) with dense lipid deposits, and cell junction (J) showing adhesion plate (s-collidinebuffered osmium, X 10,400). FIG. 7 (upper right). Electron micrograph showing portion of endothelial nucleus (N) with prominent nucleolus (Nu). Note small Golgi apparatus (G), centriole (arrow), and
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FIGs. 6 to 8. scattered fibrils (f). Mitochondria, M; endoplasmic reticulum, ER; lumen, L (s-collidinebuffered osmium, X 12,000) . FIG. 8 (lower right). Electron micrograph of lymphatic endothelium showing a cluster of fibrils (f) surrounded by numerous pinocytotic vesicles. Note large vesicles (V), cell junctions (J), and basal lamina (arrow). Smooth muscle, SM; lumen, L (cacodylate-buffered osmium, X 10,600) . 997
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FIG. 10 (left) . Higher magnification of cell junction shown in figure 6. There appears to be dense material in the intercellular space (arrow) and increased density along the cell membranes (M), suggesting an adhesion plate between the endothelial cells (E) . Note fibrils (f) and pinocytotic vesicles or caveolae (C) (s-collidine-buffered osmium, X 72,500). FIG. 11 (right). Electron micrograph illustrating gap (arro w ) at junction of endothelial cells (E) . Lumen, L (s-collidine-buffered osmium, X 20,000). FIG. 9. Electron micrograph showing central lacteal of villus from a fasting subject. This figure illustrates features shown in figures 5 and 6 and shows in addition two endothelial nuclei (N), numerous mitochondria (M), numerous vesicles (V) , multivesicular bodies (B), and several lysosomes (L). Note prominent investment of endothelial cells by collagen fibers (Co) but only occasional presence of basal lamina. Smooth muscle, SM; fibro cyte, F (cacodylate-buffered osmium, X 5300).
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FIG. 12.
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somes (heterogeneous dense bodies), multivesicular bodies, and lipid droplets were present, usually adjacent to nuclei (fig. 9). Intercellular junctions of endothelial cells varied from interlocking to edge-toedge approximations, usually without clearly defined adhesion plates (figs. 5, 6, and 8). Occasional dense regions along cell membranes with increased intercellular density were noted at cell junctions (fig. 10). There were gaps at some junctions (fig. 11). Chylomicrons were usually external to lymphatic endothelium and were not seen within open cell junctions. Occasionally, they were seen within endothelial vesicles and within lymphatic lumina. Submucosal lymphatics were similar to lymphatics of the lamina propria. However, submucosal lymphatics had a greater diameter, were usually distended, were more prominently invested by bundles of collagen fibers, were less intimately related to smooth muscle, had thicker endothelial walls, and did not contain prominent openings at cell junctions (fig. 12). Glutaraldehyde-fixed lymphatics were similar to osmium-fixed lymphatics. Capillaries were quite similar in ultrastructure to lymphatics (fig. 13). Capillaries, however, possessed a prominent basal lamina, had a fenestrated endothelium, and had pericytes. Capillary endothelial cell junctions usually had adhesion plates, though often there was only close proximity of endothelial cells similar to that seen in the usual lymphatic cell junction. Discussion
These observations have shown that the fine structure of intestinal mucosal lymphatics in man is similar to that of intestinal lymphatics in animals. 1 - 6 Lacteals of the lamina propria in man and animals had thin walls, a poorly developed discontinuous basal lamina, a close relationship to smooth muscle and connective tissue ele-
1001
ments, and cell organelles, as described. It was difficult to identify the central lacteal at light microscopy. This may have been a result of rapid emptying and collapse of the central lacteal at the time of biopsy excision. It has been observed in vivo that minimal stimuli cause the central lacteal to empty rapidly and become invisible.1 Central lacteals were dilated and more easily identified following a fat meal, as shown in this study, or following injection of serum into the muscular wall of the gut, as shown by Papp et al.l Though no direct connections were noted, collagen fibers and smooth muscle were closely apposed to lymphatic endothelial cells and appeared to perform a supporting function, as has been described in normal capillaries.21 Abluminal endothelial projections, in particular, could serve to anchor the lymphatic wall to surrounding connective tissue. Then expansions or contractions of smooth muscle would cause concomitant widening or narrowing of the vessel lumen. Papp et aP outlined the features that distinguish intestinal lymphatics from capillaries in the cat, and these same features apply in man. Lymphatic endothelium was quite similar to capillary endothelium, except that capillaries possess a well developed, continuous basal lamina, were fenestrated, and had well defined pericytes. Lymphatics of the lamina propria were centrally located, while capillaries were peripheral.2° Lymphatics tended to be irregular in shape, while capillaries were rounded. Capillaries usually had more clearly defined adhesion plates at endothelial cell junctions and did not have gaps at cell junctions. Submucosal lymphatics were largely similar to lymphatics of the lamina propria. However, submucosal lymphatics were greater in diameter and tended to remain distended, possibly because of their much greater investment by "supporting" collagen fibers. Submucosal lymphatics did
FIG. 12. Electron micrograph of submucosal lymphatic illustrating much greater investment by collagen fibers (Co), more prominent basal lamina (arrows), and overlapping and interlocking cell junctions (J). Lumen contains some precipitated material and occasional chylomicrons (Ch), though the biopsy was obtained from a fasting subject. Nucleus, N; centriole, C; Golgi apparatus, G (bicarbonate-buffered osmium, X 10,600).
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FIG. 13. Left, electron micrograph of capillary in lamina propria. Note well developed basal lamina (arrow) about the endothelial cells (E) with scattered collagen fibers externa l to the basal lamina. Pericytes (PC) are prominent and erythrocytes are present within the lumen (s-collidine-buffered osmium, X 6750). Right, higher magnification of capi llary endothelium showing fenestration (arrow). Note the dense material in th e intereellular space at cell junction (J) (X 20,000).
not have closely associated smooth muscle and did not possess prominent openings at cell junctions. The mode of entry of chylomicrons into the lymphatic lumen has not been sett1ed.1· 3 • 4 • 6 • 10 • 11 Rubin 11 and CasleySmith6 recently reviewed this subject. Rubin pointed out the paucity in which fat particles were seen entering lacteals by any route but noted that entry between gaps in endothelial cells was more common than "transport" across the endothelium.U In contrast, in the present study , chylomicrons were not seen within cell gaps, though they were occasionally seen within endothelial
vesicles. The usual absence of chylomicrons within the lymphatic lumen after fat ingestion may have been related to rapid emptying of the lacteal at the time of biopsy. Casley-Smith 6 has suggested that lack of adhesion plates and the presence of a poorly developed basal lamina facilitated separation of lymphatic endothelia to allow passage of particles. He further suggested that these open junctions acted as inlet valves and "force pumps" with muscular contraction. Occasional lipid droplets noted within lymphatic walls may well be metabolized in situ and are not necessarily evidence of endothelial transport. 22
December 1966
INTESTINAL MUCOSAL LYMPHATIC IN MAN
Summary and Conclusions
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