THE ANATOMICAL RECORD 233~547-554(1992)
Forms of Lung Lymphatics: A Scanning Electron Microscopic Study of Casts DEAN E. SCHRAUFNAGEL Departments of Medicine and Pathology, University of Illinois at Chicago
ABSTRACT In a recent study, rats given monocrotaline underwent angiogenesis on their pleural surfaces. The rats also had novel structures in their bronchovascular bundles that were detected by scanning electron microscopy of vascular casts. These vessels could have been either new blood capillaries or dilated lymphatic capillaries. To determine if these structures were lymphatics or new blood vessels, specimens from animals that were undergoing angiogenesis were compared to those that were not. Finding similar structures in normal animals would imply that they were lymphatic. The second purpose of this work was to describe the three-dimensional anatomy of the lymphatics of the lung. Cast lymphatics were found in most lungs with edema or angiogenesis, but were rare in other conditions. The vascular structures in question were found in animals not undergoing angiogenesis and were, therefore, lymphatic. Additionally, scanning electron microscopy of casts showed several distinct forms of lymphatics in the lung. Prelymphatics are tissues planes beneath the pleura and around bronchovascular structures. They join reservoir, conduit or tubulo-saccular lymphatics. Reservoir lymphatics are broad ribbon-like structures with textured surfaces and small laterally branching pouches. They occur on the pleural surface, are closely linked with prelymphatics, and join conduit lymphatics. Conduit lymphatics are tubular structures that may contain valves, twist and go great distances without accepting tributaries. On the pleural surface, they may wind around blood vessels and vary greatly in diamater. Sacculo-tubular lymphatics surround arteries, veins and bronchioles. They have thin walls with wide saccular segments. They may be so dense that they form cylinders around the vessels or airways. Different forms of lung lymphatics suggest different function and potential. 0 1992 Wiley-Liss, Inc.
In a recent study of lung vascular casts by scanning electron microscopy, I showed that rats given the pyrrolizidine alkaloid, monocrotaline, underwent angiogenesis on their pleural surfaces. The new vessels were typical fully differentiated arteries and veins. In addition, I found previously unreported, unusually shaped capillaries around arteries that could have been either novel blood capillaries or dilated lymphatic capillaries (Schraufnagel, 1990a). Scanning electron microscopy of casts is a valuable tool to study the threedimensional ultrastructure of small vessels, but images of neither lymphatic nor developing blood vessels of the lung had been reported previously, so that one could not be certain what the structures were. To find out if they were lymphatic or new blood vessels, specimens from animals that were not undergoing angiogenesis were compared to those from the monocrotaline study. The animals that were not undergoing angiogenesis were normal rats given a head blow to produce neurogenic pulmonary edema (Schraufnagel and Patel, 1990). If similar structures could be found in animals that were not undergoing angiogenesis, I would conclude that they were lymphatic. I also sought to describe the three-dimensional anatomy of the lymphatics of the lung. To do this I screened cast specimens 0 1992 WILEY-LISS, INC
from other studies to chart lymphatic shapes and associations. MATERIALS AND METHODS
Specimens for the group with angiogenesis were taken from a previous study of rats given monocrotaline (Schraufnagel, 1990a). They were compared with specimens from a study of normal rats that received a blow to the head to produce neurogenic pulmonary edema (Schraufnagel and Patel, 1990). In addition, several hundred micrographs of cast lung vasculature from other published (Schraufnagel 1989a,b; Schraufnagel et al., 1986; Schraufnagel and Schmid 1988a,b,c, 1989) and pilot studies were reviewed to search for lymphatic casts. These studies had more than 150 rats, although not all lungs cast well enough to study lymphatics. The angiogenic group had six animals. The methods have been described in detail (Schrauf-
Received July 30, 1991; accepted January 9, 1992. Address reprint requests to Dean E. Schraufnagel, M.D., Section of Respiratory and Critical Care Medicine, Department of Medicine MIC 787, University of Illinois a t Chicago, P.O. Box 6998, Chicago, IL 60680-6998.
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nagel, 1987,1990b).In brief, all animals were SpragueDawley rats weighing about 180-400 g. In the study of angiogenesis, animals received 80 mg/kg of monocrotaline (Transworld Chemical, Washington, DC) and were killed 19 or 20 days later. Each animal was anesthetized with pentobarbital. The abdomen was opened and the caudal vena cava and aorta were cannulated with a 20 gauge catheter. Warmed heparinized saline infused into the vena cava until the aortic effluent was clear. Twenty ml of partially polymerized methylmethacrylate (Mercox, Ladd Research Industries, Burlington, VT) diluted 4 parts to 1 with n-methylmethacrylate monomer (SPI-Chem, West Chester, PA) was mixed with a catalyst and injected with a hand-held syringe through the vena cava to fill the pulmonary vasculature over one minute. The animals with neurogenic pulmonary edema were normal similar rats that did not receive monocrotaline. They were cast with undiluted Mercox. Just after their lungs were filled, but before the resin set, they received a sharp blow to the head. Light microscopy of animals that received a similar blow to the head, but were not cast, showed proteinaceous and hemorrhagic pulmonary edema (Schraufnagel and Patel, 1990). In all animals, the plastic was allowed to harden for 1hour. One piece of tissue was sent for light microscopy and the rest of the lungs were placed in a sodium hydroxide solution until the tissue corroded. The casts were rinsed and cut with scissors or razor blades into pieces about 1 mm thick. The specimens were fastened to aluminum studs with double-sided tape or silver cement, sputter-coated with palladium-gold, and viewed with a JEOL, (Tokyo) JSM-35C, scanning electron microscope. The accelerating voltage was 15 kV. The working distance was 15 mm. RESULTS
Both the animals with angiogenesis and those with neurogenic edema had extensive and diverse lymphatic casts, although some animals in both groups also had none. The only other animals that regularly had cast lymphatics were those with prolonged vascular rinsing with saline before casting (Schraufnagel and Schmid, 1988a). The lymphatics in all groups were structurally similar. The bronchovascular structure in question was found in the lungs with pulmonary edema, indicating that it was lymphatic and not neovascular. There was no evidence for new blood vessels in the animals with pulmonary edema. The lungs that had cast lymphatics that could be seen with scanning electron microscopy also had dilated lymphatics that could be seen with light microscopy. Scanning electron microscopy showed many forms of lymphatics in the lungs. Casts from different areas had distinct structures. The interstitial spaces or tissue planes that receive fluid from capillaries or alveoli and the interstitial space beneath the pleura have been calledprelymphatics by von Hayek (1960)and others. In the casting preparation, these spaces filled well and had a characteristic appearance. At low magnification, they were films of plastic contained by the visceral pleura (Fig. 1).Although they could nearly cover the lung, they were usually found as patches along the pleura and occasionally around large vessels. They were never around an individual alveolus in the lung
Fig. 1. The pleural surface of a cast at low magnification shows a sheet o f plastic contained within the visceral pleura. It is easily separable from the cast o f the blood capillaries. No plastic was found in the parietal pleura or in the thoracic cavity, indicating that the plastic is just beneath the pleura. These sheets were found in both the animals given monocrotaline (the group undergoing angiogenesis) and those with neurogenic pulmonary edema and rarely in saline control animals, which this animal was. Casts o f the tissue planes have been called prelymphatics because they join lymphatics. Original magnification was 20 x . Bar = 1,000 km.
parenchyma. The casts of prelymphatics were marked with connective tissue strands across their surface (Figs. 2, 3). Although prelymphatics occasionally drained directly into conduit lymphatics (Fig. 41, they usually connected to other structures, not previously reported, called reservoir lymphatics. These flat ribbon-like structures, about 15-40 pm in width, had small pouches branching laterally from their long axes (Fig. 5 ) . Their leaf-like side branches ended blindly. They did not have the imprints of connective tissue strands on their surface, but the surface was textured (Fig. 6). Although the term prelymphatic was reserved for planar structures, and reservoir lymphatics had to have a linear dimension, it was often difficult to distinguish between the two because of the gradual transition from one to the other. The rough surfaces of prelymphatics and reservoir lymphatics also distinguished them from blood capillaries. Reservoir lymphatics connected to the conduit lymphatic vessels (Fig. 7). The conduit lymphatic vessels were usually round on the pleural surface (Fig. 8) and often flattened within the lung. On the pleural surface, they were about the same size as the small blood vessels they tracked, but their diameters varied several-fold, from less than 5 pm to more than 35 pm, within a few hundred micrometers of length. They were often tortuous, sometimes helical, and wound around blood vessels (Fig. 9). The conduit vessels within the lung were usually more delicate and had less variation in diameter than the pleural ducts. Conduit lymphatics rarely accepted tributaries or gave off branches, although an example of octopus-like branching is shown in Figure
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Fig. 2. This is a higher magnification of a p r e l y m p h a t i c cast of the pleural surface of an animal that received monocrotaline. The surface is indented by strands of connective tissue. The gradual transition from prelymphatics to reservoir lymphatics often makes it dificult to distinguish the two. Original magnification was 100 x . Bar = 100 pm.
Fig. 4. Although most prelymphatic casts connected to reservoir lymphatics, this prelymphatic (p) space attached to a conduit lymphatic (c).The rat had pulmonary edema. Original magnification was 600 x . Bar = 10 pm.
Fig. 3. A more magnified view of the prelymphatic cast of the animal shown in Figure 2 illustrates its textured surface. If a tubular character could be detected, it would be classified as a reservoir lymphatic. Original magnification was 220 x . Bar = 100 km.
Fig. 5. Reservoir lymphatics, on the left, have flat laterally budding structures that distinguish them from the blood capillaries on the right (B). This micrograph was taken near Figure 3 on the same specimen. Original magnification was 220 x . Bar = 100 km.
10. Often the casts of the lymphatics within the lung had longitudinal grooves and joints (Fig. 11). Within the lung, these conduit vessels often coursed in the bronchovascular bundle where they connected to, but remained separate from, tubulo-saccular lymphatics. Around large blood vessels and airways were broad tubulo-succular lymphatics (Figs. 12-15). These differed from the other forms by being large, sac-like arrangements that surrounded a vessel or airway. The saccules could be more than 10 times wider than conduit lymphatics and were more heterogenous than the reservoir lymphatics. Around certain vessels they were sparse, but in other areas they were extremely dense. The dense lymphatics formed a hollow cylinder if the inner blood vessel cast could be removed (Fig. 14). It
was one of these lymphatics t h a t was pictured in Figure 6 of the previous study of angiogenesis (Schraufnagel, 1990a). In the monocrotaline animals, the lymphatics around the arteries appeared to be more abundant than around the veins (Fig. 12), but in the animals with pulmonary edema, it appeared to be the opposite. The lymphatics around the airway had similar shapes to those around the blood vessels (Fig. 13). The size of the saccular component of these lymphatics ranged widely from 10 pm to more than 100 km, yet their walls were thin. In the monocrotaline animals, the lymphatic casts appeared to have more trapped cells, although lymphocyte remains could be found in all groups (Fig. 15).
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Fig. 6.A higher magnification micrograph of the reservoir lymphatics of Figure 5 shows a closely packed, rather tubular structure with a textured surface. Its morphology suggests it may have absoqtive, reservoir and transport functions. Original magnification was 660 x . Bar = 10 pm.
Fig, 7. The reservoir lymphatics (r) drain into a conduit lymphatic (c). The rat had pulmonary edema. Original magnification was 780 X . Bar = 10 pm.
DISCUSSION
Although this is the first demonstration by scanning electron microscopy of lung lymphatic casts, much of this has already been described. Miller (1947), von Hayek (1960), Lauweryns (1971, 1974) and many others have studied lung lymphatics by light and electron microscopy. Bastianini’s (1967a) illustrations made from serial reconstructions of the light microscopic images of lymphatics more than two decades ago were amazingly similar to the perivascular structures shown here. He found the lymphatics in hyaline membrane disease and noncardiac pulmonary edema had similar structures (Bastianini, 1967a,b, 1970). I found the lymphatic casts in all lungs were nearly the same. The conduit lymphatics around pleural blood vessels were more common in the group with angiogenesis but large blood vessels were not found on the pleura of the group with noncardiogenic edema.
Fig. 8. Small segments of prezymphatics (p) lie near a conduit lymphatic channel. The long conduit has frequent indentations (arrows) that may be caused by valves. The other vessels are blood capillaries. This animal received monocrotaline. Original magnification was 100 x . Bar = 100 wm.
Fig. 9. The same specimen shown in Figure 6 has a tubular conduit lymphatic (arrowheads) winding around the vein on the pleural surface. Note the variation in the diameter of the lymphatic. Original magnification was 180 x . Bar = 100 pm.
Lymphatics of the lung can be cast only in conditions that produce excess lymph. In such conditions, large lymphatics also can be seen by light microscopy, but without a perturbation, they can hardly be seen by any means. This suggests that the normal state of lymphatics is collapsed and that these methods only study the dilated state. In angiogenesis, forming blood vessels may have incomplete basement membranes that allow the resin to move into the interstitial spaces. In head injury, the resin may be driven through the blood vessel walls by increased intravascular pressure or it may escape because of increased vascular permeability. Prolonged rinsing with hypo-osmotic saline (Schraufnagel and Schmid, 1988a1, avoiding fixation before casting (Schraufnagel and Schmid, 1988b), and using a lower viscosity resin (Weiger et al., 1986; Steeber et al., 19871, have been reported to promote lymphatic casting, although I found that dilution of methacrylate is
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Fig. 10. Conduit lymphatic channels within the lung are flatter, often twisted, vessels that are not connected to blood vessels at this level. Six branches of a lymphatic join at one place (curved arrow) in this animal with neurogenie pulmonary edema. Original magnification was 400 x . Bar = 10 bm.
not necessary to produce good lymphatic casts in these abnormal lung conditions. The pressure at which the methacrylate is injected in these whole animal models appears to have little or no influence on whether lymphatics were cast, although it is not possible to measure the pressure in the capillaries when casting. The pressure in the syringe does not reflect the capillary pressure because of the resistance of the heart and extensively branched vasculature to the viscous resin (Lametschwandtner et al., 1990). If the capillary pressure is too high, the thin (0.5 pm) alveolar-capillary membrane will rupture, spill resin into the alveoli and cast them. Cast alveoli are easily recognizable and also almost never occur in normal lungs. The pressure at the exit port of the fluid column, the aorta, is less than 5 cm of water (Schraufnagel, 1990b). When studying angiogenesis in monocrotaline animals, I considered that the perivascular structures might be newly formed blood capillaries. Finding similar structures in the group with edema establishes them as lymphatic instead of blood spaces. It is not surprising that these structures were not discovered before the monocrotaline study, because lymphatic casts are rare in most experimental models. Lymphatics have many forms. None look like normal blood vessels, but remodeled or newly formed blood vessels could have caused confusion. Prelymphatic, reservoir, and saccular lymphatics are not difficult to distinguish from blood vessels because they lack tubular shape. The textured surface of reservoir lymphatics is caused by irregular projections into the lymphatic vessel lumen (Lauweryns and Baert, 1977). Conduit lymphatic vessels are tubular and may be similar in size to blood vessels, but their variation in diameter and lack of branches and tributaries sets them apart. On the pleural surface, round conduit lymphatics meander and wrap themselves around blood vessels. Conduit lymphatic vessels do not join neighboring blood capillaries and they have frequent indentations. Prelymphatics are tissue spaces that expand with a f h i d burden or destruction of downstream lymphatics.
Fig. 11. a: The bends in the cast of a conduit lymphatic (L) in the lung are probably the site of valves. Original magnification was 720 X . Bar = 10 pm. b: A groove runs in its long axis. This rat had prolonged vascular rinsing. Original magnification was 1,800x . Bar = 10 pm.
They have been described in the brain (Casely-Smith Jr. et al., 1976), tongue (Castenholz, 1984) and other organs. Rodbard and Taller (1969) found the interstitial casts were characteristic for different tissues and that injected fluid flowed from them into the lymphatics. The figures show how this connection is made. Casts of pleural prelymphatics address the controversy of whether they are in the subpleural tissue or the pleura (von Hayek, 1960; Okada et al., 1979). I found that the plastic sheets were always exterior to the blood capillaries but never spilled into the thoracic cavity. The pleural prelymphatic space is close to alveoli and could easily accept fluid from them. Reservoir lymphatics were continuous with prelymphatics. Although lung lymphatics have been cast by many investigators (Miller, 1947; Karpf, 1965; Pennell, 1966; Lauweryns, 1971; Okada et al., 1979; Rodrigues Grande et al., 1983), they have not been studied with scanning electron microscopy as they have in other organs (Kobayashi et al., 1976; Castenholz, 1986; Yamamoto and Phillips, 1986; Ohtani, 1987; Ohtani and Murakami, 1990). Although lymphatics in other organs have similarities to lung lymphatics, the many forms found in the lung are distinctive. By studying
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Fig. 12. a: This vein is surrounded by tubulo-saccular lymphatics (wavy arrows) that are large and heterogeneous in shape. The animal received monocrotaline. Original magnification was 72 x . Bar = 100 pm. b: Blood capillaries (B) surrounding a tributary vein are small compared to lymphatic capillaries (wavy arrow). An alveolar cast is marked with A. Original magnification was 260 x . Bar = 100 p n .
lung lymphatic casts with light microscopy and radiography, Lauweryns (1974) found blind endings and a wide polyhedral meshwork on the pleural surface. He considered the club-like outpocketings to be the beginnings of the lymphatics (Lauweryns, 1971). These structures appear similar to reservoir lymphatics. These lymphatics could keep alveoli dry by holding fluid until the conduit lymphatic traffic lessened. Although I can only speculate on the function of the different lymphatic forms from this anatomic study, the size of the reservoir lymphatics suggests they may have storage capacity. Their fronds suggest they may have absorptive function and their linearity suggests they are itinera for lymph. The storage concept of reservoir and tubulo-saccular lymphatics fits data of lymph accumulation in the lung (Havill and Gee, 1984) and the great potential of the lymphatics fits the findings of Paino et al. (1989). The difference in roundness of the pleural and parenchymal conduit lymphatics may have been caused by different pressure on the casts when they were formed. The nonuniform diameter of lymphatics that others have noted (Lauweryns, 1971), is mainly in the round conduit forms. The lymphatic walls have actin
Fig. 13. a: This tubulo-saccular lymphatic (1) that was part of a bronchiolar wall has airspaces (A) and a pulmonary artery (a)nearby. Capillaries with the bronchial pattern (arrow) and alveolar pattern (arrowhead) are in the background. The animal had prolonged vascular rinsing. Original magnification was 72 x . Bar = 100 pm. b: A higher magnification shows the tubulo-saccular lymphatic capillaries (1)are similar to those around the veins. Original magnification was 180 x . Bar = 100 pm.
and myosin (Leak and Jamuar, 1983),so muscular contraction could cause the narrowing. Small pouches located proximal to valves also could cause widening (Leak and Jamuar, 1983). Several investigators have found that lymphatics that flank arteries have large diameters (Miller, 1947; Bastianini, 1967a,b; Leak and Jamuar, 1983; Albertine et al., 1982) and are more important than those around veins. This study found great variation in the perivascular lymphatics and suggests that the method of producing the lymph and displaying the lymphatics determines their relative size. It also shows that lymphatics have complicated structures. These diverse structures may have diverse functions. ACKNOWLEDGMENTS
I thank Dr. Maitreya Thakkar, Dr. Taral Patel, Aloisa Schmid, and Jaime Llopart Basterra, and the Electron Microscopy Facility of the Research Resources Center, University of Illinois a t Chicago, for assistance and use of the electron microscope. This project was
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sponsored by a grant from the University of Illinois Campus Research Board. LITERATURE CITED
Fig. 14. The lymphatics andprelymphutics in the vessel wall were cast. The artery in the center (a)was removed to show the lymphatics as a hollow cylinder. An airway (circle)is nearby. The inner surface of the cylinder has cross striations corresponding to connective tissue strands seen on light microscopy suggesting it is prelymphatic. Other parts of the wall, especially the branches, have a succulot u b u l a r structure. Original magnification was 160 x . Bar = 100 pm.
Fig. 15. a: The perivenous sacculo-tubular lymphatics (arrows)of lungs with prolonged rinsing are less dense than those shown in the other figures. The differences may relate to the conditions under which the lymphatics were filled. In the background are alveolar blood capillaries (arrowhead). Original magnification was 110 X . Bar = 100 pm. b A higher magnification shows many cell fossils (arrowheads). This lymphatic is less dense and has more blind endings that the tubulo-saccular ones shown in the other figures. Original magnification was 400 X . Bar = 10 pm.
Albertine, K.H., J.P. Wiener-Kronish, P.J. Roos, and N.C. Staub 1982 Structure, blood supply, and lymphatic vessels of the sheep’s visceral pleura. Am. J . Anat., 165:277-294. Bastianini, A. 1967a Ricostruzioni grafiche della trama linfatica polmonare nell’organo normale ed in condizioni patologiche e sperimentali. Atti. Accad. Fisiocrit. Siena, 16:1470-1480. Bastianini, A. 1967b Sulla morfologia microscopica della trama vascolare linfatica del polmone umano. Atti. Accad. Fisiocrit. Siena, 16:1341-1363. Bastianini, A. 1970 Osservazioni sulla morfologia microscopica e l’istotopografia dei vasi linfatici del polmone umano. Boll. SOC. Ital. Biol. Sper., 43t1567-1570. Casley-Smith, J.R., E. Foldi-Borcsok, and M. Foldi 1976 The prelymphatic pathways of the brain a s revealed by cervical lymphatic obstruction and the passage of particles. Brit. J. Exp. Pathol., 57r179-188. Castenholz, A. 1984 Morphological characteristics of initial lymphatics in the tongue as shown by scanning electron microscopy. Scanning Electron Microsc., 3t1343-1352. Castenholz, A. 1986 Corrosion cast technique applied in lymphatic pathways. Scanning Electron Microsc., 2t599-605. Havill, A.M., and M.H. Gee 1984 Role of interstitium in clearance of alveolar fluid in normal and injured lungs. J. Appl. Physiol. Respirat. Environ. Exercise Physiol., 57:l-6. Karpf, V.A. 1965 Das innere LymphgefaCjsystem der Lunge. Anat. . Anz. Bd., 116:442-251. Kobayashi, S., H. Osatake, and Y. Kashima 1976 Corrosion casts of lymphatics. Arch. Histol. Jap., 39:177-181. Lametschwandtner, A., U. Lametschwandtner, and T. Weiger 1990 Scanning electron microscopy of vascular corrosion casts-techniques and applications: Updated review. Scanning Microsc., 4: 889-941. Lauweryns, J.M. 1971 The blood and lymphatic microcirculation of the lung. Cardiopulmonary Pathol. Ann., 6t365-415. Lauweryns, J.M. 1974 Morphological studies of the blood and lymphatic microcirculation of the lung. Acta. Cardiol. Suppl., 19: 157-167. Lauweryns, J.M., and J.H. Baert 1977 Alveolar clearance and the role of the pulmonary lymphatics. Am. Rev. Respir. Dis., 115t625683. Leak, L.V., and M.P. Jamuar 1983 Ultrastructure of pulmonary lymphatic vessels. Am. Rev. Respir. Dis., 128tS59S65. Miller, W.S. 1947 The Lung. Charles Thomas, Springfield IL. Ohtani, 0.1987 Three-dimensional organization of lymphatics and its relationship to blood vessels in rabbit small intestine. Cell Tissue Res., 248:365-374. Ohtani, O., and T. Murakami 1990 Organization of the lymphatic vessels and their relationships to blood vessels in rabbits Peyer’s patches. Arch. Histol. Cytol., 53:(suppl) 155-164. Okada, Y., M. Ito, and Ch. Nagaishi 1979 Anatomical study of the pulmonary lymphatics. Lymphology, 12t118-124. Paino, G., G. Scala, and L. Avallone 1989 Su alcune caratteristiche morfostrutturali del circolo linfatico pleurico del maiale. Boll. SOC.Ital. Biol. Sper., 65:289-294. Pennell, T.C. 1966 Anatomic study of the peripheral pulmonary lymphatics. J. Thor. Cardiovas. Surg., 52t629-634. Rodrigues Grande, N., J. Ribeiro, M. Soares, and E. Carvalho 1983 The lymphatic vessels of the lung: Morphological study. Acta. Anat., I I5:302-309. Rodbard, S., and S. Taller 1969 Plastic casts of extracapsular spaces and lymphatic vessels. Med. Exp., 19:65-70. Schraufnagel, D.E., 1987 Microvascular casting of the lung. A stateof-the-art review. Scanning Microsc., 1~1733-1747. Schraufnagel, D.E. 1989a Microvascular casting of the lung: Bronchial versus pulmonary filling. Scanning Microsc., 3t575-578. Schraufnagel, D.E. 1989b Ranking corrosion efficiency: A Latin square study on rat lung microvascular corrosion casts. Scanning Microsc., 3:299-304. Schraufnagel, D.E. 1990a Monocrotaline-induced angiogenesis: Differences between bronchial and pulmonary vasculature. Am. J. Pathol., 137t1083-1090. Schraufnagel. D.E. 1990b Microcorrosion castinn. In: Electron Microscopy oyf the Lung. D.E. Schraufnagel, ed. Marcel Dekker, New York, p 257-297. Schraufnagel, D.E., and K.R. Pate1 1990 Sphincters in pulmonary
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veins: An anatomic study in rats. Am. Rev. Respir. Dis., 141: 721-726. Schraufnagel, D.E., and A. Schmid 1988a Microvascular casting of the lung: Vascular lavage. Scanning Microsc., 2t1017-1020. Schraufnagel, D.E., and A. Schmid 1988b Microvascular casting of the lung: Effects of various fixation protocols. J. Electron Microsc. Tech., 8:185-191. Schraufnagel, D.E., and A. Schmid 1988c Capillary structure in elastase-induced emphysema. Am. J . Pathol., 130:126-135. Schraufnagel, D.E., and A. Schmid 1989 Pulmonary capillary density in rats given monocrotaline: A cast corosion study. Am. Rev. Respir. Dis., 140:1405-1409. Schraufnagel, D.E., D.N. Mehta, R. Harshbarger, K. Treviranus, and N.S. Wang 1986 Capillary remodeling in bleomycin-induced pulmonary fibrosis. Am. J. Pathol., 125:97-106.
Steeber, D.A., C.M. Erickson, K.C. Hodde, and R.M. Albrecht 1987 Vascular changes in popliteal lymph nodes due to antigen challenge in normal and lethally irradiated mice. Scanning Microsc., 1:831-839. von Hayek, H. 1960 The Human Lung. Hafner, New York. Weiger, T., A. Lametschwandtner, and P. Stockmayer 1986 Technical parameters of plastics (Mercox CL-2B and various methylmethacrylates) used in scanning electron microscopy of vascular corrosion casts. Scanning Electron Microsc., 1243-252. Yamamoto, K., and M.J. Phillips 1986 Three-dimensional observation of the intrahepatic lymphatics by scanning electron microscopy of corrosion casts. Anat. Record, 214t67-70.
Forms of Lung Lymphatics: A Scanning Electron Microscopic Study of Casts DEAN E. SCHRAUFNAGEL Departments of Medicine and Pathology, University of Illinois at Chicago
ABSTRACT In a recent study, rats given monocrotaline underwent angiogenesis on their pleural surfaces. The rats also had novel structures in their bronchovascular bundles that were detected by scanning electron microscopy of vascular casts. These vessels could have been either new blood capillaries or dilated lymphatic capillaries. To determine if these structures were lymphatics or new blood vessels, specimens from animals that were undergoing angiogenesis were compared to those that were not. Finding similar structures in normal animals would imply that they were lymphatic. The second purpose of this work was to describe the three-dimensional anatomy of the lymphatics of the lung. Cast lymphatics were found in most lungs with edema or angiogenesis, but were rare in other conditions. The vascular structures in question were found in animals not undergoing angiogenesis and were, therefore, lymphatic. Additionally, scanning electron microscopy of casts showed several distinct forms of lymphatics in the lung. Prelymphatics are tissues planes beneath the pleura and around bronchovascular structures. They join reservoir, conduit or tubulo-saccular lymphatics. Reservoir lymphatics are broad ribbon-like structures with textured surfaces and small laterally branching pouches. They occur on the pleural surface, are closely linked with prelymphatics, and join conduit lymphatics. Conduit lymphatics are tubular structures that may contain valves, twist and go great distances without accepting tributaries. On the pleural surface, they may wind around blood vessels and vary greatly in diamater. Sacculo-tubular lymphatics surround arteries, veins and bronchioles. They have thin walls with wide saccular segments. They may be so dense that they form cylinders around the vessels or airways. Different forms of lung lymphatics suggest different function and potential. 0 1992 Wiley-Liss, Inc.
In a recent study of lung vascular casts by scanning electron microscopy, I showed that rats given the pyrrolizidine alkaloid, monocrotaline, underwent angiogenesis on their pleural surfaces. The new vessels were typical fully differentiated arteries and veins. In addition, I found previously unreported, unusually shaped capillaries around arteries that could have been either novel blood capillaries or dilated lymphatic capillaries (Schraufnagel, 1990a). Scanning electron microscopy of casts is a valuable tool to study the threedimensional ultrastructure of small vessels, but images of neither lymphatic nor developing blood vessels of the lung had been reported previously, so that one could not be certain what the structures were. To find out if they were lymphatic or new blood vessels, specimens from animals that were not undergoing angiogenesis were compared to those from the monocrotaline study. The animals that were not undergoing angiogenesis were normal rats given a head blow to produce neurogenic pulmonary edema (Schraufnagel and Patel, 1990). If similar structures could be found in animals that were not undergoing angiogenesis, I would conclude that they were lymphatic. I also sought to describe the three-dimensional anatomy of the lymphatics of the lung. To do this I screened cast specimens 0 1992 WILEY-LISS, INC
from other studies to chart lymphatic shapes and associations. MATERIALS AND METHODS
Specimens for the group with angiogenesis were taken from a previous study of rats given monocrotaline (Schraufnagel, 1990a). They were compared with specimens from a study of normal rats that received a blow to the head to produce neurogenic pulmonary edema (Schraufnagel and Patel, 1990). In addition, several hundred micrographs of cast lung vasculature from other published (Schraufnagel 1989a,b; Schraufnagel et al., 1986; Schraufnagel and Schmid 1988a,b,c, 1989) and pilot studies were reviewed to search for lymphatic casts. These studies had more than 150 rats, although not all lungs cast well enough to study lymphatics. The angiogenic group had six animals. The methods have been described in detail (Schrauf-
Received July 30, 1991; accepted January 9, 1992. Address reprint requests to Dean E. Schraufnagel, M.D., Section of Respiratory and Critical Care Medicine, Department of Medicine MIC 787, University of Illinois a t Chicago, P.O. Box 6998, Chicago, IL 60680-6998.
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nagel, 1987,1990b).In brief, all animals were SpragueDawley rats weighing about 180-400 g. In the study of angiogenesis, animals received 80 mg/kg of monocrotaline (Transworld Chemical, Washington, DC) and were killed 19 or 20 days later. Each animal was anesthetized with pentobarbital. The abdomen was opened and the caudal vena cava and aorta were cannulated with a 20 gauge catheter. Warmed heparinized saline infused into the vena cava until the aortic effluent was clear. Twenty ml of partially polymerized methylmethacrylate (Mercox, Ladd Research Industries, Burlington, VT) diluted 4 parts to 1 with n-methylmethacrylate monomer (SPI-Chem, West Chester, PA) was mixed with a catalyst and injected with a hand-held syringe through the vena cava to fill the pulmonary vasculature over one minute. The animals with neurogenic pulmonary edema were normal similar rats that did not receive monocrotaline. They were cast with undiluted Mercox. Just after their lungs were filled, but before the resin set, they received a sharp blow to the head. Light microscopy of animals that received a similar blow to the head, but were not cast, showed proteinaceous and hemorrhagic pulmonary edema (Schraufnagel and Patel, 1990). In all animals, the plastic was allowed to harden for 1hour. One piece of tissue was sent for light microscopy and the rest of the lungs were placed in a sodium hydroxide solution until the tissue corroded. The casts were rinsed and cut with scissors or razor blades into pieces about 1 mm thick. The specimens were fastened to aluminum studs with double-sided tape or silver cement, sputter-coated with palladium-gold, and viewed with a JEOL, (Tokyo) JSM-35C, scanning electron microscope. The accelerating voltage was 15 kV. The working distance was 15 mm. RESULTS
Both the animals with angiogenesis and those with neurogenic edema had extensive and diverse lymphatic casts, although some animals in both groups also had none. The only other animals that regularly had cast lymphatics were those with prolonged vascular rinsing with saline before casting (Schraufnagel and Schmid, 1988a). The lymphatics in all groups were structurally similar. The bronchovascular structure in question was found in the lungs with pulmonary edema, indicating that it was lymphatic and not neovascular. There was no evidence for new blood vessels in the animals with pulmonary edema. The lungs that had cast lymphatics that could be seen with scanning electron microscopy also had dilated lymphatics that could be seen with light microscopy. Scanning electron microscopy showed many forms of lymphatics in the lungs. Casts from different areas had distinct structures. The interstitial spaces or tissue planes that receive fluid from capillaries or alveoli and the interstitial space beneath the pleura have been calledprelymphatics by von Hayek (1960)and others. In the casting preparation, these spaces filled well and had a characteristic appearance. At low magnification, they were films of plastic contained by the visceral pleura (Fig. 1).Although they could nearly cover the lung, they were usually found as patches along the pleura and occasionally around large vessels. They were never around an individual alveolus in the lung
Fig. 1. The pleural surface of a cast at low magnification shows a sheet o f plastic contained within the visceral pleura. It is easily separable from the cast o f the blood capillaries. No plastic was found in the parietal pleura or in the thoracic cavity, indicating that the plastic is just beneath the pleura. These sheets were found in both the animals given monocrotaline (the group undergoing angiogenesis) and those with neurogenic pulmonary edema and rarely in saline control animals, which this animal was. Casts o f the tissue planes have been called prelymphatics because they join lymphatics. Original magnification was 20 x . Bar = 1,000 km.
parenchyma. The casts of prelymphatics were marked with connective tissue strands across their surface (Figs. 2, 3). Although prelymphatics occasionally drained directly into conduit lymphatics (Fig. 41, they usually connected to other structures, not previously reported, called reservoir lymphatics. These flat ribbon-like structures, about 15-40 pm in width, had small pouches branching laterally from their long axes (Fig. 5 ) . Their leaf-like side branches ended blindly. They did not have the imprints of connective tissue strands on their surface, but the surface was textured (Fig. 6). Although the term prelymphatic was reserved for planar structures, and reservoir lymphatics had to have a linear dimension, it was often difficult to distinguish between the two because of the gradual transition from one to the other. The rough surfaces of prelymphatics and reservoir lymphatics also distinguished them from blood capillaries. Reservoir lymphatics connected to the conduit lymphatic vessels (Fig. 7). The conduit lymphatic vessels were usually round on the pleural surface (Fig. 8) and often flattened within the lung. On the pleural surface, they were about the same size as the small blood vessels they tracked, but their diameters varied several-fold, from less than 5 pm to more than 35 pm, within a few hundred micrometers of length. They were often tortuous, sometimes helical, and wound around blood vessels (Fig. 9). The conduit vessels within the lung were usually more delicate and had less variation in diameter than the pleural ducts. Conduit lymphatics rarely accepted tributaries or gave off branches, although an example of octopus-like branching is shown in Figure
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Fig. 2. This is a higher magnification of a p r e l y m p h a t i c cast of the pleural surface of an animal that received monocrotaline. The surface is indented by strands of connective tissue. The gradual transition from prelymphatics to reservoir lymphatics often makes it dificult to distinguish the two. Original magnification was 100 x . Bar = 100 pm.
Fig. 4. Although most prelymphatic casts connected to reservoir lymphatics, this prelymphatic (p) space attached to a conduit lymphatic (c).The rat had pulmonary edema. Original magnification was 600 x . Bar = 10 pm.
Fig. 3. A more magnified view of the prelymphatic cast of the animal shown in Figure 2 illustrates its textured surface. If a tubular character could be detected, it would be classified as a reservoir lymphatic. Original magnification was 220 x . Bar = 100 km.
Fig. 5. Reservoir lymphatics, on the left, have flat laterally budding structures that distinguish them from the blood capillaries on the right (B). This micrograph was taken near Figure 3 on the same specimen. Original magnification was 220 x . Bar = 100 km.
10. Often the casts of the lymphatics within the lung had longitudinal grooves and joints (Fig. 11). Within the lung, these conduit vessels often coursed in the bronchovascular bundle where they connected to, but remained separate from, tubulo-saccular lymphatics. Around large blood vessels and airways were broad tubulo-succular lymphatics (Figs. 12-15). These differed from the other forms by being large, sac-like arrangements that surrounded a vessel or airway. The saccules could be more than 10 times wider than conduit lymphatics and were more heterogenous than the reservoir lymphatics. Around certain vessels they were sparse, but in other areas they were extremely dense. The dense lymphatics formed a hollow cylinder if the inner blood vessel cast could be removed (Fig. 14). It
was one of these lymphatics t h a t was pictured in Figure 6 of the previous study of angiogenesis (Schraufnagel, 1990a). In the monocrotaline animals, the lymphatics around the arteries appeared to be more abundant than around the veins (Fig. 12), but in the animals with pulmonary edema, it appeared to be the opposite. The lymphatics around the airway had similar shapes to those around the blood vessels (Fig. 13). The size of the saccular component of these lymphatics ranged widely from 10 pm to more than 100 km, yet their walls were thin. In the monocrotaline animals, the lymphatic casts appeared to have more trapped cells, although lymphocyte remains could be found in all groups (Fig. 15).
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Fig. 6.A higher magnification micrograph of the reservoir lymphatics of Figure 5 shows a closely packed, rather tubular structure with a textured surface. Its morphology suggests it may have absoqtive, reservoir and transport functions. Original magnification was 660 x . Bar = 10 pm.
Fig, 7. The reservoir lymphatics (r) drain into a conduit lymphatic (c). The rat had pulmonary edema. Original magnification was 780 X . Bar = 10 pm.
DISCUSSION
Although this is the first demonstration by scanning electron microscopy of lung lymphatic casts, much of this has already been described. Miller (1947), von Hayek (1960), Lauweryns (1971, 1974) and many others have studied lung lymphatics by light and electron microscopy. Bastianini’s (1967a) illustrations made from serial reconstructions of the light microscopic images of lymphatics more than two decades ago were amazingly similar to the perivascular structures shown here. He found the lymphatics in hyaline membrane disease and noncardiac pulmonary edema had similar structures (Bastianini, 1967a,b, 1970). I found the lymphatic casts in all lungs were nearly the same. The conduit lymphatics around pleural blood vessels were more common in the group with angiogenesis but large blood vessels were not found on the pleura of the group with noncardiogenic edema.
Fig. 8. Small segments of prezymphatics (p) lie near a conduit lymphatic channel. The long conduit has frequent indentations (arrows) that may be caused by valves. The other vessels are blood capillaries. This animal received monocrotaline. Original magnification was 100 x . Bar = 100 wm.
Fig. 9. The same specimen shown in Figure 6 has a tubular conduit lymphatic (arrowheads) winding around the vein on the pleural surface. Note the variation in the diameter of the lymphatic. Original magnification was 180 x . Bar = 100 pm.
Lymphatics of the lung can be cast only in conditions that produce excess lymph. In such conditions, large lymphatics also can be seen by light microscopy, but without a perturbation, they can hardly be seen by any means. This suggests that the normal state of lymphatics is collapsed and that these methods only study the dilated state. In angiogenesis, forming blood vessels may have incomplete basement membranes that allow the resin to move into the interstitial spaces. In head injury, the resin may be driven through the blood vessel walls by increased intravascular pressure or it may escape because of increased vascular permeability. Prolonged rinsing with hypo-osmotic saline (Schraufnagel and Schmid, 1988a1, avoiding fixation before casting (Schraufnagel and Schmid, 1988b), and using a lower viscosity resin (Weiger et al., 1986; Steeber et al., 19871, have been reported to promote lymphatic casting, although I found that dilution of methacrylate is
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Fig. 10. Conduit lymphatic channels within the lung are flatter, often twisted, vessels that are not connected to blood vessels at this level. Six branches of a lymphatic join at one place (curved arrow) in this animal with neurogenie pulmonary edema. Original magnification was 400 x . Bar = 10 bm.
not necessary to produce good lymphatic casts in these abnormal lung conditions. The pressure at which the methacrylate is injected in these whole animal models appears to have little or no influence on whether lymphatics were cast, although it is not possible to measure the pressure in the capillaries when casting. The pressure in the syringe does not reflect the capillary pressure because of the resistance of the heart and extensively branched vasculature to the viscous resin (Lametschwandtner et al., 1990). If the capillary pressure is too high, the thin (0.5 pm) alveolar-capillary membrane will rupture, spill resin into the alveoli and cast them. Cast alveoli are easily recognizable and also almost never occur in normal lungs. The pressure at the exit port of the fluid column, the aorta, is less than 5 cm of water (Schraufnagel, 1990b). When studying angiogenesis in monocrotaline animals, I considered that the perivascular structures might be newly formed blood capillaries. Finding similar structures in the group with edema establishes them as lymphatic instead of blood spaces. It is not surprising that these structures were not discovered before the monocrotaline study, because lymphatic casts are rare in most experimental models. Lymphatics have many forms. None look like normal blood vessels, but remodeled or newly formed blood vessels could have caused confusion. Prelymphatic, reservoir, and saccular lymphatics are not difficult to distinguish from blood vessels because they lack tubular shape. The textured surface of reservoir lymphatics is caused by irregular projections into the lymphatic vessel lumen (Lauweryns and Baert, 1977). Conduit lymphatic vessels are tubular and may be similar in size to blood vessels, but their variation in diameter and lack of branches and tributaries sets them apart. On the pleural surface, round conduit lymphatics meander and wrap themselves around blood vessels. Conduit lymphatic vessels do not join neighboring blood capillaries and they have frequent indentations. Prelymphatics are tissue spaces that expand with a f h i d burden or destruction of downstream lymphatics.
Fig. 11. a: The bends in the cast of a conduit lymphatic (L) in the lung are probably the site of valves. Original magnification was 720 X . Bar = 10 pm. b: A groove runs in its long axis. This rat had prolonged vascular rinsing. Original magnification was 1,800x . Bar = 10 pm.
They have been described in the brain (Casely-Smith Jr. et al., 1976), tongue (Castenholz, 1984) and other organs. Rodbard and Taller (1969) found the interstitial casts were characteristic for different tissues and that injected fluid flowed from them into the lymphatics. The figures show how this connection is made. Casts of pleural prelymphatics address the controversy of whether they are in the subpleural tissue or the pleura (von Hayek, 1960; Okada et al., 1979). I found that the plastic sheets were always exterior to the blood capillaries but never spilled into the thoracic cavity. The pleural prelymphatic space is close to alveoli and could easily accept fluid from them. Reservoir lymphatics were continuous with prelymphatics. Although lung lymphatics have been cast by many investigators (Miller, 1947; Karpf, 1965; Pennell, 1966; Lauweryns, 1971; Okada et al., 1979; Rodrigues Grande et al., 1983), they have not been studied with scanning electron microscopy as they have in other organs (Kobayashi et al., 1976; Castenholz, 1986; Yamamoto and Phillips, 1986; Ohtani, 1987; Ohtani and Murakami, 1990). Although lymphatics in other organs have similarities to lung lymphatics, the many forms found in the lung are distinctive. By studying
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Fig. 12. a: This vein is surrounded by tubulo-saccular lymphatics (wavy arrows) that are large and heterogeneous in shape. The animal received monocrotaline. Original magnification was 72 x . Bar = 100 pm. b: Blood capillaries (B) surrounding a tributary vein are small compared to lymphatic capillaries (wavy arrow). An alveolar cast is marked with A. Original magnification was 260 x . Bar = 100 p n .
lung lymphatic casts with light microscopy and radiography, Lauweryns (1974) found blind endings and a wide polyhedral meshwork on the pleural surface. He considered the club-like outpocketings to be the beginnings of the lymphatics (Lauweryns, 1971). These structures appear similar to reservoir lymphatics. These lymphatics could keep alveoli dry by holding fluid until the conduit lymphatic traffic lessened. Although I can only speculate on the function of the different lymphatic forms from this anatomic study, the size of the reservoir lymphatics suggests they may have storage capacity. Their fronds suggest they may have absorptive function and their linearity suggests they are itinera for lymph. The storage concept of reservoir and tubulo-saccular lymphatics fits data of lymph accumulation in the lung (Havill and Gee, 1984) and the great potential of the lymphatics fits the findings of Paino et al. (1989). The difference in roundness of the pleural and parenchymal conduit lymphatics may have been caused by different pressure on the casts when they were formed. The nonuniform diameter of lymphatics that others have noted (Lauweryns, 1971), is mainly in the round conduit forms. The lymphatic walls have actin
Fig. 13. a: This tubulo-saccular lymphatic (1) that was part of a bronchiolar wall has airspaces (A) and a pulmonary artery (a)nearby. Capillaries with the bronchial pattern (arrow) and alveolar pattern (arrowhead) are in the background. The animal had prolonged vascular rinsing. Original magnification was 72 x . Bar = 100 pm. b: A higher magnification shows the tubulo-saccular lymphatic capillaries (1)are similar to those around the veins. Original magnification was 180 x . Bar = 100 pm.
and myosin (Leak and Jamuar, 1983),so muscular contraction could cause the narrowing. Small pouches located proximal to valves also could cause widening (Leak and Jamuar, 1983). Several investigators have found that lymphatics that flank arteries have large diameters (Miller, 1947; Bastianini, 1967a,b; Leak and Jamuar, 1983; Albertine et al., 1982) and are more important than those around veins. This study found great variation in the perivascular lymphatics and suggests that the method of producing the lymph and displaying the lymphatics determines their relative size. It also shows that lymphatics have complicated structures. These diverse structures may have diverse functions. ACKNOWLEDGMENTS
I thank Dr. Maitreya Thakkar, Dr. Taral Patel, Aloisa Schmid, and Jaime Llopart Basterra, and the Electron Microscopy Facility of the Research Resources Center, University of Illinois a t Chicago, for assistance and use of the electron microscope. This project was
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sponsored by a grant from the University of Illinois Campus Research Board. LITERATURE CITED
Fig. 14. The lymphatics andprelymphutics in the vessel wall were cast. The artery in the center (a)was removed to show the lymphatics as a hollow cylinder. An airway (circle)is nearby. The inner surface of the cylinder has cross striations corresponding to connective tissue strands seen on light microscopy suggesting it is prelymphatic. Other parts of the wall, especially the branches, have a succulot u b u l a r structure. Original magnification was 160 x . Bar = 100 pm.
Fig. 15. a: The perivenous sacculo-tubular lymphatics (arrows)of lungs with prolonged rinsing are less dense than those shown in the other figures. The differences may relate to the conditions under which the lymphatics were filled. In the background are alveolar blood capillaries (arrowhead). Original magnification was 110 X . Bar = 100 pm. b A higher magnification shows many cell fossils (arrowheads). This lymphatic is less dense and has more blind endings that the tubulo-saccular ones shown in the other figures. Original magnification was 400 X . Bar = 10 pm.
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