Characteristics of Bovine Lung as Observed by Scanning Electron Microscopy ' A. T. MARIASSY, C . G. PLOPPER AND D. L. DUNGWORTH Department o f Veterinary Pathology, School o f Veterinary Medicine, University o f California, Davis, California a n d Letterman A r m y Institute o f Research, Presidio o f S a n Francisco, California
ABSTRACT The surface characteristics of the intrapulmonary airways and alveoli of the lungs from 12 young adult cattle examined by the scanning elec-
tron microscope are described. When compared with similar studies of the lungs of various other mammalian species, the bovine lung generally resembles that of other mammals except for several important features: (1) Alveolar pores of Kohn are small and extremely rare; ( 2 ) Alveolar macrophages are seldom seen in alveolar air spaces; ( 3 ) Interlobular septa completely separate the lung into distinct lobules. MATERIALS AND METHODS The normal mammalian respiratory system has proven to be a highly suitable subFresh lungs were obtained from twelve ject for morphological studies employing cross-bred beef cattle, approximately 20 the scanning electron microscope. A num- months of age, ( 6 neutered males, 4 intact ber of descriptions exist for the common males, and 2 females), a t a local abbatoir. laboratory species, including the hamster The middle lobe from each steer, a cranial (Nowell and Tyler, '71; Kuhn and Finke, lobe from each intact male, and the middle '72), rat (Kuhn and Finke, '72; Parkinson and cranial lobes from each of the cows and Stephens, '73), mouse (Greenwood were processed for examination. After canand Holland, '72; Kuhn and Finke, '72; nulation of the lobar bronchus, each lobe Andrews, '74), guinea pig (Dahlgren et al., was fixed overnight at 4°C by airway per'72) and rabbit (Holma, '69; Wang et al., fusion a t 30 cm water pressure with cac'73). Among larger species, the dog (Gro- odylate-buffered formaldehyde-glutaraldeniowski et al., '72), the horse (Nowell and hyde (Karnovsky, '65) adjusted to 550 Tyler, '71; Nowell et al., '71), and non- milliosmoles per liter. Approximately 30 human primates (Greenwood and Holland, samples, 1 X 1 X 0.5 cm in size, were '73; Castleman et al.,'75) and m a n (Wang taken from each lobe, dehydrated with and Thurlbeck, '70) have received atten- graded solutions of ethanol followed by tion. While ultrastructural studies of the amyl acetate, and critical point dried with lungs of normal adult cattle (Epling, CO, (Anderson, '51). Dried specimens were '64a,b) and calves (Rybicka et al., '74a,b) mounted on stubs, double coated with silhave been performed by transmission elec- ver and gold, and examined with a scantron microscopy, to the best of our knowl- ning electron microscope (ETEC Autoscan). edge the surface morphology of only one RESULTS ruminant species, newborn lamb (Tyler et Bronchi al., '71), has been described. This report presents a brief description of the surface Bronchi are identified by their large Iucharacteristics of bovine lung as seen by minal diameters and by the cut surfaces scanning electron microscopy. Its primary of their walls which contain a layer of purpose is to illustrate several distinctive epithelium supported by thick bands of anatomic features that are especially well smooth muscle, dense connective tissue, revealed by this method of examination bronchial glands, and plates of cartilage. and that are important for an understand- The epithelium is composed of ciliated and ing of pathophysiological responses of bo- ~Received Sept. 9, '74. Accepted Mar. 18, '75. vine lungs. 1 Supported in part by NIH Grant ES HL 00628. ANAT. REC., 183: 13-26.
13
14
A. T. MARIASSY, C. G. PLOPPER AND D. L. DUNGWORTH
nonciliated cells. Ciliated cells predominate over the majority of the surface, but among them are interspersed single nonciliated cells (fig. 1). Dense aggregations of long uniform cilia, with microvilli distributed around their bases, project from the surfaces of the ciliated cells. The mucosal surface of the nonciliated cells is covered by shorter microvilli and does not protrude into the lumen. In nonciliated cells with few microvilli, large pits or pores open onto the surface. These pores sometimes contain secretory material fixed in situ (fig. 1). Actively-secreting nonciliated cells have very few microvilli, as opposed to quiescent non-secreting cells which are covered by a dense population of short, stubby microvilli. Bronchioles The terminal (non-respiratory ) bronchioles are differentiated from bronchi by their smaller diameter and thinner walls, which lack cartilage and glands. The epithelium is supported by thin bands of smooth muscle and connective tissue (fig. 2). In contrast to bronchi, the bronchiolar mucosa is composed of approximately equal numbers of ciliated and nonciliated cells (fig. 3). The ciliated cells have slightly shorter cilia than those in the bronchi. Bronchiolar ciliated cells also differ from their bronchial counterparts by having shorter and more numerous microvilli. The luminal surface of the nonciliated bronchiolar cell has an oval perimeter and appears to have a granular surface, with few very short microvilli. The central portion of the cell frequently protrudes into the airway. The cell boundaries are easily discernible by virtue of the contrast provided by villi on adjacent ciliated cells. Respiratory bronchioles are defined by the presence of occasional alveolar outpocketings in their walls. In contrast to the well-developed system of terminal bronchioles, respiratory bronchioles are short and poorly alveolarized (fig. 4). The proportion of nonciliated to ciliated cells increases in the respiratory bronchioles. Ciliated cells become few in number; their cilia are shorter, less abundant and more disorderly. The apical protrusions of nonciliated cells are most prominent at the
bronchiolar junction with the respiratory epithelium (fig. 5).
Alveoli The pulmonary parenchyma is composed of polyhedral alveolar spaces whose walls are formed by the thin interalveolar septa. Three cell types are observed in alveoli. The majority of the alveolar surface is covered by the flattened, smooth-surfaced squamous (type I ) epithelial cells (fig. 6). Small projections can be seen scattered on the surfaces of these cells. The second type of epithelial cell, the granular (type 11) pneumonocyte, usually lines only a small portion of the septum. Its surface protrudes into the alveolar lumen and is covered by many microvilli. Openings or pits on the surface of the type I1 cells are rarely seen. The granular pneumonocytes are particularly numerous in alveoli adjacent to interlobular septa (fig. 6 ) and in alveoli close to bronchioles. Boundaries between epithelial cells are marked by a prominent seam (fig. 6). The third cell, the pulmonary alveolar macrophage, was very rarely observed. A notable characteristic of alveoli is the usual almost total lack of interalveolar pores (of Kohn) in the interalveolar septa (fig. 7). Their frequency is approximately one per ten septal faces and when present they appear as small ellipsoidal openings penetrating the alveolar walls. In only one of the 12 lungs is there an atypical finding of more numerous small pores (fig. 8). Interlobular septa The interlobular septa reported by others from subgross studies of bovine lung are readily apparent (fig. 2). They are formed of interconnected sheets of connective tissue of various thicknesses extending from peribronchial regions to the pleura, subdividing the pulmonary parenchyma throughout the substance of the lobes into separate polyhedral lobules. Even where the septa are insubstantial, the separation between adjacent lobules is complete. The connective tissue is formed into loosely arranged, thin apposed laminae running in a plane of each septum. Blood vessels and lymphatics are observed in thicker septal regions.
SEM OF BOVINE LUNG DISCUSSION
This study demonstrates that, in general, bovine intrapulmonary respiratory surfaces resemble those of other mammalian species previously reported. The epithelium of airways is a mixture of ciliated and nonciliated cells, with the former predominating in bronchi and the latter in bronchioles. In the bronchi, the nonciliated mucous goblet cell has short microvilli in the non-secretory phase, as has been observed in rodents (Greenwood and Holland, ’72) and macaques (Castleman et al., ’75). We did not observe the smooth, domed surfaces found in equine lungs (Nowell and Tyler, ’71). Also, brush cells (Watson and Brinkman, ’64; Meyrick and Reid, ’68) were not observed in bronchi, bronchioles or alveoli, but more extensive studies will be needed before a decision can be made on whether or not they occur at all in bovine lungs. The bronchiolar epithelial admixture of ciliated and nonciliated cells seems to be a common feature of all mammalian lungs thus far described. The role of the nonciliated cells and the closeness of their resemblance to human Clara cells (Cutz and Conen, ’71) remains to be determined. In addition, we have confirmed that bovine lungs have short or nonexistent respiratory bronchioles, as reported on the basis of subgross studies (McLaughlin et al., ’61). With respect to features of alveolar epithelial cells, we did not observe the large pits in the surface of type I1 cells as shown to be present in equine lung by Nowell and Tyler (’71). Reasons for the difference are purely speculative until information is available on surfactant synthesis and turnover in the two species. Three morphologic features of the parenchymal tissue of bovine lung are in sharp contrast with the pulmonary parenchyma of other mammals thus far studied by SEM. First, interalveolar pores of Kohn are extremely uncommon in the lungs of young adult cattle. We interpret the rarity of pores in 11 of 12 lungs to be the typical situation for this age group. The presence of moderate numbers of small pores in one of the 12 lungs represents an atypical finding, possibly resulting from previous pulmonary damage. That there had been earlier damage to the lung was indicated by the existence of abnormal deformations
15
and folds in the walls of some of its bronchioles. The second characteristic morphologic feature is that interlobular septa are well delineated and completely isolate pulmonary lobules, as previously indicated by subgross studies (McLaughlin et al., ’61) and by physiological studies of Van Allen et al. (’31). The third feature is the rarity of alveolar macrophages in alveolar lumens. A similar conclusion was reached by Rybicka et al. (’74a) after their examination of lungs of calves by transmission electron microscopy. Rybicka et al. (‘74a,b) also observed numerous cells in the pulmonary capillaries of normal calves which they felt, on the basis of ultrastructural characteristics and evidence of phagocytic capability, were more suitably designated “intravascular macrophages” than simply blood monocytes. It is tempting to speculate that the relationship between scarcity of alveolar macrophages and abundance of cells within pulmonary capillaries already having features of macrophages is not fortuitous. That is to say, the intravascular cells might represent a more readily recruitable pool of macrophages than usual to compensate for the scarcity of resident alveolar macrophages. If this interpretation is correct, the intravascular macrophages described by Rybicka et al. (’74b) represent cells comprising a portion of the intermediate lung maturation compartment proposed by Bowden (’71) to be where blood monocytes undergo differentiation into functional alveolar macrophages. These special features have important implications with regard to the pathophysiology and pathology of the bovine lung. Collateral ventilation within and among pulmonary lobules is severely limited or absent because of the scarcity of pores of Kohn and the completeness of the interlobular septa (Van Allen et al., ’31). Van Allen and colleagues also showed that focal pulmonary atelectasis was more prone to occur in the absence of collateral ventilation. Lack of collateral ventilation is also likely both to hamper compensation for inequalities of ventilation associated with obstructions of small airways and to reduce the efficiency of mechanical expulsion of intraluminal obstructions. Consequences of
16
A. T. MARIASSY, C. G. PLOPPER AND D. L. DUNGWORTH
the latter in the case of intrabronchiolar accumulations of exudate would be to impair the combatting of a n incipient bronchopneumonia or the resolution of a fully developed inflammatory process. The paucity of alveolar macrophages would also appear to put the bovine lung at a disadvantage with respect to resistance to aerogeneous bacterial infections since maintenance of bacterial sterility of alveolar parenchyma is primarily ascribed to these cells (Green, '73). The completeness of the interlobular septa, and the apparent ease with which the component connective tissue laminae can be separated, correlate well with the frequency with which interstitial emphysema is found in damaged lungs of cattle following periods of severely labored breathing, particularly in association with acute (atypical) interstitial pneumonia (Jubb and Kennedy, '70). A final point of concurrence between these findings and our observations of the response of the bovine lung to injury, is that the hyperplasia and hyDertrophy of type I1 epithelial cells (epithelialization ) which can be extensive as early as three to four days after widespread alveolar damage is most pronounced i n peribronchiolar and paraseptal regions (Dungworth). These are the two regions i n which the type I1 cells are most numerous in the normal bovine lung. LITERATURE CITED Anderson, T. F. 1951 Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci., 13: 130-134. Andrews, P. M. 1974 A scanning electron microscopic study of the extrapulmonary respiratory tract. Am. J. Anat., 139: 399-424. Bowden, D. H. 1971 The alveolar macrophage. Current Top. Pathol., 55: 1-36. Castleman, W. L., D. L. Dungworth and W. S. Tyler 1975 Intrapulmonary airway morphology in three species of monkeys. A correlated scanning and transmission electron microscopic study. Am. J. Anat., 142(1): 107-121. Cutz, E., and P. E. Conen 1971 Ultrastructure and cytochemistry of Clara cells. Am. J. Path., 62: 127-142. Dahlgren, S. E., H. Dalen and T. Dalhamm 1972 Ultrastructural observations on chemically induced inflammation in guinea pig trachea. Virchows Arch. Abt. B. Zellpath., 1 1 : 211-223. Dungworth, D. L. Unpublished observations. Epling, G. P. 1964a Electron microscopy of the bovine lung; The normal blood-air barrier. Am. J. Vet. Res., 25: 679-689.
196413 Electron microscopy of the bovine lungs: Lattice and lamellar structures i n the alveolar lumen. Am. J. Vet. Res., 25: 14241430. Green, G. M. 1973 Lung defense mechanisms. Med. Clin. North Am., 57(3): 547-562. Greenwood, M. F., and P. Holland 1972 The mammalian respiratory tract surface. A scanning electron microscopic study. Lab. Invest., 27: 296-304. 1973 Scanning electron microscopy of the normal and BCG-stimulated primate respiratory tract. J. Reticuloendo. SOC.,13: 183-192. Groniowski, J., M. Walski and W. Biczysko 1972 Application of scanning electron microscopy for studies of the lung parenchyma. J. Ultrastructure Res., 38: 473481. Holma, B. 1969 Scanning electron microscopic observation of particles deposited in the lung. Arch. Environ. Health, 18: 330-339. Jubb, K. V. F., and P. C. Kennedy 1970 Pathology of domestic animals. Second ed. Academic Press, Inc., New York, p. 182. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A138A. Kuhn, C., 111, and E. H. Finke 1972 The topography of the pulmonary alveolus: Scanning electron microscopy using different fixations. J. Ultrastruct. Res., 38: 161-173. McLaughlin, R. F., W. S. Tyler and R. 0. Canada 1961 A study of the subgross pulmonary anatomy in various mammals. Am. J. Anat., 108: 149-165. Meyrick, B., and L. Reid 1968 The alveolar brush cell in rat lung - a third pneumonocyte. .J. Ultrastruct. Res., 23: 71-80. Nowell, J. A., J. R. Gillespie and W. S. Tyler 1971 Scanning electron microscopy of chronic pulmonary emphysema: A study of the equine model. In: Scanning Electron Microscopy/l971. Part I. 0. Johari, ed. IIT Research Institute, Chicago, pp. 297-304. Nowell, J. A., and W. S. Tyler 1971 Scanning electron microscopy of the surface morphology of mammalian lungs. Am. Rev. Resp. Dis., 103: :313-328. Parkinson, D. R., and R. J. Stephens 1973 Morphological surface changes in the terminal bronchiolar region of NOz-exposed rat lung. Environ. Res., 6: 37-51. Rybicka, K., B. D. T. Daly, J. J. Migliore and J. C. Norman 1974a Ultrastructure of pulmonary alveoli of the calf. Am. J. Vet. Res., 35: 213222. 1974b Intravascular macrophages in normal calf lung. An electron microscopic study. Am. J. Anat., 139: 353-368. Tyler, W. S., A. A. de Lorimier, A. G. Manus and J. A. Nowell 1971 Surface morphology of hypoplastic and normal lungs from newborn lambs. Scanning Electron Microscopy/l971. Part I. 0. Johari, ed. IIT Research Institute, Chicago, pp. 305-312. Van Allen, C. M., G. E. Lindskog and H. G . Richter 1931 Collateral respiration. Transfer of
SEM OF BOVINE LUNG air collaterally between pulmonary lobules. J. Clin. Invest., 10: 559-590. Wang, N. S., H. W. Taeusch, W. M. Thurlbeck and M. E. Avery 1973 A combined scanning and transmission electron microscopic study of alveolar epithelial development of the fetal rabbit lung. Am. J. Path., 73: 365-376.
17
Wang, N. S., and W. M. Thurlbeck 1970 Scanning electron microscopy of the lung. Human Path., I: 227-231. Watson, J. H. L., and G. L. Brinkman 1964 Electron microscopy of the epithelial cells of normal and bronchitic human bronchus. Am. Rev. Resp. Dis., 90: 851-866.
PLATE 1 EXPLANATION O F FIGURES
18
1
Bronchial epithelium showing predominance of ciliated cells with interspersed nonciliated cells. Secretory material emerging from the latter has been fixed in situ. x 4,600.
2
Pulmonary parenchyma with segment of a lobule completely delineated by interlobular septa and a n adjacent portion of a terminal bronchiole. Note the delicacy of the two septa on the left (arrow) and the artifactual separation of the more substantial septum on the right (double arrows). x 28.
SEM OF BOVINE LUNG A. T. Mariassy, C. G. Plopper and D. L. Dungworth
PLATE 1
19
PLATE 2 EXPLANATION O F FIGURES
20
3
Bronchiolar epithelium from terminal bronchiole. The ciliated cells with short cilia and microvilli intermingle with the nonciliated cells. The border of the latter is distinctly delineated by the short stubby microvilli of the former. The apical portion of the nonciliated cell protrudes into the airway lumen. x 3,200.
4
Transition of respiratory bronchiole ( r b ) to alveolar ducts ( a d ) . Note the extremely poor alveolarization of the respiratory bronchiole, represented here by only a few shallow alveolar outpocketings (arrows). x 130.
SEM OF BOVINE LUNG A. T . Mariassy, C. G. Plopper and D. L. Dungworth
PLATE 2
21
PLATE 3 EXPLANATION O F FIGURES
5
Area of transition from bronchiolar to alveolar epithelium. Note the protruding surfaces of the nonciliated cells and the large number of type I1 epithelial cells (11). x 1,500.
6 Alveolar epithelium adjacent to interlobular septum illustrating large number of type I1 epithelial cells (11) and seams at epithelial cell junctions (arrow). x 1,870.
22
SEM OF BOVINE LUNG A. T. Mariassy, C . G . Plopper and D. L. Dungworth
PLATE 3
23
PLATE 4 EXPLANATION O F FIGURES
7
Typical group of alveoli illustrating almost complete absence of interalveolar pores of Kohn. x 290.
8 Atypical occurrence of small pores in one of the 12 lungs examined noted as depicted here. Their frequency and size is still appreciably smaller than observed in other species studied. x 440.
24
SEM OF BOVINE LUNG A. T. Mariassy, C . G. Plopper and D. L. Dungworth
PLATE 4
25
ABSTRACT The surface characteristics of the intrapulmonary airways and alveoli of the lungs from 12 young adult cattle examined by the scanning elec-
tron microscope are described. When compared with similar studies of the lungs of various other mammalian species, the bovine lung generally resembles that of other mammals except for several important features: (1) Alveolar pores of Kohn are small and extremely rare; ( 2 ) Alveolar macrophages are seldom seen in alveolar air spaces; ( 3 ) Interlobular septa completely separate the lung into distinct lobules. MATERIALS AND METHODS The normal mammalian respiratory system has proven to be a highly suitable subFresh lungs were obtained from twelve ject for morphological studies employing cross-bred beef cattle, approximately 20 the scanning electron microscope. A num- months of age, ( 6 neutered males, 4 intact ber of descriptions exist for the common males, and 2 females), a t a local abbatoir. laboratory species, including the hamster The middle lobe from each steer, a cranial (Nowell and Tyler, '71; Kuhn and Finke, lobe from each intact male, and the middle '72), rat (Kuhn and Finke, '72; Parkinson and cranial lobes from each of the cows and Stephens, '73), mouse (Greenwood were processed for examination. After canand Holland, '72; Kuhn and Finke, '72; nulation of the lobar bronchus, each lobe Andrews, '74), guinea pig (Dahlgren et al., was fixed overnight at 4°C by airway per'72) and rabbit (Holma, '69; Wang et al., fusion a t 30 cm water pressure with cac'73). Among larger species, the dog (Gro- odylate-buffered formaldehyde-glutaraldeniowski et al., '72), the horse (Nowell and hyde (Karnovsky, '65) adjusted to 550 Tyler, '71; Nowell et al., '71), and non- milliosmoles per liter. Approximately 30 human primates (Greenwood and Holland, samples, 1 X 1 X 0.5 cm in size, were '73; Castleman et al.,'75) and m a n (Wang taken from each lobe, dehydrated with and Thurlbeck, '70) have received atten- graded solutions of ethanol followed by tion. While ultrastructural studies of the amyl acetate, and critical point dried with lungs of normal adult cattle (Epling, CO, (Anderson, '51). Dried specimens were '64a,b) and calves (Rybicka et al., '74a,b) mounted on stubs, double coated with silhave been performed by transmission elec- ver and gold, and examined with a scantron microscopy, to the best of our knowl- ning electron microscope (ETEC Autoscan). edge the surface morphology of only one RESULTS ruminant species, newborn lamb (Tyler et Bronchi al., '71), has been described. This report presents a brief description of the surface Bronchi are identified by their large Iucharacteristics of bovine lung as seen by minal diameters and by the cut surfaces scanning electron microscopy. Its primary of their walls which contain a layer of purpose is to illustrate several distinctive epithelium supported by thick bands of anatomic features that are especially well smooth muscle, dense connective tissue, revealed by this method of examination bronchial glands, and plates of cartilage. and that are important for an understand- The epithelium is composed of ciliated and ing of pathophysiological responses of bo- ~Received Sept. 9, '74. Accepted Mar. 18, '75. vine lungs. 1 Supported in part by NIH Grant ES HL 00628. ANAT. REC., 183: 13-26.
13
14
A. T. MARIASSY, C. G. PLOPPER AND D. L. DUNGWORTH
nonciliated cells. Ciliated cells predominate over the majority of the surface, but among them are interspersed single nonciliated cells (fig. 1). Dense aggregations of long uniform cilia, with microvilli distributed around their bases, project from the surfaces of the ciliated cells. The mucosal surface of the nonciliated cells is covered by shorter microvilli and does not protrude into the lumen. In nonciliated cells with few microvilli, large pits or pores open onto the surface. These pores sometimes contain secretory material fixed in situ (fig. 1). Actively-secreting nonciliated cells have very few microvilli, as opposed to quiescent non-secreting cells which are covered by a dense population of short, stubby microvilli. Bronchioles The terminal (non-respiratory ) bronchioles are differentiated from bronchi by their smaller diameter and thinner walls, which lack cartilage and glands. The epithelium is supported by thin bands of smooth muscle and connective tissue (fig. 2). In contrast to bronchi, the bronchiolar mucosa is composed of approximately equal numbers of ciliated and nonciliated cells (fig. 3). The ciliated cells have slightly shorter cilia than those in the bronchi. Bronchiolar ciliated cells also differ from their bronchial counterparts by having shorter and more numerous microvilli. The luminal surface of the nonciliated bronchiolar cell has an oval perimeter and appears to have a granular surface, with few very short microvilli. The central portion of the cell frequently protrudes into the airway. The cell boundaries are easily discernible by virtue of the contrast provided by villi on adjacent ciliated cells. Respiratory bronchioles are defined by the presence of occasional alveolar outpocketings in their walls. In contrast to the well-developed system of terminal bronchioles, respiratory bronchioles are short and poorly alveolarized (fig. 4). The proportion of nonciliated to ciliated cells increases in the respiratory bronchioles. Ciliated cells become few in number; their cilia are shorter, less abundant and more disorderly. The apical protrusions of nonciliated cells are most prominent at the
bronchiolar junction with the respiratory epithelium (fig. 5).
Alveoli The pulmonary parenchyma is composed of polyhedral alveolar spaces whose walls are formed by the thin interalveolar septa. Three cell types are observed in alveoli. The majority of the alveolar surface is covered by the flattened, smooth-surfaced squamous (type I ) epithelial cells (fig. 6). Small projections can be seen scattered on the surfaces of these cells. The second type of epithelial cell, the granular (type 11) pneumonocyte, usually lines only a small portion of the septum. Its surface protrudes into the alveolar lumen and is covered by many microvilli. Openings or pits on the surface of the type I1 cells are rarely seen. The granular pneumonocytes are particularly numerous in alveoli adjacent to interlobular septa (fig. 6 ) and in alveoli close to bronchioles. Boundaries between epithelial cells are marked by a prominent seam (fig. 6). The third cell, the pulmonary alveolar macrophage, was very rarely observed. A notable characteristic of alveoli is the usual almost total lack of interalveolar pores (of Kohn) in the interalveolar septa (fig. 7). Their frequency is approximately one per ten septal faces and when present they appear as small ellipsoidal openings penetrating the alveolar walls. In only one of the 12 lungs is there an atypical finding of more numerous small pores (fig. 8). Interlobular septa The interlobular septa reported by others from subgross studies of bovine lung are readily apparent (fig. 2). They are formed of interconnected sheets of connective tissue of various thicknesses extending from peribronchial regions to the pleura, subdividing the pulmonary parenchyma throughout the substance of the lobes into separate polyhedral lobules. Even where the septa are insubstantial, the separation between adjacent lobules is complete. The connective tissue is formed into loosely arranged, thin apposed laminae running in a plane of each septum. Blood vessels and lymphatics are observed in thicker septal regions.
SEM OF BOVINE LUNG DISCUSSION
This study demonstrates that, in general, bovine intrapulmonary respiratory surfaces resemble those of other mammalian species previously reported. The epithelium of airways is a mixture of ciliated and nonciliated cells, with the former predominating in bronchi and the latter in bronchioles. In the bronchi, the nonciliated mucous goblet cell has short microvilli in the non-secretory phase, as has been observed in rodents (Greenwood and Holland, ’72) and macaques (Castleman et al., ’75). We did not observe the smooth, domed surfaces found in equine lungs (Nowell and Tyler, ’71). Also, brush cells (Watson and Brinkman, ’64; Meyrick and Reid, ’68) were not observed in bronchi, bronchioles or alveoli, but more extensive studies will be needed before a decision can be made on whether or not they occur at all in bovine lungs. The bronchiolar epithelial admixture of ciliated and nonciliated cells seems to be a common feature of all mammalian lungs thus far described. The role of the nonciliated cells and the closeness of their resemblance to human Clara cells (Cutz and Conen, ’71) remains to be determined. In addition, we have confirmed that bovine lungs have short or nonexistent respiratory bronchioles, as reported on the basis of subgross studies (McLaughlin et al., ’61). With respect to features of alveolar epithelial cells, we did not observe the large pits in the surface of type I1 cells as shown to be present in equine lung by Nowell and Tyler (’71). Reasons for the difference are purely speculative until information is available on surfactant synthesis and turnover in the two species. Three morphologic features of the parenchymal tissue of bovine lung are in sharp contrast with the pulmonary parenchyma of other mammals thus far studied by SEM. First, interalveolar pores of Kohn are extremely uncommon in the lungs of young adult cattle. We interpret the rarity of pores in 11 of 12 lungs to be the typical situation for this age group. The presence of moderate numbers of small pores in one of the 12 lungs represents an atypical finding, possibly resulting from previous pulmonary damage. That there had been earlier damage to the lung was indicated by the existence of abnormal deformations
15
and folds in the walls of some of its bronchioles. The second characteristic morphologic feature is that interlobular septa are well delineated and completely isolate pulmonary lobules, as previously indicated by subgross studies (McLaughlin et al., ’61) and by physiological studies of Van Allen et al. (’31). The third feature is the rarity of alveolar macrophages in alveolar lumens. A similar conclusion was reached by Rybicka et al. (’74a) after their examination of lungs of calves by transmission electron microscopy. Rybicka et al. (‘74a,b) also observed numerous cells in the pulmonary capillaries of normal calves which they felt, on the basis of ultrastructural characteristics and evidence of phagocytic capability, were more suitably designated “intravascular macrophages” than simply blood monocytes. It is tempting to speculate that the relationship between scarcity of alveolar macrophages and abundance of cells within pulmonary capillaries already having features of macrophages is not fortuitous. That is to say, the intravascular cells might represent a more readily recruitable pool of macrophages than usual to compensate for the scarcity of resident alveolar macrophages. If this interpretation is correct, the intravascular macrophages described by Rybicka et al. (’74b) represent cells comprising a portion of the intermediate lung maturation compartment proposed by Bowden (’71) to be where blood monocytes undergo differentiation into functional alveolar macrophages. These special features have important implications with regard to the pathophysiology and pathology of the bovine lung. Collateral ventilation within and among pulmonary lobules is severely limited or absent because of the scarcity of pores of Kohn and the completeness of the interlobular septa (Van Allen et al., ’31). Van Allen and colleagues also showed that focal pulmonary atelectasis was more prone to occur in the absence of collateral ventilation. Lack of collateral ventilation is also likely both to hamper compensation for inequalities of ventilation associated with obstructions of small airways and to reduce the efficiency of mechanical expulsion of intraluminal obstructions. Consequences of
16
A. T. MARIASSY, C. G. PLOPPER AND D. L. DUNGWORTH
the latter in the case of intrabronchiolar accumulations of exudate would be to impair the combatting of a n incipient bronchopneumonia or the resolution of a fully developed inflammatory process. The paucity of alveolar macrophages would also appear to put the bovine lung at a disadvantage with respect to resistance to aerogeneous bacterial infections since maintenance of bacterial sterility of alveolar parenchyma is primarily ascribed to these cells (Green, '73). The completeness of the interlobular septa, and the apparent ease with which the component connective tissue laminae can be separated, correlate well with the frequency with which interstitial emphysema is found in damaged lungs of cattle following periods of severely labored breathing, particularly in association with acute (atypical) interstitial pneumonia (Jubb and Kennedy, '70). A final point of concurrence between these findings and our observations of the response of the bovine lung to injury, is that the hyperplasia and hyDertrophy of type I1 epithelial cells (epithelialization ) which can be extensive as early as three to four days after widespread alveolar damage is most pronounced i n peribronchiolar and paraseptal regions (Dungworth). These are the two regions i n which the type I1 cells are most numerous in the normal bovine lung. LITERATURE CITED Anderson, T. F. 1951 Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci., 13: 130-134. Andrews, P. M. 1974 A scanning electron microscopic study of the extrapulmonary respiratory tract. Am. J. Anat., 139: 399-424. Bowden, D. H. 1971 The alveolar macrophage. Current Top. Pathol., 55: 1-36. Castleman, W. L., D. L. Dungworth and W. S. Tyler 1975 Intrapulmonary airway morphology in three species of monkeys. A correlated scanning and transmission electron microscopic study. Am. J. Anat., 142(1): 107-121. Cutz, E., and P. E. Conen 1971 Ultrastructure and cytochemistry of Clara cells. Am. J. Path., 62: 127-142. Dahlgren, S. E., H. Dalen and T. Dalhamm 1972 Ultrastructural observations on chemically induced inflammation in guinea pig trachea. Virchows Arch. Abt. B. Zellpath., 1 1 : 211-223. Dungworth, D. L. Unpublished observations. Epling, G. P. 1964a Electron microscopy of the bovine lung; The normal blood-air barrier. Am. J. Vet. Res., 25: 679-689.
196413 Electron microscopy of the bovine lungs: Lattice and lamellar structures i n the alveolar lumen. Am. J. Vet. Res., 25: 14241430. Green, G. M. 1973 Lung defense mechanisms. Med. Clin. North Am., 57(3): 547-562. Greenwood, M. F., and P. Holland 1972 The mammalian respiratory tract surface. A scanning electron microscopic study. Lab. Invest., 27: 296-304. 1973 Scanning electron microscopy of the normal and BCG-stimulated primate respiratory tract. J. Reticuloendo. SOC.,13: 183-192. Groniowski, J., M. Walski and W. Biczysko 1972 Application of scanning electron microscopy for studies of the lung parenchyma. J. Ultrastructure Res., 38: 473481. Holma, B. 1969 Scanning electron microscopic observation of particles deposited in the lung. Arch. Environ. Health, 18: 330-339. Jubb, K. V. F., and P. C. Kennedy 1970 Pathology of domestic animals. Second ed. Academic Press, Inc., New York, p. 182. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A138A. Kuhn, C., 111, and E. H. Finke 1972 The topography of the pulmonary alveolus: Scanning electron microscopy using different fixations. J. Ultrastruct. Res., 38: 161-173. McLaughlin, R. F., W. S. Tyler and R. 0. Canada 1961 A study of the subgross pulmonary anatomy in various mammals. Am. J. Anat., 108: 149-165. Meyrick, B., and L. Reid 1968 The alveolar brush cell in rat lung - a third pneumonocyte. .J. Ultrastruct. Res., 23: 71-80. Nowell, J. A., J. R. Gillespie and W. S. Tyler 1971 Scanning electron microscopy of chronic pulmonary emphysema: A study of the equine model. In: Scanning Electron Microscopy/l971. Part I. 0. Johari, ed. IIT Research Institute, Chicago, pp. 297-304. Nowell, J. A., and W. S. Tyler 1971 Scanning electron microscopy of the surface morphology of mammalian lungs. Am. Rev. Resp. Dis., 103: :313-328. Parkinson, D. R., and R. J. Stephens 1973 Morphological surface changes in the terminal bronchiolar region of NOz-exposed rat lung. Environ. Res., 6: 37-51. Rybicka, K., B. D. T. Daly, J. J. Migliore and J. C. Norman 1974a Ultrastructure of pulmonary alveoli of the calf. Am. J. Vet. Res., 35: 213222. 1974b Intravascular macrophages in normal calf lung. An electron microscopic study. Am. J. Anat., 139: 353-368. Tyler, W. S., A. A. de Lorimier, A. G. Manus and J. A. Nowell 1971 Surface morphology of hypoplastic and normal lungs from newborn lambs. Scanning Electron Microscopy/l971. Part I. 0. Johari, ed. IIT Research Institute, Chicago, pp. 305-312. Van Allen, C. M., G. E. Lindskog and H. G . Richter 1931 Collateral respiration. Transfer of
SEM OF BOVINE LUNG air collaterally between pulmonary lobules. J. Clin. Invest., 10: 559-590. Wang, N. S., H. W. Taeusch, W. M. Thurlbeck and M. E. Avery 1973 A combined scanning and transmission electron microscopic study of alveolar epithelial development of the fetal rabbit lung. Am. J. Path., 73: 365-376.
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Wang, N. S., and W. M. Thurlbeck 1970 Scanning electron microscopy of the lung. Human Path., I: 227-231. Watson, J. H. L., and G. L. Brinkman 1964 Electron microscopy of the epithelial cells of normal and bronchitic human bronchus. Am. Rev. Resp. Dis., 90: 851-866.
PLATE 1 EXPLANATION O F FIGURES
18
1
Bronchial epithelium showing predominance of ciliated cells with interspersed nonciliated cells. Secretory material emerging from the latter has been fixed in situ. x 4,600.
2
Pulmonary parenchyma with segment of a lobule completely delineated by interlobular septa and a n adjacent portion of a terminal bronchiole. Note the delicacy of the two septa on the left (arrow) and the artifactual separation of the more substantial septum on the right (double arrows). x 28.
SEM OF BOVINE LUNG A. T. Mariassy, C. G. Plopper and D. L. Dungworth
PLATE 1
19
PLATE 2 EXPLANATION O F FIGURES
20
3
Bronchiolar epithelium from terminal bronchiole. The ciliated cells with short cilia and microvilli intermingle with the nonciliated cells. The border of the latter is distinctly delineated by the short stubby microvilli of the former. The apical portion of the nonciliated cell protrudes into the airway lumen. x 3,200.
4
Transition of respiratory bronchiole ( r b ) to alveolar ducts ( a d ) . Note the extremely poor alveolarization of the respiratory bronchiole, represented here by only a few shallow alveolar outpocketings (arrows). x 130.
SEM OF BOVINE LUNG A. T . Mariassy, C. G. Plopper and D. L. Dungworth
PLATE 2
21
PLATE 3 EXPLANATION O F FIGURES
5
Area of transition from bronchiolar to alveolar epithelium. Note the protruding surfaces of the nonciliated cells and the large number of type I1 epithelial cells (11). x 1,500.
6 Alveolar epithelium adjacent to interlobular septum illustrating large number of type I1 epithelial cells (11) and seams at epithelial cell junctions (arrow). x 1,870.
22
SEM OF BOVINE LUNG A. T. Mariassy, C . G . Plopper and D. L. Dungworth
PLATE 3
23
PLATE 4 EXPLANATION O F FIGURES
7
Typical group of alveoli illustrating almost complete absence of interalveolar pores of Kohn. x 290.
8 Atypical occurrence of small pores in one of the 12 lungs examined noted as depicted here. Their frequency and size is still appreciably smaller than observed in other species studied. x 440.
24
SEM OF BOVINE LUNG A. T. Mariassy, C . G. Plopper and D. L. Dungworth
PLATE 4
25
