Microscopic and submicroscopic anatomy of the parabronchi air sacs and respiratory space of the budgerigar (Melopsittacus undulatus).код для вставкиСкачать
THE AMERICAN JOURNAL OF ANATOMY 177:221-242 (1986) Microscopic and Submicroscopic Anatomy of the Parabronchi, Air Sacs, and Respiratory Space of the Budgerigar (Melopsittacus undulatus) JEROME H. SMITH, JUDY L. MEIER, CHERYL LAMKE, P.J.G. NEILL, AND EDITH D. BOX Department of Pathology and Laboratory Medicine, College of Medicine, Texas A and M University, College Station, Texas 77843-1114(J.H.S., I?J.G.N.); and Departments of Pathology (J.L M., C.L) and Microbiology (E.D.B.), University of Texas Medical Branch, Galweston, Texas 77550 ABSTRACT The normal microscopic and submicroscopic structure of the lower respiratory tract of the budgerigar (Melopsittacus undulatus)is described and compared with other birds and mammals. Granular (type 11)pneumocytes are confined to linings of air sacs, parabronchi, and their atria; however, their secretions (surfactant) cover the surfaces of the infundibula and respiratory space. Infundibula extend from the atria and give rise to the air capillaries, which branch and anastomose freely with those of adjacent infundibula and other parabronchi (interparabronchial septa are not found). Infundibula and the respiratory labyrinth are lined by a continuous epithelium of squamous pneumocytes, whose perikarya are concentrated in the infundibula and whose peripheral cytoplasm is markedly attenuated. The squamous pneumocytes of the respiratory labyrinth share a basal lamina with the blood capillaries that they envelop. In 1978 the protozoan Sarcocystis falcatula was discovered to complete its asexual schizogony in many families of birds (Duszynski and Box, 1978; Box and Smith, 1982; Box et al., 1984). This breadth of intermediate host range was remarkable, considering that previously described Sarcocystis species had single genera as intermediate hosts. Thus its development and comparative pathology in different avian families needed study. Early generations of the sporozoan develop within specific pulmonary vessels and produce distinctive pulmonary lesions whose description demanded precise anatomic definition of normal pulmonary structures. Review of the literature at that time revealed careful descriptions and illustrations of air passages down to parabronchi (Krause, 1922; Akester, 1960; King, 1966; Evans, 1969; King and Atherton, 1970; Lasiewski, 1972) and brief notes regarding the ultrastructure of the respiratory anatomy of some birds (Tyler et al., 1961; Pattle and Hopkinson, 1963; Tyler and Pangborn, 1964; Fugiwara et al., 1970), but there was no integrated comprehensive description of the microscopic and s u b 0 1986 ALAN R. LISS, INC. microscopic structure of the budgerigar (Melopsittacus undulatus) respiratory system. MATERIALS AND METHODS Light and electron microscopy Six adult budgerigars were sacrificed by COZ inhalation and the anterior neck was immediately opened. An 18-gauge hypodermic needle was inserted and tied into the upper trachea, and the lungs were inflated with cold (0-4 "C) half-strength Karnovsky's solution (Karnovsky, 1965) until fixative flowed freely (without bubbles) out of the ruptured thoracic, abdominal, and cervical air sacs. After 15 min the lungs were removed and placed in cold (0-4°C) half-strength Karnovsky's solution for 30 min, then cut into 0.5- to 1.0-mm slices and 1.0-mm cubes, and fixed for 1-2 h r more. Blocks were postfixed , in ethanol, and embedin 0 ~ 0 4 dehydrated ded in Polybed 812. Thick (1pm) sections for light microscopy were stained with 1% toluidine blue in 1% sodium borate. Thin sections Received April 18,1983. Accepted May 25,1986. 222 J.H. SMITH ET AL smooth muscle fascicles defined the adventitia of the parabronchi (Figs. 4,5 ) , and basement membranes of blood capillaries abutted on the abluminal surface of this adventitia. The upper portion of the sides of the atria was buttressed by the parabronchial muscle fascicles (Figs. 4, 5). A fornix was formed below these fascicles where the sides and floor of the atria joined; this recess extended for a short distance beneath the abluminal RESULTS margins of the muscle fascicles (Fig. 4). Atrial General epithelial cells covered the sides, filled the While light microscopy of 4-to 5-pm-thick fornices, and lined the floor of the atrium. paraffin sections afforded insufficient resolu- The atrial epithelium was supported by a tion for detailed study, examination of tolui- basal lamina through which coursed colladine blue-stained semithin (1-pm) plastic gen fibers, peripheral processes of fibrosections allowed definition of parabronchi blasts, and rare smooth muscle cells (Figs. 4, and their atria, air sacs, infundibula, and the 5, 12). The atrial cell (Figs. 4-8) varied from squarespiratory space. Vessels consisted of arteries, arterioles, two orders of capillaries, pri- mous to cuboid. Distal processes of atrial cells mary and secondary venules, and veins. extended over the luminal surface of the parTerminal secondary bronchi were lined by abronchial smooth muscle fascicles and mucociliary columnar epithelia with some formed junctional complexes with the s q u a nuclear pseudostratification (Smith et al., in mous cells lining infundibula (Fig. 12). The press). Where secondary bronchi and para- apical surface of some cells had numerous bronchi connected, cuboid cells covered the microvilli, but others were smooth. The latjunction (Fig. 1); these cells like bronchial eral margins were undulating and connected basal cells contained tonofibrils and like to adjacent cells with junctional complexes atrial cells contained myelinoid bodies. Tu- (Figs. 8, lo), but maculae to the basal lamina bular parabronchi with shallow atria coursed were rarely seen. These junctional complexes through the pulmonary parenchyme and were principally composed of long gap juncwere perforated by infundibular ostia in tions that were often reinforced by maculae depths of the atria (Fig. 2). Both longitudinal adherentia subapically. Occasionally, apparand cross sections of parabrohchi showed a ent zonulae occludentes were observed within scalloped profile with atria forming the evag- the gap junctions (Fig. 10). The ovoid nucleus inations of the lumina (Fig. 3). The structure frequently had a corrugated margin. There of the atrial and parabronchial walls differed was marked variation in the clumping of hetslightly, but both differed markedly from erochromatin at the nuclear membrane, and more proximal and distal respiratory linings. the euchromatin had moderate density. NuThe wall of the parabronchus (Figs. 3-8) cleoli were not prominent. Mitochondria were was composed of widely dispersed fibroblasts, collagen fibers, and occasional elastic fibers surmounted by fascicles of smooth muscle (Figs. 3-5). It was lined by extensions of the peripheral cytoplasm of atrial cells. A layer of myelinoid material of varying depth overlay this epithelial surface. Junctional com- Fig. 1. Electron micrograph of the junction of a secplexes joined these cells at their lateral ondary bronchus (S)parabronchus (PI. The thick fibromargins. The basal plasma membrane rested muscular wall of the secondary bronchus is composed of smooth muscle cells (s) and collagen (D and is surupon the basal lamina surrounding subja- mounted by an attenuated process of a cell with tonocent smooth muscle cells; this basal lamina filaments (black-on-white arrow), resembling a bronchial occasionally contained collagen fibrils. All basal cell; but a myelinoid body (as in atrial cells) is also collagen fibrils observed in the budgerigar’s seen @lack arrow). The cytoplasm of an adjacent atrial with an overlying myelinoid (surfactant) layer (white lung measured 42 & 2.3 nm in diameter and cell arrows) is seen. A thick basal lamina with collagen fiwere banded with a periodicity of 62.5 If- 5.9 bers separates the smooth muscle from the epithelium. nm. The fibroelastic layer subjacent to the x 21,600. were stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed with a Philips EM-200 transmission electron microscope. All measurements are given as the mean ~ f -1.0 standard error of the mean unless otherwise specified. Anatomic terms used conform to standard terminology (King, 1979) except where indicated. ULTRASTRUCTURE OF BUDGERIGAR LUNG 223 224 J.H. SMITH ET AL. ULTRASTRUCTURE OF BUDGERIGAR LUNG ovoid with tubular cristae. Granular endoplasmic reticulum and polysomes were abundant. Microfilaments, both 5 and 8-10 nm in diameter, were frequent but were not aggregated into tonofibrils. The most remarkable feature of these cells was the prominent myelinoid bodies (Figs. 6-8). These bodies disgorged their contents onto the apical surface, where the contents appeared to spread and form the fibrillar or myelinoid coat of both the parabronchi and the respiratory space (Figs. 6-8). Another common cytoplasmic inclusion was an electron-lucent cleft (Figs. 7, 8) which was not membrane-bound; these elongate “splinters” often intersected, rarely contained loosely concentric membranous profiles, and occasionally fused to myelinoid bodies. Some atrial cells (less than 10%) were larger, more globular, and more “watery” than the “dense” atrial cells. Heterochromatin condensation at the nuclear membrane was more uniform and narrower in these atrial cells, and the euchromatin was more electron lucent. Nucleoli were often prominent. Granular endoplasmic reticulum, polysomes, and microfilaments were widely separated. Mitochondria were larger and less numerous than in “dense” cells. Large myelinoid bodies and cleft inclusions were numerous. Fig. 2. Photomicrograph of cut surface of budgerigar lung. Longitudinal sections through parabronchi (p) show shallow atria with infundibular ostia (black-on-white arrow). Infundibula (black arrow) radiate from parabronchi into the respiratory labyrinth (1). Half-strength Karnovsky fixation, unstained, x 50. Fig. 3. Photomicrograph of two longitudinally oriented parabronchi (P) and intervening respiratory space. Muscular wall of parabronchus is noted above lower P, and the atria (A) are well defined. Several infundibula (arrows) extend into the respiratory labyrinth. No fibrous septum separates the respiratory space of the two parabronchi. Veins (V) course through the respiratory labyrinth perpendicular to the long axis of the parabronchi. Toluidine blue, x 320. Fig. 4. Low-magnification view of wall of parabronchus with an atrium (A); atrial floor is noted (small arrow) as well as atrial wall (large arrow). A prominent fascicle of smooth muscle (s) defines the wall of the parabronchus and is overlain by processes of atrial cells. Note that atrial cells (a) extend into the fornix formed beneath the fascicle of smooth muscle. x 2,400. 225 A i r sacs Some parabronchi emptied into air sacs within the thorax. These thin-walled structures were lined by cells that were identical to the atrial cells but were more flattened (Fig. 9). All stages in formation of the myelinoid inclusions were seen, including multivesicular bodies and disgorgement of myelinoid contents t o form the surface coating over the apical plasma membranes (Fig. 10).Polysomes, granular endoplasmic reticulum, and mitochondria were abundant. The apical plasma membrane was smooth or had small microvilli. Adjacent cells had undulant or oblique lateral surfaces attached by long junctional complexes (Fig. 10). The basal plasmalemma had myriads of pinocytotic and coated vesicles; and the epithelia rested on a thin basement membrane, but hemidesmosomes were rare. The mesenchymal wall of the air sacs was composed principally of two to five layers of fibrocytes, abundant collagen fibers, and elastic fibers oriented parallel to the surface. Intermittent smooth muscle fascicles were noted (Fig. 9). Respiratory space (Figs. 2, 3, 11-19) As seen in the dissecting microscope (Fig. 21, the floor or base of the atrium was perforated by numerous small ostia that opened into elongate wedges whose diameter decreased progressively from their ostia; these were the infundibula of the respiratory space (Figs. 3,ll-13). The structure of the infundibulum was slightly different from numerous air passages that originated from it. When parabronchi were expanded and atria were distended, the ostia of the infundibula were open (Fig. 11);but when atrial muscle was contracted and the atria were shallow, the ostia appeared closed (Fig. 11,inset). The infundibulum was lined by distal processes of squamous pneumocytes whose perikarya were usually located near the ostium (Figs. 11, 13).The basal lamina of the atria extended to the margin of the ostia and turned perpendicular to the atrial floor to form the wall of the infundibulum (Fig. 12). There often were redundant, globular deposits of basal lamina matrix at its reflection down the infundibulum (Fig. 12). Near the ostium, the infundibular basal lamina contained a few collagen fibers; but these progressively disappeared distally (Figs. 12, 13). The infundibular basement membrane had a thickness that was similar to that in the 226 J.H. SMITH ET AL. Fig. 5. Wall and floor of distended atrium. Smooth muscle (s) defining the wall of a parabronchus is noted in the upper right. Abundant collagen fibers (f, right) are noted along the atrial side wall finteratrial septum), which is overlain by atrial cells (a). One atrial cell con- tains numerous myelinoid inclusions (it. Collagen fibers (f, below) are less frequent in the atrial floor. An artifactually extravasated nucleated erythrocyte lies in the atrial lumen (A). x 8,600. ULTRASTRUCTURE OF BUDGERIGAR LUNG 227 Fig. 6. Electron micrograph of atrial floor with atrial (black-on-white arrow) and fibroblasts (0. x 15,400. cells (a) containing laminated myelinoid inclusions (9. Fig, 7. Electron micrograph of atrial cell with myeliOne of these has spread along the surface of the cell noid inclusions (i) and cleft-like inclusions (c). The super(black arrows). Numerous mitochondria (m) are noted. ficial myelinoid layer (arrows) has a fibrillar appearance. Subjacent to the atrial cells, there are collagen fibers x 70,000. Fig. 8. Atrial surface showing portions of four atrial plexes (arrows). X 18,400. cells. The surface is covered by a fibrillar myelinoid Fig. 9. This portion of the wall of a thoracic air sac is layer, and the superficial plasma membrane has numer- composed principally of leiomyocytes (s),elastin (el, and ous microvilli. The cytoplasm contains cleft-like (c) and collagen fibers (0. A lining of flattened cuboid to squamyelinoid (i) inclusions as well as numerous mitochon- maus cells with numerous mitochondria (m) and myelidria (m). Adjacent cells are linked by junctional corn- noid inclusions (i) is seen. x 13,600. Fig. 10. High-magnification view of cells lining air sac. The prominent microvillous surface is partly COV. ered by a myelinoid surfactant layer (large arrows). Multivesicular bodies (mb), laminated myelinoid inclusions (i), and polysomes are numerous. Mitochondria (m) contain tubular cristae (black-on-white arrow). One inclu- sion appears to be disgorging its contents to the surface (small black arrows). Adjacent cells have apical junctional complexes that principally consist of long gap junctions with an apparent zonula occludens (arrowhead). x 46,000. 230 J.H. SMITH ET AL. Fig. 11. Ostia (asterisks) of infundibula (I)and atrium (A). Atrial wall is present at small arrows. Infundibular ostia and respiratory labyrinth are open and distended. Squamous pneumocytes (arrowheads) are noted at the lip of a n infundibulum and along the infundibula. x 3,710. Inset: Low-power electron micrograph of a nearly closed infundibular ostium with prominent cuboid atrial cells (a). x 3,100. Fig. 12. Higher magnification of junction of atrium (A) and infundibular ostium (I) seen in Figure 11. A smooth muscle cell (s)underlies the attenuated process of the atrial cell (a). Numerous collagen fibers (white arrows) are noted in the basal lamina under this atrial cell. This basal lamina has redundant folds (black ar- rows). The squamous pneumocyte (p) lies on a basal lamina with few collagen fibers (white arrowhead). The pneumocyte’s processes extend down the infundibular wall and along the lip of the ostium. Erythrocyte-filled capillaries (B) lie along the abluminal surface of the atrial and infundibular wall. X 22,400. Fig. 13. Junction of infundibulum 0) and “air capillary” (1). The squamous pneumocyte (p) extends its cytoplasm over the infundibular wall to line the air capillary (arrow). The basal lamina of the infundibulum is thick but becomes attenuated as it continues as the basal lamina of the air capillary. No collagen fibers are seen in this basal lamina from the depths of the infundibulum. x 8,600. Fig. 14. The squamous pneumocyte (p) within the respiratory labyrinth conforms to the angle of two adjacent blood capillaries 03). Pneumocyte cytoplasm extends in long attenuated processes (arrows) over the capillary basal lamina. The blood capillaries are lined by a continuous endothelium. 1, “air capillary.” X 13,000. ULTRASTRUCTURE OF BUDGERIGAR LUNG 233 Fig. 15. Two blood capillaries intersect with endothe- toplasm of' two pneumocytes is shown at broad arrow lial perikarya (E) in approximation. Note marked atten- (and inset). X 12,000. Inset: The apical interface of the uation (slender arrows) of capillary endothelium, basal two pneumocytes is closed by a gap junction with a lamina, and squamous pneumocyte opposite the endo- subjacent macula adherens forming a typical epithelial thelial cell nuclei. Junction of expanded peripheral cy- junctional complex. X 50,400. 234 J.H. SMITH ET AL. ULTRASTRUCTURE OF BUDGERIGAR LUNG atria but was nearly twice that of the respiratory passages (air capillaries) emanating from it (Fig. 13). The abluminal surface of the infundibular basement membrane abutted capillaries and was overlain by distal extensions of squamous pneumocytes. Multiple respiratory passages (air capillaries) branched off the infundibula at acute or obtuse angles (Figs. 3,11,13).These passages varied markedly in diameter and shape of profile. They branched and anastomosed freely to form the labyrinth that is the respiratory space. They were lined by the markedly attenuated processes of squamous pneumocytes, which were applied over the basement membranes of capillaries, the abluminal aspects of atrial and infundibular walls, and the adventitia of larger vessels. The squamous pneumocytes (Figs. 11-18) completely enveloped the air capillaries and infundibula and formed junctional complexes with atrial cells (Fig. 12) and with each other (Fig. 15). These junctional complexes con- Fig. 16. High-magnification view of air-blood barrier. The erythrocyte (r) lies within the capillary lumen separated from the apical plasma membrane of the endothelial cell. Pinocytotic vesicles appear to fuse with both apical and basal endothelial plasma membranes. The basal endothelial plasma membrane lies on the basal lamina, which is markedly attenuated. Squamous pneumocytic cytoplasm is extremely attenuated and barely separates its apical and basal trilaminate plasma membranes; at two points it appears that the inner leaflets of the apical and basal pneumocytic plasma membranes fuse (slender arrows). A thin myelinoid surfactant layer (broad arrows) rests upon the surface of the apical plasma membrane of the pneumocyte. X 104,000. Fig. 17. Air-blood barrier. The capillary lumen (bottom) is lined by a continuous endothelium that rests upon the basal lamina. The apical and basal trilaminate plasma membranes of the squamous pneumocyte are barely separated. The trilaminate myelinoid surfactant layer (slender arrow) forms a bleh (broad arrow) in the lumen of the respiratory space. The pneumocytic membrane appears everted at the bleb. x 130,000. Fig. 18. Air-blood barrier. The erythrocyte (below) is barely separated from the endothelial cell plasma membrane. Numerous pinocytotic vesicles appear to have joined the apical and basal plasma membranes of endothelial cell (arrows), suggesting formation of a channel. x 121,000. 235 sisted of apical gap junctions with subjacent zonulae or maculae adherentia (Fig. 14). Their nuclei and perinuclear cytoplasm were most frequently seen along the infundibula (Figs. 11, 13) but also were found within the respiratory labyrinth (Figs. 14, 15). In the infundibula, these perikarya were usually flattened; but in the air passages they were globose or angular as they conformed to the corners formed by branching or intersecting blood vessels. Their nuclei resembled those of endothelial cells and were round to ovoid or compressed and crescentiform with scalloped margins and prominent irregular clumping of heterochromatin at the nuclear membrane. Nucleoli were not prominent. The cytoplasm contained polysomes and small ovoid mitochondria with lamellar cristae, but granular and agranular reticulum were sparse. Microfilaments were not prominent, and pinocytosis from any surface was rare. The basal plasmalemma rested on the basement membrane common to both the pneumocyte and endothelial cell, but no hemidesmosomes were seen (Fig. 14). Basal lamina were not observed subjacent to squamous pneumocytes, where “air capillaries” abutted on atrial, arterial, or venous adventitia. The apical plasma membrane was overlain by a myelinoid layer (Figs. 16-18); sections perpendicular to the surface usually showed this myelin to be one or more trilaminar membranes with occasional breaks or submembranous bullae (Fig. 17). Sections oblique or parallel to the surface, however, revealed a fibrillar or tubular myelin structure. Throughout the respiratory space the cytoplasm between the apical and basal plasma membrane of the pneumocyte was markedly attenuated (Figs. 14-18), often less than 10 nm; occasionally the inner leaflets of the apical and basal plasmalemma appeared to fuse (Fig. 16). Apical and basal plasma membranes diverged to include more cytoplasm at the periphery where junctions between squamous cells occurred (Fig. 15). The other components of the respiratory space were blood capillaries, their basal lamina, their contents, and their afferent and efferent vessels. Cells serving as “mesangial cells” were not identified. The capillaries possessed continuous endothelia differing in no way from previous descriptions (Fawcett, 1981) of continuous endothelia (Figs. 11-21). Their cytoplasm was thinned adjacent to the, attenuation of pneumocytic processes but not to the same extent as that of the pneumo- 236 J.H. SMITH ET AL. Fig. 19. Junction of wall of arteriole (lower left, and small muscular artery (upper right). The arterial and arteriolar lumens are lined by continuous endothelia (arrows). The wall of the arteriole is made up of a single layer of smooth muscle cells (s), whereas that of the small muscular artery contains several layers of smooth muscle cells. N o internal or external elastic layer is noted in either vessel. A markedly attenuated adventitia is seen. Air and blood capillaries abut upon this adventitia. Outlined rectangle is enlarged in Figure 20. x 12,300. ULTRASTRUCTURE OF BUDGERIGAR LUNG 237 Fig. 20. Higher magnification of area indicated in Fig. 21. Secondary venular wall showing continuous Figure 19, showing continuous endothelia (E), smooth endothelium (E), fibroblast processes 0,and banded Colmuscle media (s),and absence of elastics. Fibroblasts (0 lagen fibers (arrows) with only occasional leiomyocyte and collagen (arrow) of adventitia abut directly on air 0 ) processes noted. Air (1) and blood (B) capillaries abut and blood (B) capillaries. x 14,700, directly on the adventitia. X 14,800. 238 J.H. SMITH ET AL cytes (Figs. 16-18). Marked pinocytotic activity was evident at apical and basal surfaces, often forming apparent channels from apex to base (Fig. 18); and cells were joined by gap junctions. As noted before, the endothelial cells shared their basal lamina with the pneumocytes. The basal lamina conformed to the blood capillaries’ conformation; thus, the air capillary shape became the complement of the adjacent blood capillaries by default. The basal lamina was thinner in areas of endotheliaVpneumocytic cytoplasmic attenuation and thicker near endothelial perikarya. Rarely, a collagen fibril was seen within the basal lamina where the blood capillaries approximated. Endothelial perikarya of approximated blood capillaries most frequently backed upon each other, rather than locating opposite the point of capillary conjunction (Fig. 15). The blood-gas barrier was composed (from air space to capillary lumen) of a myelinoid layer (8.1 f 0.7 nm), apical pneumocytic plasmalemma (7.2 f 0.2 nm), pneumocytic cytoplasm (0-10.0 f 1.3 nm), basal pneumocytic plasma membrane (7.1 f 0.3 nm), basal lamina (9.0 f 2.4 to 41.4 f 4.0; range = 0-69 nm), endothelial basal plasma membrane (7.8 f 0.3 nm), endothelial cytoplasm (14.3 f 3.5 to 88.2 f 12.4 nm; range = 0147 nm, excepting channels), and endothelial apical plasmalemma (7.8 f 0.3 nm). The resulting interface was 61-178 nm thick, a very narrow separation between air and blood. The major afferent and efferent blood uessels of the capillary ran midway between the parabronchi. Arteries appeared to course parallel to the axis of the parabronchi, and veins ran perpendicular or at angles to the major arteries. The right and left pulmonary arteries had the structure of an elastic artery, whereas most of the “major” arteries at the parabronchial level looked like arterioles being characterized by an internal elastic lamina, smooth muscular media, absence of external elastic lamina, and scant adventitia. Branches of these small arteries emerged at obtuse angles (with respect to blood flow) and had two or three layers of smooth muscle cells, no elastic laminae, continuous endothelia, and a narrow adventitia with collagen fibers and occasional fibroblasts (Figs. 19,201. These in turn branched at nearly perpendicular angles to give a vessel with a single layer of circumferentially oriented smooth muscle cells, continuous endothelia, and ru- dimentary collagenous adventitia (Figs. 19, 20). These in turn gave rise to 20-pm-diameter capillaries (precapillaries) that branched into 10-pmcapillaries. Several of these small capillaries anastomosed to form two orders of venules (20-30 pm and 40-50 pm in diameter) that had continuous endothelia, basal lamina, no smooth muscle (or occasional, widely dispersed, single smooth muscle cells), occasional pericytes, and scant collagen fibers (Fig. 21). These emptied at nearly right angles into secondary veins that consisted of a continuous endothelium, one or two layers of smooth muscle cells and/or pericytes, and scant collagen. Primary veins were similar but had several layers of pericytes and smooth muscle cells and modest adventitial collagen. Significantly, no connective tissue septa enveloped the vessels coursing between the parabronchi. Thus, the respiratory labyrinth emanating from one parabronchus was continuous with that of the adjacent parabronchi. DISCUSSION The essential features of the budgerigar lower respiratory tract (parabronchialatrial-infundibular and respiratory labyrinthine) structure are schematically presented in Figure 22. The light microscope morphology of the parabronchi of various species of birds including the budgerigar has been described (Krause, 1922; King and Molony, 1971; Duncker, 1971, 1974; Lasiewski, 1972; Dubach, 1981; Evans, 1982; Drescher and Welsch, 1983). The atria of the budgerigar parabronchi are shallower and less distinctly separable from the parabronchus proper than in other birds. Interatrial septi are less substantial. Interparabronchial connective tissue septi are practically absent. It is uncertain whether this difference represents evolutionary divergence in families and orders of birds or is merely a matter of the size of the bird. Nevertheless, the aseptate nature of the budgerigar parabronchial system permits potential free exchange of gas (or liquid) between adjacent parabronchi via the respiratory labyrinth. The fine structure of the avian parabronchi and the atrial cells, but not that of the budgerigar, have also been described (Pattle and Hopkinson, 1963; Tyler and Pangborn, 1964; Petrik and Riedel, 1968a,b; Lambson and Cohn, 1968; Akester and Mann, 1969; King and Molony, 1971; Powell and Mazzone, 1983; 239 ULTRASTRUCTURE OF BUDGERIGAR LUNG 22 SMOOTH MUSCLE INTERATRIAL SEPTUM FIBROBLAST GRANULAR PNEUMOCYTES INFUNDIBULAR OSTlA BLOOD CAPILLARIES AIR CAPILLARY OSTIA INFUNDIBULUM PROCESS Of SOUAMOUS PNEUMOCYTE SOUAMOUS PNEUMOCYTES Fig. 22. Schematic representation of parabronchial atrium, infundibulum, and adjacent respiratory labyrinth. Drescher and Welsch, 1983). The morphologic similarity of the atrial cell and type 2 (granular) pneumocytes of the mammalian lung (Weibel, 1973; Kuhn, 1976; Sorokin, 1977) has been noted in chickens (Tyler and Pangborn, 1964) and geese (Lambson and Cohn, 1968); these authors postulated that the atrial cell gave rise t o the continuous osmiophilic laminated membrane which coated not only parabronchi and atria but also the surfaces of the respiratory labyrinth. Other workers, however, have demonstrated in essence the same findings (ie., the continuous osmiophilic layer and the atrial cells with laminated myelinoid inclusions in fetal and newborn chicks as well as adult chickens, sparrows, and pigeons) but have come to the conclusion that the material was produced by squamous pneumocytes and phagacytosed by cells lining the parabronchi and atria (Petrik and Riedel, 1968a,b). Additionally, this myelinoid material has been postulated to be the surfactant material in turkeys (Fugiwara et al., 1970) and in chickens (Pat- tle and Hopkinson, 1963; Hylka and Doneen, 1982). Carlson and Beggs (1973) described similar cells lining the abdominal air sacs of chickens. In the budgerigar, parabronchi, atria, and thoracic air sacs are lined by these granular cells; and there is convincing morphologic evidence of active secretion of these myelinoid granules to form the osmiophilic trilaminar membrane that overlies all airways distal to the secondary bronchi, including parabronchi and their atria, air sacs, and resFiratory spaces. It is notable in the budgerigar that these cells are found nowhere else in the respiratory lining; thus, there is sequestration of granular pneumocytes remote from the gasexchange areas in contrast to the mammalian lung where granular pneumocytes are distributed throughout alveolar ducts and alveoli (Weibel, 1973; Kuhn, 1976; Sorokin, 1977). Standard nomenclature (King, 1979) indicates that the appropriate term for the cell lining the atria is a “granular cell”; the pres- 240 J.H. SMITH ET AL. ent authors believe that the term “granular pneumocyte,” “type 2 pneumocyte,” or “atrial cell” would be more appropriate, since it would avoid confusion with “granular cells” of the trachea and bronchi, which are APUD, neuroendocrine cells with dense-core granules. In the budgerigar, there are apparently two populations of granular pneumocytes. A minority of granular pneumocytes are somewhat larger and have larger, more ovoid nuclei, prominently “active” nucleoli, and less dense cytoplasm; these are presumably differentiating postmitotic cells, whereas the more common granular pneumocytes with more dense cytoplasm are mature differentiated cells. Another function of granular pneumocytes in mammalian lungs appears to be re-epithelialization of alveolar ducts and alveoli after damage to the squamous pneumocytes (Kuhn, 1976); a similar function of granular pneumocytes in the budgerigar lung, with proliferation of lucent granular pneumocytes and extension of granular pneumocytes down infundibula and into the respiratory labyrinth has been observed in the reparative phase of squamous pneumocytic injury (Smith, Meier, Neill, and Box, unpublished data). While myelinoid inclusions have been noted in avian and mammalian granular pneumocytes, we have not seen descriptions of the cleft-like electron-lucent inclusions. Their spicular shape suggests a crystalline composition, but the low electron density suggests a lipid, soluble in the dehydrating and clearing agents used in the plastic impregnation process. It is tempting to speculate that these bodies may be cholesterol or some other sterol precursor of “surfactant,” since some of these non-membrane-boundinclusions appear to be fusing with the myelinoid inclusions. The fibromuscular wall, granular pneumocytic lining, and myelinoid coating of the thoracic air sacs suggest that these structures are comparable to, if not embryologically derived from, the parabronchi and their atria. The relatively avascular wall of these sacs would seem to be a poor locus of gas exchange between blood and inspired air. The rich investment of “surfactant” over the surfaces of these air sacs suggests that the surfactant may function more to decrease friction and facilitate air flow through the small airways rather than to facilitate gas exchange. Alternatively, this surfactant layer of the air sacs may act as a reserve for the respiratory labyrinth, which produces no myelin in the budgerigar. Krause described infundibula at the light microscopic level in 1922, but they had been largely omitted from descriptions of avian lung anatomy until the pioneering work of Duncker (1971, 1974). The infundibula provide an intermediate passage between the large airways and the respiratory labyrinth, and their ostia probably control air entry to and exit from the respiratory labyrinth (in concert with dilatatioddistention of parabronchi and their atria). Their structure is distinct from either the atria or the air capillaries in that the principal component of their walls is a thickened basal lamina, yet infundibula share an epithelium composed of squamous pneumocytes with the remainder of the respiratory labyrinth. In the budgerigar, squamous pneumocytic perikarya are concentrated in the infundibula. These squamow pneumocytes appear comparable to squamous pneumocytes of mammals (type 1 or membranous pneumocytes) (Weibel, 1973; Kuhn, 1976; Sorokin, 1977). The squamous pneumocytes of the infundibula and respiratory labyrinth in the budgerigar were always overlain by a myelinoid “surfactant” layer, yet we never saw myelinoid granules or complex elaborations of the surface membrane in these cells such as described by others (Petrik and Riedel, 1968a,b). Thus, we conclude that in the budgerigar this myelinoid layer originates exclusively from the granular pneumocytes of more proximal air ways. The term air capillaries seems to us a misnomer for the passages that emanate from the infundibula. “Air capillary” conjures the image of a uniformly tubular structure that is quite unlike the respiratory space of the budgerigar. These respiratory passages are markedly tortuous and variable in caliber; and they branch and anastomose with fellow passages from the same infundibulum, other infundibula, other atria, and even other parabronchi. Thus the term respiratory laby rinth seems to us a more apt, descriptive term that may have salutary functional implications. It seems difficult to reconcile this architecture with its embryologic derivation from a budding duct or the more familiar mammalian “blind-ended” alveolar structure. The negative pressure in the pleural space in mammals keeps the mammalian lungs distended into globular alveoli. Without this ULTRASTRUCTURE OF BUDGERIGAR LUNG negative pressure (as in birds, which have no pleural cavity), mammalian alveoli collapse (i.e., atelectasis) and lose their globular conformation, and their walls become contorted in ribbon-like folds; thus, we can roughly reconcile the racemose rather than globular conformation of avian respiratory units whose ostia are the infundibula. Furthermore, the freedom of intercommunication between these units can be conceptualized as a n exaggeration of alveolar pores noted in mammalian lung (pores of Cohn). While this simplistic conceptualization may be temporarily or emotionally satisfying to the microscopist, further work on the development of the avian respiratory system at a n ultrastructural resolution is indicated. Such efforts may have real value in producing understanding of the basic processes of repair in injured avian lung. The fine structure of the respiratory labyrinth has been studied extensively in the chicken (Tyler et al., 1961; Tyler and Pangborn, 1964; Petrik and Riedel, 1968a,b; King and Malony, 1971; Abdullah et al., 1982; Kazachka, 19841, in geese (Lambson and Cohn, 1968; Powell and Mazzone, 1983), in the pigeon (Bargmann and Knoop, 1961; Policard et al., 1962; Petrik and Riedel, 1968a), in the sparrow (Petrik and Riedel, 1968a; Dubach, 19811, in penguins (Drescher and Welsch, 19831, and in many species of birds including the budgerigar (Dubach, 1981; Maina and King, 1982; Maina et al., 1982). Indeed, elaborate morphometric studies with correlation to respiratory-function factors and type of flight have now been beautifully documented (Dubach, 1981; Abdullah et al., 1982; Maina and King, 1982; Maina et al., 1982; Powell and Mazzone, 1983; Drescher and Welsch, 1983) and compared with similar data from mammals and reptiles (Weibel, 1973; Meban, 1980). Our findings confirm those structural data of others and integrate the fine structure of the respiratory labyrinth with that of the infundibula, parabronchi, and their atria in a manner that permits understanding of pathologic alterations encountered in the budgerigar lung. Squamous pneumocytes of the respiratory labyrinth appear applied to the abluminal surface of the basal lamina of the blood capillaries except in the infundibula. Moreover, where “air capillaries” abut upon adventitia of atria and larger blood vessels, squamous pneumocytic basal laminae are often not observed. While the ultrastructural ontogeny of the respiratory labyrinth 241 has not been examined, regeneration of its lining after pathologic denudation shows granular pneumocytes extending pseudopods along “bare” basal lamina of intact blood capillaries. These granular pneumocytes then differentiate into squamous pneumocytes without ostensibly adding to the basal laminae that they overlie (Smith, Meier, Neill, and Box, unpublished data). This suggests that avian squamous pneumocytes possess their own basal lamina only in the infundibula. The apical surfaces of the squamous pneumocytes are overlain by a n osmiophilic trilaminar (“surfactant”) membrane. They are connected with adjacent squamous pneumocytes by typical epithelial junctional complexes, and in places their cytoplasmic matrix is so attenuated that the squamous pneumocyte consists solely of a n apical and basal plasma membrane. Attenuation of blood capillary endothelial cell cytoplasm is most marked, is remote from the perinuclear cytoplasm, and is usually associated with the complementary attenuation of the squamous pneumocytic cytoplasm and basal lamina, creating a very narrow air-blood barrier. Our estimation of the thickness of this barrier is similar to that noted by previous authors (Dubach, 1981; Maina and King, 1982; Maina et al., 1982). ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant DHHS 5ROl A1 15945 from the National Institute of Allergy and Infectious Diseases. We greatly appreciate the secretaria1 assistance of Edna Sue Davis, M. Imogene Wiley, Julie Smylie, and Lori Mohr and the advice and encouragement of Drs. E.S. Reynolds, J.S. Davis, and H.W. Sampson. This paper was presented a t the lOlst Stated Meeting of the American Ornithologists Union, New York, NY, September, 1983. LITERATURE CITED Abdullah, M.A., J.N. Maina, A.S. King, D.Z. King, and J. 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