The Respiratory Epithelium of the Lung in the Green Turtle (Chelonia Mydas L.) SALLY E

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J. Anat. (1984), 139, 2, pp. 353-370 353 With 19 figures Printed in Great Britain The respiratory epithelium of the lung in the green turtle (Chelonia mydas L.) SALLY E. SOLOMON AND MICHAEL PURTON Department of Veterinary Anatomy, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow, G61 1QH, Scotland (Accepted 26 January 1984)

INTRODUCTION Reptilian lungs range in complexity from simple sac-like structures, as in the tegu lizard Tupinambis nigropunctatus (Klemm, Gatz, Westfall & Fedde, 1979), to multi- cameral lungs, as in varanid lizards (Perry & Duncker, 1978) and in the freshwater turtle Pseudemys scripta (Perry, 1978), so that it is not possible to define a simple model of the reptilian lung. The green turtle (Chelonia mydas) spends much ofits life at sea, diving to consider- able depths and often staying there for hours at a time. It can tolerate complete anoxia, if necessary for days at a time; provides, by means of vascular shunts, for the redistribution of blood away from the lungs during submersion; and even under normal conditions exhibits the periodic breathing pattern characteristic of many reptiles (Glass & Wood, 1983). It is to be expected, therefore, that the physiological stresses imposed upon the respiratory apparatus in the green turtle by its aquatic existence would be reflected in the structure of its lungs. This study attempts to assess lung structure in the green turtle in relation to other reptiles, and to identify and discuss those morphological adaptations related to its mode of existence.

MATERIALS AND METHODS Lung tissue was obtained from six 3-4 years old farm reared green turtles (Chelonia mydas L.). The lungs were stripped manually from the carapace and a midline incision was made along the entire length of the primary bronchus to expose the numerous ramifications. Pieces of tissue were removed at all levels and processed as follows. Light microscopy Blocks of tissue 1 cm in size were fixed in buffered neutral formalin. Wax-embedded sections were stained with haematoxylin and eosin and Masson's trichrome for routine examination, and with periodic acid-Schiff to demonstrate the presence of neutral polysaccharides and glycoproteins. Scanning microscopy Tissue blocks were fixed in modified Karnovsky (2 % paraformaldehyde/2-5 % glutaraldehyde in 0-1 M sodium cacodylate buffer), washed with buffer, pH 2, and dehydrated through a graded series of acetones. The material was critically point 354 SALLY E. SOLOMON AND M. PURTON

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Fig. 1. Diagrammatic representation of the chelonian lung. T, trachea; PB, primary bronchus. Inset; secondary bronchi (SB) and bronchioles (arrow). dried in carbon dioxide and sputter coated with gold/palladium before viewing in a Philips 501 B scanning electron microscope. Transmission microscopy Blocks oftissue, 1 mm3, were fixed as for scanning microscopy, washed with buffer, dehydrated through a graded series of acetones and embedded in EMIX (epoxy- resin). Ultrathin sections were cut and stained with uranyl acetate and lead citrate, and examined with a Hitachi HS8 electron microscope at 50 kV.

RESULTS Gross anatomy In the green turtle the lungs were closely applied to the ventral surface of the ribs and carapace in the dorsal region of the body cavity. On external examination, each lung was a spongy mass covered by a thick pulmonary pleura and showed no sign of lobation. From the tracheal bifurcation, a single primary bronchus entered each lung medially at the cranial pole and passed caudally through the substance of the lung decreasing in diameter as it did so until terminating in fine branches at the caudal border of the lung (Fig. 1). The chelonian lung 355

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Numerous branches, which varied in diameter, arose from the primary bronchus as it passed through the lung tissue. These secondary bronchi themselves gave rise to a system of repeatedly branching tubes (bronchi and bronchioles) of ever decreasing diameter which passed from the centre of the lung towards the periphery. The finest terminations of the branching bronchial tree were the respiratory bronchi, respiratory bronchioles and alveolar ducts leading directly to the alveoli. .1 e chelontiant lungg 357 The nomenclature of these terminal branches is based on the histological and ultrastructural features presented below. Bronchial tree: conducting airways Primary bronchus At light microscope level, the mucous membrane was composed of a pseudostratified ciliated columnar epithelium containing numerous goblet cells resting on a thick basement membrane. Characteristically, the epithelium was thrown into regular folds (Figs. 2, 3). The lamina propria contained many smooth muscle cells and elastic fibres. There were no mucous glands in the underlying thin submucosa although intraepithelial mucous glands were observed in the mucous membrane at this level (Fig. 4). The muscularis mucosae was thick and, together with the presence ofelastic fibres, gave the mucous membrane a folded appearance. The patency of the primary bronchus was maintained by a series of cartilage rings. The mucous secreting cells when filled with secretion granules tended to squeeze the interposed ciliated cells. In the mucous cell, the nucleus was basally located and the secretion granules, which were of variable electron density, appeared to displace the remaining intracellular organelles peripherally. The apical surface was provided with short microvilli (Fig. 5). The paler ciliated cells contained variable numbers of randomly distributed mitochondria and their nuclei were centrally located. Between the two principal cell types and the basement membrane lay numerous undifferentiated cells which probably represented a reserve population. These are the subject of a separate communication. Secondary bronchi The term 'secondary' in this instance is used simply to indicate those branches of widely varying calibre arising directly from the primary bronchus at all levels. They were histologically similar to the primary bronchus with reference to the types ofcell in the respiratory epithelium but, although the latter was thrown into regular folds (Fig. 6), it was beginning to flatten out in certain areas. As observed with the scanning electron microscope, the epithelial sheet covering the primary and secondary bronchi was often interrupted by areas composed of cells exhibiting short, clumped, cilia at their luminal surface (Fig. 7). These areas were considered to be regenerating epithelium. Bronchial tree: conducting/respiratory airways Respiratory bronchi These were distinguished on the basis of (a) the presence in the wall of incomplete cartilage rings or plaques, and (b) the appearance at the luminal surface of a gaseous exchange area characterised by capillary loops lying beneath a simple squamous epithelium (Fig. 8). These exchange areas were found between the normal columnar epithelial cells which constituted the respiratory epithelium at this level. Although both ciliated and mucous cells persisted, the latter were less plentiful and more widely scattered. Respiratory bronchioles These were histologically similar to respiratory bronchi but they lacked cartilaginous support (Fig. 9). Both bronchi and bronchioles were invested with a thick band of smooth muscle external to the submucosa. 35{ SALLY E. SOLOMON AND M. PURTON

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filled with secretory products (M). The pale ciliated cells (C) contain few organelles apart from mitochondria and the centrally placed nucleus. R, reserve cell population. x 4000. The chelonian lung 359

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Alveolar ducts These arose from both the respiratory bronchi and bronchioles and opened directly into clusters of alveoli (Fig. 10). The most obvious feature of the duct wall was the broad band of smooth muscle and associated elastic fibres. At the point of origin of the alveolar duct, and where the duct opened into the alveoli, the smooth muscle appeared to form a sphincter. At this level, the epithelium was primarily of 360 SALLY E. SOLOMON AND M. PURTON

Fig. 7. Surface view of secondary bronchus. The epithelial sheet supports a population of cells with short clumped cilia (arrows). x 5000. the exchange type although isolated clumps of ciliated and mucous cells could still be distinguished (Figs. 11, 12). Bronchial tree: respiratory airways Alveoli Since the histological material examined in this study was derived from collapsed lung, the shape of the alveoli and their precise relationship to the alveolar ducts had to be treated with some caution. It did appear, however, that the alveoli formed grape-like clusters around each alveolar duct. The alveoli were thick walled and septate (Fig. 10). At each luminal surface each The chelonian lung 361

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Fig. 10. Alveolar duct (arrow) opening into alveoli (*). x 35. septum had numerous capillary loops lying beneath a simple squamous epithelium (Figs. 13, 14). Numerous elastic fibres and prominent smooth muscle bundles were scattered amongst the dense connective tissue core of each septum. Small isolated clumps ofciliated and mucous cells were distributed very sparsely along the alveolar septa (Fig. 15). Septal perforations or apertures were not observed. The epithelial layer was composed of type I and type II pneumocytes (Fig. 16) which could be identified at the ultrastructural level. The type I pneumocyte occurred either in the angle between two capillaries or overlay a capillary (Fig. 17). The nucleus was in the thickened central region and often bulged into the alveolar lumen, while the peripheral cytoplasm was attenuated into sheets covering the blood capillaries. At its extremity, each sheet was united to an adjacent type I or type II pneumocyte by a tight junction (zonula occludens). Intracellular organelles were sparse and the luminal surface, although roughly folded, did not bear microvilli. The chelonian lung 363

Fig. 11. Clumps of ciliated cells (C) still persist at the level of the alveolar duct. E, exchange epithelium. x 250. Fig. 12. Ciliated cells within the exchange epithelium. x 2500. 364 SALLY E. SOLOMON AND M. PURTON

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Fig. 15. A clump of ciliated cells (C) associated with the alveolar septa. x 2500. Fig. 16. Surface of an alveolus showing type I and type II cells; arrows indicate intercellular junctions. x 10000. 366 SALLY E. SOLOMON AND M. PURTON

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Fig. 19. Detail of the blood-air barrier. Arrow indicates micropinocytotic vesicles. AL, alveolar lumen; CL, capillary lumen. Note the presence of what is probably surfactant (S). x 2150.

The type II pneumocyte was roughly cuboidal in form (Fig. 18). The centrally placed nucleus had an irregular outline. Mitochondria were numerous, and the cytoplasm also contained many scattered ribosomes, profiles of agranular endo- plasmic reticulum and, usually, a Golgi complex. The lamellar osmiophilic inclusion bodies were probably the most conspicuous group of organelles, however. These bodies tended to lie in the apical regions of the cytoplasm, and the contents appeared to be either amorphous masses or irregular tangles of membranous material. In some instances, deposits of similar membranous material could be observed within the alveolus (Fig. 19). The apical surface of each type II pneumocyte bore short, blunt, microvilli. The pulmonary capillaries lay subjacent to the epithelial layer and usually caused it to bulge out into the lumina of the alveoli. The capillary endothelium lacked pores or fenestrations but became extremely attenuated in regions where it formed the inner layer of the blood-air barrier (Fig. 19). Micropinocytotic vesicles were found throughout the cytoplasm. In addition, densely stained bodies having round or oval profiles were commonly observed in the capillary endothelial cell (Fig. 14). The basal lamina between the endothelial and epithelial layers of the blood-air barrier was either single or double. 368 SALLY E. SOLOMON AND M. PURTON

DISCUSSION The bronchial tree The pattern of branching of the reptilian bronchial tree varies from a simple system with alveoli opening directly off a variously named central tubular lumen (Varde, 1951), bronchus (Okada et al. 1962) or axial air channel (Meban, 1978) as in the snakes, geckos and some lizards through to the system in the crocodilians and more advanced lizards, where there is secondary and tertiary branching of the bronchi with smaller and smaller branches that eventually end in alveoli (Porter, 1972). The present study shows that the green turtle falls into this latter group, although the bronchial tree pattern, with the primary bronchus entering at the cranial pole of the lung and giving rise to secondary bronchi as it passes through the lung substance before terminating at the caudal pole, is suggestive of one of the characteristic features of the avian lung.

The respiratory epithelium Although the respiratory epithelium of the turtle lung is typically vertebrate, being pseudostratified columnar with ciliated cells serving to drive the mucous blanket towards the pharynx, there are still obvious differences in the nature of the epithelium between different orders of reptiles. The large population of goblet cells, together with the extensive distribution of ciliated epithelium in the turtle, is in marked contrast to the appearance of the bronchial respiratory epithelium in the snake (Tucker, 1974). The areas of short clumped cilia observed in this study are similar to the nodular areas described by Tucker (1974); these cells, with cilia of variable length, are similar to the progenitor cells described by Kessel & Kardon (1979) which they consider to be suggestive of morphogenesis. These areas are therefore most likely areas of regenerating epithelium.

The blood-air barrier In the chelonian lung, gaseous exchange areas appear at all levels from the respiratory bronchi to the alveoli, the latter forming by far the major part of the exchange area. The capillaries which cover the surface of each alveolar septum are arranged in a very close meshed network (Figs. 13, 14) which seems to approach the form which would permit an effective 'sheet-bloodflow' of the type suggested by Sobin, Tremer & Fung (1970). Each capillary also projects markedly into the alveolar lumen, an arrangement which would allow a greater area of capillary wall to be exposed to the air and hence facilitate gaseous exchange. It is interesting to note that this double alveolar capillary supply is also found in many marine mammals, e.g. cetaceans and certain pinnipeds (Simpson & Gardner, 1972). The structure of the blood-gas barrier is obviously essential for efficient gaseous exchange. It is characterised by a simple squamous epithelium, lining the airway lumen, separated by a single or double basal lamina, from the endothelial lining of the underlying blood capillary. The epithelial lining is made up of type I and type II pneumocytes morphologically similar to those of birds and mammals. The lamellar inclusion bodies typical of the type II cells described in the present study have also been observed in the lungs of snakes, geckos and lizards (Okada et al. 1962; Meban, 1978) and are the cellular form of lung surfactant in the reptilian lung (Pattle, 1976). The chelonian lung 369 Functional considerations of lung morphology The proximal conducting and terminal exchange airways show several modifi- cations ideally suited for a rapid and vigorous exchange of gases. The patency of the conducting airways is maintained by supportive cartilage rings which surround the passages completely and extend in the form of plates into the smallest airways as far distally as the junction with the respiratory bronchioles. Additional airway support and resiliency is provided by the presence ofthick smooth muscle bands and many scattered elastic fibres in the airway wall. The walls of the terminal airways lack the cartilaginous supporting elements but have an extremely thick smooth muscle wall and elastic connective tissue. These thick muscular walls are very obvious in the alveolar ducts, from which level the smooth muscle and elastic tissue extends into the alveolar septal walls. Within the septal walls, some of the muscle fibre bundles run in the free edge of the septa and their contraction would decrease the diameter of the alveolar entrance. In this respect they resemble the circular bands of smooth muscle surrounding the mouths of mammalian alveoli (Bloom & Fawcett, 1975). By contrast in the turtle, the muscle bundles in the central region of the septa are arranged longitudinally; their contraction is probably res- ponsible for shortening the septa and thereby reducing the capacity of the alveoli. A similar distribution of smooth muscle has been noted in the lung of the green lizard (Lacerta viridis; Meban, 1978). The features exhibited by the proximal conducting airways would provide for rapid inflow and outflow of air during the respiratory cycle, whilst the characteristic features of the terminal airways and alveolar septa would facilitate not only their primary blood-gas exchange function but a rapid and forceful expulsion of air at the end of a prolonged dive (S. E. Solomon, unpublished observation). The periodic breathing found in most reptiles is especially characteristic of diving reptiles and involves a redistribution of blood away from the lungs during submersion. Such a decrease in pulmonary perfusion during breath holds is partly related to a progressive increase of pulmonary resistance (Glass & Wood, 1983). Although this obviously involves cardiovascular adjustments, contraction of the lung parenchyma, for which the structure of the terminal airways is ideally suited, may also play a role. The thick muscular and elastic walls may also contribute to the maintenance or alteration of gas pressure and volume in the lung as a result of diving, especially important as the lung serves not only as a significant oxygen store (Glass & Wood, 1983) but also to maintain negative buoyancy. The striking increase in the amount of supporting material, mainly cartilage, collagen, elastic tissue and smooth muscle, in the peripheral portion ofthe chelonian lung as compared to that of terrestrial reptiles, added to the fact that many of these features are also found in the lungs ofcetaceans and pinnipeds (Simpson & Gardner, 1972), suggests that they may be common adaptations to the physical parameters imposed on the vertebrate lung by a deep sea existence. The sphincteric narrowings of the terminal airways, producing compartmentalisation effects as observed in the porpoise (Simpson & Gardner, 1972), were not observed in the green turtle.

SUMMARY The chelonian lung exhibits reptilian, mammalian and avian features. The respiratory epithelium is typically vertebrate, i.e. pseudostratified columnar with cilia; gaseous exchange areas appear at all levels from the respiratory bronchi down I3 ANA 139 370 SALLY E. SOLOMON AND M. PURTON to the alveoli. The latter are invested with a capillary network and both type I and type II cells are present. The possible functional significance of the distribution of collagen, elastic tissue, cartilage and smooth muscle is discussed. We would like to thank the Cayman Turtle Farm (1983) Ltd for providing the research material and S. Solomon gratefully acknowledges the Mittag Trust for its financial support. We would also like to thank our colleagues Mrs E. Harrop and Mr Gordon McMillan for their technical assistance, Mr C. Paterson for preparing Figure 1 and Mrs J. McKendrick for typing the manuscript.

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