J. exp. Biol. 134, 99-117 (1988) 99Printed in Great Britain © The Company of Biologists Limited 1988

FUNCTIONAL MORPHOLOGY OF THE LUNGS OF THENILE CROCODILE, CROCODYLUS NILOTICUS:

NON-RESPIRATORY PARAMETERS

By STEVEN F. PERRY

Fachbereich 7 (Biologie), Universitdt Oldenburg, D-2900 Oldenburg,Federal Republic of Germany

Accepted 30 July 1987

SUMMARY

The complex, multicameral lungs of the Nile crocodile are characterized by rowsof tubular chambers, which in cranial and ventral lung regions are broad and sac-like. The inner surface of the chambers is enhanced by cubicles (ediculae), thecapillary-bearing walls of which are often perforated. Extrabronchial communicationamong chambers is infrequent. The ediculae end in a network of myoelastictrabeculae, which face the central lumen of the chambers. The trabecular epitheliumis similar to that of mammalian bronchi and contains isolated endocrine-like cellsbasally, whereas the edicular epithelium is similar to that of other reptiles and ofmammals. The distribution of non-vascular smooth muscle, 64% in trabeculae and36% in interedicular walls, is consistent with the hypothesis that these twoantagonistically oriented muscle groups interact to effect lung patency. The volume-specific lung compliance is similar to that of much simpler, unicameral gekko lungs,implying that lung compliance is a function of parenchymal structure and not ofprimary structural type.

INTRODUCTION

Numerous studies have dealt with the morphology of the epithelium in therespiratory partitions of reptilian lungs (Okada et al. 1964; Meban, 1977, 1978a,b;Welsch, 1979; Klemm, Gatz, Westfall & Fedde, 1979; Luchtel & Kardong, 1981;Perry, 1972, 1978, 1983; Pohunkovd & Hughes, 1985). Little attention, however, hasbeen given to those structures which bear upon the mechanical properties of suchlungs and allow the respiratory surfaces to remain exposed to ventilatory airmovement during and between breaths (Ogawa, 1920; Klemm et al. 1979; Perry,1983). This applies particularly to crocodilian lungs, for which only general, grossanatomical or developmental information is available (Milani, 1897; Broman, 1939;Duncker, 19786).

The present paper describes the gross and fine anatomy in one of the most complexstructural types of reptilian lung with respect to its dynamic function, and presents

Key words: lungs, crocodile, reptile, ultrastructure, non-respiratory epithelium, smooth muscle,compliance.

100 S. F. PERRY

hypotheses for the coupling of structure and function with respect to the support ofrespiratory surfaces during breathing.

MATERIALS AND METHODS

Gmss anatomy

The lungs of two zoo-born, juvenile Nile crocodiles, Crocodylus niloticus Laurenti(mass = 763 g and 325 g), which had recently died were fixed in situ by inflation with10% formalin, removed intact and air dried from ethanol or xylol under constantintratracheal pressure with compressed air. The dried lungs were opened laterallyand dissected by removal of lung tissue with forceps to reveal the lung chambers andtheir connection to the intrapulmonary bronchus. The results were recorded asstereo-pair photographs and as drawings. Using these data, a wire model of a lungwas constructed, which served as a basis for Fig. 1.

Microscopic anatomy and lung mechanics

Four Nile crocodiles of body mass W = 3-38kg, 3-59 kg, 3-69 kg and 5-68 kg wereobtained through legal, commercial channels and maintained in aqua-terraria on adiet consisting mainly of fish. They were killed by an intraperitoneally administeredoverdose of sodium pentobarbital (60-120mgkg"1). Immediately after death, 50 mlof blood was removed for other studies and substituted with an anticoagulant-vaso-dilating solution of 2 ml of heparin (5000 units ml"1), 25 ml of nitroprusside sodium(2-4 % in 0-9 % NaCl), 39-5 ml of dextran (0-96g in 0-9 % NaCl) and 6 ml of distilledwater. Before the body was opened, two volume—pressure diagrams were obtainedusing the method of Perry & Duncker (1978, 1980) to determine lung volumes.Residual lung volume (VLr) was determined as the amount of air which could bewithdrawn from the lungs of the anaesthetized, supine animal after equilibration toatmospheric pressure without exceeding an intratracheal pressure of — lOcmh^O(1 CIT1H2O = 98' 1 Pa). Maximal lung volume ( VL,,,) was the amount of air required toraise the intratracheal pressure from —10 to +20cmH2O. These arbitrary limits wereset to avoid damaging the lungs for subsequent electron microscopic examination.

The body cavity was opened ventrally and two volume-pressure diagrams wereobtained for the exposed lungs of three specimens. The body cavity was then suturedand the lungs were fixed in situ by intratracheal instillation of 0-75 VL,,, of cold (4°C)glutaraldehyde (3 % glutaraldehyde in 0-05 mol 1~' phosphate buffer, pH 6-8). After3 h of fixation, the lungs were removed intact and their length and displacementvolume determined according to the method of W'eibel (1970/71). Each lung wasthen cut transversely into 10—12 slices. Samples for transmission electron mi-croscopy were taken from centrally (proximally) and peripherally (distally) locatedsites in either the right or the left lung. These samples were maintained overnight incold (4°C) phosphate buffer (pH70, 350mosmoll~') for postfixation in 1%0 1 moll"1 phosphate-buffered OsO4 at 0°C, alcohol dehydration and Epon embed-ding the following day. The remaining tissue was postfixed in Bouin's fluid,

Cmcodile lungs 101

dehydrated through graded alcohols and embedded in paraffin wax. These prep-arations were planed using a sliding microtome and the blocks were exposed toalcoholic methylene blue, revealing the cut surfaces of the embedded tissue (Perry,1981a).

Morphometric evaluation of the paraffin preparations was carried out with the aidof a dissecting microscope and using stereological methods for multicameral lungs(Perry, 19816). Volumetric analysis of tissue components employing stereologicalpoint- and intersection-counting techniques (Weibel, 1979) was performed onphotomontages of toluidine-blue-stained, semi-thin sections at a final magnificationof 625X.

The volumes of all tissue elements were calculated separately for proximal anddistal sampling sites of each lung slice. The sums of these values, calculated for bothlungs of each test animal, are presented in Table 1.

Electron micrographs were obtained from uranyl acetate, lead citrate contrastedthin sections using a Zeiss EM 9 or Zeiss EM 109 electron microscope.

Symbols and definitions

The hierarchy and definition of symbols used in the designation of anatomicalstructures are as follows. The symbols are used as subscripts.

L, lung; L consists ofNP, tissue-free central lumen of lung or of lung chambers; the central lumen is

arbitrarily separated from the parenchyma by a line connecting the larger trabeculae.P, parenchyma: that portion of the lung in which air spaces are surrounded by

partitions and their associated trabeculae; P consists ofNt, air spaces.t, tissue component; t consists of:

B, large blood vessels.A, septa: wall-like, flattened structures connecting trabeculae (NA) with the

general inner surface of the lung or of intercameral septa. The central leaflet consistsof a collagenous matrix in which non-vascular smooth muscle, blood vessels andnerves are found. A network of capillaries in which gas exchange occurs covers thesurface; there are separate networks on opposing surfaces. A consists of c, m, e and u(see below).

NA, trabeculae: structures supporting free edges of septa. They are composedof a central core of smooth muscle and elastic tissue. Large trabeculae possess a non-respiratory, ciliated epithelium facing the central lumen (NP) and a respiratoryepithelium on the abluminal surface. NA and A consist of

c, content of capillaries on partitions or trabeculae;m, non-vascular smooth muscle in partitions or trabeculae;e, non-respiratory epithelium;u, other components (not c, m or e) of partitions or trabeculae.

102 S. F. PERRY

Other symbols are:C, compliance (mlcml-^O"1): CLB total compliance,

CB body wall compliance,CL, lung compliance;

VL, displacement volume of excised, fixed lungs (ml);VLr, resting lung volume (see text);VLm, maximal lung volume (see text);W, body mass (kg).

RESULTS

Descriptive morphology

Gross anatomy

The lungs, together with the heart (including pericardium and the great vessels)and the oesophagus occupy the cranial half of the body cavity. Each lung lies in aseparate, closed pleural space, bordered laterally by the dorsal ribs and intercostalmusculature, dorsally by the vertebral column, medially by the mediastinum, andventrally by the sternal ribs. The lungs, particularly in the dorsal half, tend to fusewith the parietal pleural surface.

Right and left lungs are mirror images of each other reflected in the mediosagittalbody plane. Each represents approximately a truncated cone, with its base lyingagainst the liver and its cranial apex extending ventrally into the base of the neck,between the shoulder girdle and the oesophagus.

As in other crocodilians (Duncker, 19786) the lungs are multicameral, consistingof a variable number of chambers (camera), each of which connects independentlywith an unbranched, intrapulmonary bronchus (Fig. 1). The cranial half of theintrapulmonary bronchus (solid outline in Fig. 1) is cartilage reinforced. It displaysthree rows of orifices which supply four dorsal chambers, four lateral chambers andthree ventral chambers (Fig. 1). A medial row is lacking. The rows of orifices do notrun parallel to the long axis of the intrapulmonary bronchus, but instead form a left-hand spiral in the left lung (right-hand in the right lung). Hence the first lateralchamber opens into the lateral aspect of the intrapulmonary bronchus, but the orificeof the fourth lateral chamber is dorsal (Fig. 1).

The first dorsal chamber is rudimentary. The remaining chambers in the dorsalrow are long, tubular structures. Together with a cluster of medial chambers whichoriginate at the end of the cartilage-reinforced portion of the intrapulmonarybronchus, they form a dorsomedial lung lobe (Fig. 1).

The cavernous first lateral chamber extends cranially from its bronchial entranceand, together with its large ventral and lateral branches, forms the lung apex(Fig. 1). Lateral chambers 2-4 make up the deep lateral and superficial dorsalportions of the lung along the middle third of its length.

In the middle third of the lung the three ventral chambers comprise the sac-likeventrolateral and ventromedial lung regions. The first two ventral chambers thendouble back dorsally and form the superficial lateral portion of the lung (Fig. 1).

Crocodile lungs 103

Fig. 1. Semi-schematic representation of the left lung of Crocodylus niloticus in lateralview (A) and medial view (B), showing the distribution of chambers. Chambersonginating in the cartilage-reinforced portion of the intrapulmonary bronchus (outlinedby solid lines) occur in spiralling rows and are indicated: dorsal row, D1-D4; lateral row,L1-L4; ventral row, V1-V3. In the cartilage-free portion of the intrapulmonarybronchus only the major, tubular chambers are indicated by letters, indicating theirdorsal (D' and D"), ventral (V and V") or lateral (L') origin. At the transition betweenthe two portions of the intrapulmonary bronchus a cluster of four medial chambers(M1-M4) originates. Other, smaller chambers are numerous and irregular in occurrence.The intrapulmonary bronchus ends in a terminal chamber (T). The drawing is basedupon the dissection of dried specimens from two C. niloticus and upon comparison withsimilar preparations from C. porosus and Caiman crocoddus. The right lung does notdiffer substantially from a mirror image of the left.

The caudal half of the intrapulmonary bronchus lacks cartilage and its chambersare not continuous with the rows described above. In addition to the cluster of medialchambers mentioned above, five long, tubular chambers and the sac-like terminalchamber supply the superficial portions, while a large number of irregularlydistributed outgrowths (bronchial niches) supply the deep lung parts (Fig. 1).

The inner surface of the chambers is elaborated with a system of cubicles(ediculae; Duncker, 19786). The free edges of the ediculae face the central lumen ofthe chambers and are supported by a system of stout trabeculae, as described foramphibian and reptilian lungs (Goniakowska-Witaliriska, 1986; Graper, 1931). Theinteredicular septa, which bear the respiratory capillaries, are often perforated,whereas the intercameral septa, which separate adjacent chambers, rarely show suchperforations.

104 S. F . PERRY

Microscopic anatomy

The interedicular partitions in the Nile crocodile consist of a central leaflet ofcollagenous connective tissue and smooth muscle. They bear on each surface aseparate network of capillaries which rarely communicate across the central leaflet(Fig- 2).

The epithelium of the respiratory surfaces (Fig. 2) contains type 1 and type 2pneumocytes (Campiche, 1960), similar to those in turtles (Perry, 1972; Meban,1977).

The trabecular epithelium is composed primarily of ciliated cells and seroussecretory cells (Figs 2, 3). On the ciliated cells, short microvilli outnumber thekinocilia by 7 to 1 and at the base of the microvilli horseshoe-shaped invaginations ofthe plasmalemma are observed (Fig. 3). The cilia display prominent, striatedrootlets; the longer, terminal rootlet extending at approximately 45° from the tip ofthe basal body and the shorter rootlet inserting perpendicularly on the middle of thebasal body (Figs 3,6).

The ciliated cells and secretory cells are joined at their apical surface by ajunctional complex (Fig. 3) and exhibit laterally interdigitating processes. Themicrovilli and mitochondria of these two cell types are similar but more numerous inthe ciliated cells. The serous secretory cells, however, are packed with large granulesof moderate electron density (Fig. 3). Scattered glycogen deposits are observed inboth cell types.

Endocrine-like cells (elc) are often found singly or in small groups at the base ofthe trabecular epithelium, where they form desmosomes with the ciliated cells(Fig. 6). Typical of theelcs are dense-core vesicles (Fig. 7), large, oval mitochondriaand abundant smooth-surfaced endoplasmic reticulum. The often dilated cisternaeof the latter give these cells a lacey, bright appearance (Figs 6, 7). elcs have not beenobserved to contact the free trabecular surface in the Nile crocodile.

Beneath the basement lamina of the trabecular epithelium, capillaries and nervesare found in the connective tissue, which envelops the 'myoelastic' core of thetrabecula (Fig. 2). The smooth muscle of the core (Fig. 4) is characterized byprominent fusiform densities. Amorphous elastic tissue deposits between the smoothmuscle cells are embedded in a fine, fibrous or electron-lucent, multivesicular matrix(Fig. 5).

The connective tissue sheath of the trabecula is continuous with the central leafletof the interedicular partition. It supports a double capillary net (Fig. 2) and containssmooth muscle (Fig. 2), which is ultrastructurally indistinguishable from that of thetrabecula. The interedicular muscle bundles are oriented perpendicular to thetrabecula (Fig. 8). At the base of the trabecula a subtrabecular vein and anaccompanying peri vascular lymph space are often present (not illustrated).

Quantitative moiphology

The results of the volumetric analysis of the lungs are summarized in Table 1. Theaverage pulmonary displacement volume (VL), which is the reference volume for

Crocodile lungs 105

sc

Fig. 2. Semi-thin cross-section of a trabecula and an interedicular partition in Cmcodylusniloticus. In the trabecula the smooth muscular core (tsm), subepithelial connectivetissue (ct), subepithelial capillary (sec) and non-respiratory epithelium with secretory(sc) and ciliated (cc) cells can be recognized. Respiratory capillaries (rc) are present notonly on the partitions but also on the trabecula. Squamous, type 1 (El) and more cubical,type 2 (E2) epithelial cells as well as smooth muscle (ism) are indicated in theinteredicular partition. Stained with toluidine blue. Scale bar, 10j<m.

106 S. F. PERRY

morphometric calculations, is 109 ml kg . In the three smaller specimens(W = 3-6kg) VL per kg body mass is greater than in the larger, 5-7-kg specimen.Also, the smaller animals tend to have a greater parenchymal volume in relation tobody mass (48 ml kg"1) than the larger one (39 ml kg"1). Other parameters aresimilar for small and large specimens.

Crocodile lungs 107

The volume of lung tissue is only approximately 4 ml kg"1. The remainder(105 ml kg"1) is air, of which 63 ml kg"1 is the central lumina of the chambers and theintrapulmonary bronchus, with the remaining 42 ml kg"1 in the parenchymal airspaces (ediculae).

The interedicular septa make up some 68% of the total tissue volume. Theremaining lung tissue consists of trabeculae and large blood vessels (13 % and 19%,respectively). The major components of the septa are connective tissue (57 %) andblood (36%). This represents 93 % of the connective tissue and 96% of the blood(exclusive of major vessels) in the lung.

Non-vascular smooth muscle makes up 7 % of the interedicular septa but 55 % ofthe trabeculae. However, since the absolute volume of the septa is five times that ofthe trabeculae, 36% of the total non-vascular pulmonary smooth muscle is in theinteredicular septa (for specimen 3; 47 %). The largest specimen also has the lowestpercentage of muscle in the septa: 24%.

The regional distribution of components of the septa is shown in Fig. 9. Thisindicates that only the cranial third of the lung (which consists primarily of the sac-like, first lateral chamber) appears to differ from the rest. Here blood makes upbetween 20 and 33 % of the total volume compared with 38—50 % in the caudal two-thirds.

In general, sampling sites distal to the intrapulmonary bronchus tend to yieldrelatively more blood and often less smooth muscle than proximal sites. Theproportion of connective tissue is also greater distally than proximally. Thesedifferences tend to be most pronounced cranially (see Fig. 9), and are most clearlyseen when the (perhaps variable) blood content is subtracted.

Lung volumes and mechanics

The residual lung volume (VLr) is 18mlkg"1, which represents only 13% of themaximal lung volume (VLm) and 1-8% of the body volume (Fig. 9; Table 1).

Fig. 3. Electron micrograph of the apical portion of ciliated cells (cc\ and ccT) and aserous secretory cell (sc) of the trabecular epithelium. Both cell types possess similarmicrovilli (mv), glycogen clusters (g) and mitochondria (m), and are connected by aterminal, junctional complex (jc). Possible vesicle formation in a ciliated cell is indicatedby arrowheads; intercellular vesicles of various sizes are indicated by v. n indicates thenucleus. Kinocilia (kc) with prominent rootlets (cr) are characteristic of ciliated cells;secretory granules (sg) of moderate electron density, of the secretory cells. //, laterallabyrinth. Scale bar, OSfim.

Fig. 4. Electron micrograph of a transversely sectioned trabecular smooth muscle cell,showing the nucleus («) and prominent fusiform densities (fd) on the cell membrane andin the cytoplasm, g indicates glycogen deposits in a neighbouring cell. In the intercellularspace a fine, fibrous matrix (/*), collagen (co) and amorphous elastic tissue deposits (el) aswell as an electron-lucent field (•) are visible. Scale bar, 0 5 um.

Fig. 5. Electron micrograph of amorphous elastic tissue deposits in the trabecular core.Associated with them are fibrous deposits (/) and electron-lucent areas such as thoseindicated in Fig. 4. Upon closer inspection, the latter appear vesicular (arrowheads), sin,smooth muscle cells; el, elastic tissue deposits. Scale bar, 0-2 um.

108 S. F. PERRY

The compliance of the lungs and body wall together is 1-23 mlcmH20 ' 100 g '(±0-26s.D.) or, standardized against VLr, 071 mlcmHzO"1 ml"1 (±0-06s.D.). Thelungs, with a value of 7-39mlcmH2O~'100g~' (±2-06s.D.) are more than four

Crocodile lungs 109

times as compliant as the body wall. The VLr-standardized lung compliance is4-32mlcmH2O"1mr1 (±M8s'.D.).

DISCUSSION

Studies of development and comparative anatomy of crocodilian lungs carried outduring the nineteenth century (for references see Milani, 1897; Broman, 1939) tendto be fragmentary, but stress the basic similarity of lung structure in alligators,caymans and crocodiles. Only Broman (1939) traced the development of the lungs ina single species (Alligator mississippiensis) through a period sufficient to explain theorigin of the chambers. He describes a row of large dorsal chambers which give rise attheir bases to medial and lateral subdivisions. These subdivisions later communicateas separate chambers with the intrapulmonary bronchus.

During late foetal development the cranial portion of the lung rotates so that thechambers which were originally dorsal later communicate with the lateral aspect ofthe intrapulmonary bronchus. These chambers may represent the lateral row in theyoung Nile crocodile (Fig. 1). The medial and lateral chamber rows of the foetalalligator would then represent the dorsal and ventral rows, respectively, in thepresent study.

Broman (1939) reported that the caudal portion of the alligator lung developsmuch more slowly than does the cranial portion, thus explaining the lack ofcontinuity between cranial and caudal rows of chambers. A similar process isexpected in the Nile crocodile.

Unfortunately no developmental study similar to that of Broman (1939) exists forthe Nile crocodile. Since it is not justifiable to apply nomenclature based upon lungdevelopment of an alligator to a crocodile, a provisional nomenclature for chambersbased only on the present findings is employed here (see Fig. 1).

The only original gross anatomical work explicitly concerning the lungs of the Nilecrocodile is a brief description by Lereboullet (1838), later paraphrased by Cuvier(1840). Both descriptions are included verbatim in Milani, 1897. Lereboullet

Fig. 6. Electron micrograph of an endocrine-like cell, showing numerous dense corevesicles (dcv), large mitochondria (in) and dilated cisternae of smooth endoplasmicreticulum (cs). n is the nucleus and a desmosome to a ciliated cell is encircled. In theoverlying ciliated cell subterminal and terminal ciliary rootlets (cr\ and crZ, respectively)and microvilli (wit) are indicated, c, subepithelial capillary; he, kinociha. The rectangleencases the area enlarged in Fig. 7. Scale bar, 10|im.

Fig. 7. Detail from Fig. 6. Beginning with a dense body (db) and progressing up to theleft are coincidentally dense core vesicles in progressive stages of maturation(arrowheads), dcv indicates fully mature dense core vesicles; m, a mitochondrion. Scalebar, 0-2fim.

Fig. 8. Total preparation of an interedicular partition in side view, showing the trabecula(/), a blood vessel (bv) and strands of interedicular smooth muscle (•) orientedperpendicular to the trabecula. Glutaraldehyde and osmium tetroxide fixed, Eponembedded. Scale bar, 50/im.

110 S. F. PERRY

Table 1. Volumetric parameters of the lungs in the Nile crocodile

Parameter

Body mass

Lung volume

Parenchyma

ParenchymaltissueTotal

Large bloodvessels

Partitions

Blood

Connectivetissue

Muscle

Epithelium*

Trabeculae

Blood

Connectivetissue

Muscle

Epithelium*

Symbol

W

V L / W

V P / W

Vt/W

Vt/W

V B / W

VP

VPb

VPU

vpm

VP.

VNP

VNPb

V N P U

VNPm

VNP,

Unit

kg

ml kg"1

ml kg"1

ml kg"1

I

3-48

121-3

47-428

4-514

0-820

3-070

0-872

1-971

0-271

0-002

0-624

0031

0-205

0-329

0-059

II

3-59

112-4

50018

4-282

0-932

2-814

1-335

1-345

0-115

0019

0-536

0-068

0-124

0-293

0-050

SpecimenIII

3-69

105-5

47-264

3-956

0-596

2-883

0-853

1-772

0-254

0-004

0-477

0-036

0-102

0-292

0-047

IV

5-68

98-2

39-367

3-805

0-841

2-418

0-990

1-328

0088

0-012

0-545

0061

0138

0-277

0069

1—III

3-59(0-105)1131(7-92)48-291

(1-638)

4-251(0-280)

0-783(0-171)

2-395(0-924)

1-020(0-273)

1-6%(0-320)

0-213(0-086)

0-008(0-009)

0-546(0-074)

0-045(0-020)

0-144(0-054)

0-305(0-071)

0 052

All

411(1-050)109-4(9-85)

46060(4-658)

4139(0-319)

0-797(0143)

2-796(0-274)

1-013(0-223)

1-604(0-319)

0-182(0-094)

0-009(0-008)

0-546(0-060)

0049(0-018)

0142(0044)

0-298(0-022)

0-056(0-006) (0-010)

• Non-respiratory epithelium.Numbers in parentheses indicate standard deviations.Boldfaced type indicates reference values and derived values.Units for all parameters within parenchymal tissue are ml kg- i

counted only five chambers: an anterior chamber (LI in Fig. 1), three furtherchambers (probably D2 + L2 + V1, D3 + L3 + V2, D4 + L4 + V3) and a posteriorchamber (the cartilage-free portion of the intrapulmonary bronchus and itsassociated chambers).

The presence of a small number of large chambers cranially is a common feature ofmost multicameral lungs. Similarly, the reduction of cartilagenous support in thecaudal portion of the intrapulmonary bronchus is common not only to crocodilians,

Percentage

100

75

50

25

Non-respiratoryepithelium

Capillaries

Large bloodvessels

Connectivetissue

Distal

'Proximal M u s c l e

25

62

Withblood

Crocodile lungs

1 0

111

1-4

94

30

60

10

92

30

47

55

Withoutblood

Withoutblood

Withblood

Withoutblood

Cranial Middle Caudal

Fig. 9. Graphical representation of the distribution of components of interedicularpartitions in the Nile crocodile lung. Numbers indicate the percentage values for eachcomponent. The first column in each pair represents all components, while in the secondcolumn the (perhaps variable) capillary blood volume has been eliminated. Samplingsites near the intrapulmonary bronchus (proximal) and near the lung wall (distal) arecompared using the sign test: ** indicates significant difference at the 1 % level, * at the5% level.

but also to monitor lizards and to many chelonians (Milani, 1894, 1897; Graper,1931; Kirschfeld, 1970).

Peculiar to the crocodilian lung, however, is the tendency for monopodal - asopposed to dichotomous - branching of the chambers, as well as the tendency for thisbranching to occur at the bases rather than at the tips of the chambers (Milani, 1897;Broman, 1939). The closest affinity in these respects is seen in the pattern offormation of the secondary bronchi of the avian lung (Locy & Larsell, 1916; Perry,1987). Further similarities between crocodilian and foetal avian lungs are the smallnumber of cranial chambers (secondary bronchi in birds), their tendency to occur ina spiral row or rows, the lack of a medial row of cranial chambers and the largenumber of relatively loosely ordered caudal chambers. Furthermore, the tendency ofcrocodilian lung chambers to form arching, tubular structures is reminiscent ofdeveloping avian secondary bronchi and parabronchi (Duncker, 1978a). It ispossible to construct an approximation of the avian lung—air-sac system from thecrocodilian structural type: sac-like cranial, ventral and caudal chambers become airsacs, dorsal or medial chamber rows arch caudally (with shortening of the proto-

112 S. F. PERRY

Lung and body wall

10 15Pressure (cmH2O)

20

Fig. 10. Volume-pressure diagram for the lungs and body wall (solid line) and lungsalone (dashed line) for a single Nile crocodile (mass 3595 g). Each curve represents themean of two trials. The distance along the volume axis from the starting point (single-headed arrow) to the turning point of the solid line represents Vl^. Note that VLr

represents only 14 % of the total lung volume. Furthermore, the intrapulmonary pressurerise upon opening the body wall (pressure difference between the single-headed anddouble-headed arrows) is very small, as is the amount of air in excess of VL,. that can beextracted from the exposed lungs.

avian thorax), their chamber walls deepen to parabronchi which meet terminally inthe plane of anastomosis with their counterparts from the caudal lung regions, andthe parabronchial lumina become contiguous through perforations. Only theblood-air-capillary net remains exclusively avian.

Although the present data are not suitable for construction of an allometricregression curve, the displacement volume of the lungs in the three small specimens(W = 3-6kg) is 15% greater per unit body mass than that of the large specimen(W = 5-7kg). The proportion of total lung volume devoted to parenchyma(38—44 %) is similar in all specimens, suggesting that the fixation conditions were thesame and that the tendency towards smaller lungs in larger animals is real. Tenney &Tenney (1970) reported that the lung volume in reptiles in general increases inproportion to \\ '3, whereas studies on single species indicate that the exponent ofthis relationship is closer to 1-0 (Hughes, 1977; Perry, 1978, 1983).

Crocodile lungs 113

Compared with terrestrial lizards, the Nile crocodile has very small lungs:YrLr = 1-8 ml 100g~\ as opposed to 4-4, 6-3, 16-0 and 246 ml 100g~' in the teju, thetokay gekko, the savanna monitor and the European chameleon, respectively (Perry& Duncker, 1978; Milsom & Vitalis, 1984). These differences may relate to thepossibly species-dependent relevance of VLr to survival.

VLr represents that lung volume at which the elastic contraction of the lungs is inequilibrium with the relaxed condition of the skeletomuscular pulmonary encase-ment. Although this condition is useful as a reproducible standard for comparison oflung volumes in different species, its functional significance is unclear.

Most reptiles - including crocodilians — display an intermittent breathing pattern(Glass & Johansen, 1979; Glass & Wood, 1983; Naifeh, Huggins & Hoff, 1970,1971a,b,c) in which the breathing phase begins with expiration. VLr could representa minimal respiratory pause volume, below which an initial expiration is no longereffective. Inspection of Fig. 10 reveals that the equilibrium point from which thestatic, closed-chest volume-pressure diagram begins is at the end of the linearportion of the deflation curve, and that VL,. is only a small portion of the total lungvolume. The constancy of the Nile crocodile's lung compliance over long portions ofthe volume—pressure curve, however, suggests that this species may be capable ofbreathing over a wide range of residual volume states, depending on the meanbuoyancy desired. Thus, although Vi . in the Nile crocodile lies well below that ofnon-crocodilian reptiles, the range of lung volumes available for breathing overlapswith that of many lizards (Perry, 1983).

In spite of marked differences in lung structure in crocodiles and lizards, the staticlung compliance in the crocodile (3-9 ml cmH20~' ml Vi^."1) is very similar to that ofthe tokay gekko and the savanna monitor (4-1 and 3-2-3-3 ml cmH20~' ml VLr~',respectively) (Perry & Duncker, 1978; Milsom & Vitalis, 1984). The highlyparenchymatous unicameral lungs of the teju and the emerald lizard show a lowercompliance (2-4 and l-Smlcml-^O"1 ml VLr~', respectively) (R. M. Jones, unpub-lished results; Perry & Duncker, 1978) and the sac-like chameleon lung is morecompliant (5-7 mlcmH2O~'ml VLr"') (Perry & Duncker, 1978).

The importance of these static compliance values in total work of breathing inthese species awaits direct measurement of dynamic compliance in living specimens.Recent studies (Milsom & Vitalis, 1984; Bartlett, Mortola & Doll, 1986) indicate thatthe major resistance to breathing in reptiles is in the body wall or in theextrapulmonary airways. The body wall compliance in the crocodile is calculated as1 / C B = l/CLB-l/CLtobeO-SSmlcmHzCr'rnlVL/-1. This is similar to the valuereported by Perry & Duncker (1978) for the gekko, but nearly four times greater thanvalues based upon dynamic measurements in the same species (Milsom & Vitalis,1984). The role of body wall compliance in crocodilian breathing mechanics,however, is uncertain because of their unique breathing mechanism discussed below.

Gans & Clark (1976) reported that in Caiman crocodilus both external and internalintercostal muscles tend to be simultaneously active during breathing. The intercos-tal musculature thus serves to stiffen the body wall while the m. diaphragmaticusaffects inspiration by retracting the liver, thus displacing the lungs caudally.

114 S. F. PERRY

Preliminary data from electromyographic and X-ray cinematographic studies in theNile crocodile imply a similar breathing mechanism in this species (B. Jutsch,personal communication). Further studies will elucidate details of this breathingmechanism.

Of particular relevance to the present study are the anatomical possibilities forgross lung movement and thus for ventilation of respiratory surfaces. The presenceof deep costal impressions, as well as the attachment of the lungs to the parietalpleura, suggests that the lungs do not slide, but rather stretch to accompany livermovement. The stretching of the lungs into the broad, caudal region of their conical,pleural cavities would widen the thin-walled caudal and ventral regions and draw airinto the arching distal regions of the tubular chambers, as in the mammalian lung(Loring & de Troyer, 1985).

Air can escape from the chambers during expiration by reversed flow, or possiblythrough intercameral perforations to neighbouring chambers. Patency of thechamber walls during expiration may be ensured by dynamic interplay of antagon-istic smooth muscle in the trabeculae and in the interedicular walls.

In the Nile crocodile 14% of the lung tissue is non-vascular, smooth muscle, ofwhich 64% is located in the trabeculae (see Fig. 9). Contraction of the trabeculaewould tend to raise the intrapulmonary pressure and deepen the parenchyma atthe expense of the central lumina of the chambers. The remaining 36% of theintrapulmonary smooth muscle is oriented approximately perpendicular to thetrabeculae and lies in the central leaflet of the interedicular walls (see Fig. 8). Itscontraction would thus lower the parenchyma and widen the central lumina.

The mechanism controlling and coordinating the activity of intrapulmonarysmooth muscle remains to be physiologically demonstrated. We were unable todemonstrate direct innervation of the smooth muscle, and recent observations in thered-eared turtle imply the involvement of endocrine-like cells (elc) in that species(Scheuermann, de Groodt-Lasseel, Stilman & Meisters, 1983; Scheuermann, deGroodt-Lasseel & Stilman, 1984). It is proposed that these cells release indolaminesor catecholamines in direct response to hypoxia or to nervous stimulation. In contrastto the red-eared turtle, the elcs of the crocodilian trabecular epithelium do notcontact the free surface. In addition, although abundant in the trabecular epi-thelium, they have not been observed in the interedicular tissue. In the teju lung, inwhich non-trabecular and trabecular smooth muscle are equally abundant, elcs havebeen demonstrated only in the equivalent of interedicular tissue (S. F. Perry &U. Aumann, in preparation), whereas in the water snake, Nemdia sipedon, theyoccur in domed structures on the trabeculae (S. F. Perry, unpublished obser-vations).

It is tempting to suggest that the microvilli-rich, ciliated epithelial cells of thetrabeculae may serve in more than lung clearance. The presence of a lateral labyrinthis consistent with the hypothesis that these cells may be active in fluid resorption, asproposed for similar cells in the mammalian airway epithelium (Rhodin, 1966;Wilson, Plopper & Hyde, 1984). elc secretions could be carried with the fluid flow tosubepithelial capillaries or across the smooth muscle to subtrabecular lymph spaces.

Crocodile lungs 115

The author gratefully acknowledges the technical assistance of Iris Zaehle, SabineWillig and Christine Ivanisi, the dactylographic assistance of Angelika Sievers andthe generous financial support of the Deutsche Forschungsgemeinschaft throughgrants Pe 267/1 and Pe 267/2-3.

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