Morphometric and histological analysis of the lungs of - NCBI

J. Anat. (1978), 125, 3, pp. 527-553 527With 22 figuresPrinted in Great Britain

Morphometric and histological analysis of the lungs ofSyrian golden hamsters

ANN R. KENNEDY, ARTHUR DESROSIERS, MARGARETTERZAGHI* AND JOHN B. LITTLE

Department of Physiology, Harvard School of Public Health,Boston, Massachusetts 02115

(Accepted 1 March 1977)

INTRODUCTION

Syrian golden hamsters have been used extensively in lung carcinogenesis studiessince they have a very low incidence of spontaneous lung tumours (Shubik et al.1962) and chronic respiratory diseases (Montesano, Saffiotti, & Shubik, 1970),unlike other small mammals. In many respects the hamster lung is similar to thehuman lung; however, there are substantial morphometric and histological differ-ences which suggest that regional exposures and reactions to carcinogens may differin these species.Human lung morphometry (Horsfield & Cumming, 1968; Parker, Horsfield &

Cumming, 1971; Raabe et al. 1976b; Weibel & Gomez, 1962; Weibel, 1963a),anatomy and histology (Bloom & Fawcett, 1968; Rhodin, 1974; Sorokin, 1970a,1973) have been well documented, with occasional references to differences betweensmall mammals. There is currently, however, no comprehensive analysis of eitherthe hamster lung or the lungs of other small mammals. The anatomy of the hamsterlung has been discussed by Magalhaes (1968) and Schwarze & Michel (1959-60),and occasional specific characteristics have been described, such as the distributionof glands (Kleinerman, 1972), the pattern of vasculature (Kleinerman, 1972), thefrequency distribution of cell types in the trachea (Boren et al. 1974; Kaufman et al.1972), and the ultrastructure of tracheal epithelial cells (Harris et al. 1971). Nomorphometry data on the hamster lung have been available; however, such a studyhas been published recently (Raabe et al. 1976a, b).The scarcity of existing information about the hamster lung prompted our study

of various parameters related to current lung carcinogenesis studies. As the epi-thelium is the site of origin of most human lung cancers, numbers and types of epi-thelial cells in the various regions of the lungs have been determined using 1 ,umplastic (glycol methacrylate) and electron microscopic sections. These data werecorrelated with the frequencies and sizes of airways in the lung, using measurementsfrom histological sections, fresh lungs and casts of hamster lungs. A standardterminology for hamster lung histology is presented, and comparisons are madebetween hamster and human lung morphometric and histological data.

* Current address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee.

ANN R. KENNEDY AND OTHERS

MATERIALS AND METHODS

AnimalsAdult male (approximately 125 g), random bred Syrian golden hamsters were ob-

tained from Dennen Animal Industries, Gloucester, Mass. They were fed Purina chow.

MorphometryA schematic drawing of the hamster lung is shown in Figure 1. Measurements

(length and diameter) of extralobular airways were made on fresh lungs and casts oflungs. Lung casts (Fig. 2) were made with Medical Silastic 382 Elastomer (DowCorning) which, when mixed with Catalyst M, becomes a silicone rubber. Thetechniques employed were described by Frank & Yoder (1966). Lungs were fixedat approximately 90 % of maximum inflation. Peripheral airways had clearly beencompletely filled with silicone rubber, because the respiratory portion of the lung(respiratory bronchioles to the alveolar sacs) could be seen extending from theterminal bronchioles in the casts (Fig. 2). The generation number as well as thelengths and diameters of airways (secondary bronchi to terminal bronchioles)within lobes of the lungs were determined from casts by dissection with the aid of adissecting microscope. An airway was taken to be a successive generation from aparent airway only if it noticeably decreased in size (diameter) as compared with theparent airway.Measurements for the lengths and diameters of the trachea and main bronchi on

five fresh lungs and five casts were similar, so it was assumed that measurementsmade on successive generations in lung casts would be comparable to those of freshlungs at the same degree of inflation. Diameter measurements of the large airwaysmade on fresh lungs were also comparable to the measurements made on histologicalsections of five lungs embedded in plastic (glycol methacrylate). We have carried outtwo studies to determine the effects of fixation and embedding on lung structuresusing our plastic section technique, since several investigators have reported that thedimensions of lung structures alter during the preparation of lungs for paraffinsections (Dunhill, 1962; Matsuba & Thurlbeck, 1971; Weibel, 1963b). The effectof fixation was studied by the water displacement method described by Dunhill(1962), in which the volume fixation constant is determined from the expression:volume of fresh lung/volume of fixed lung. Three fresh lungs were removed fromhamsters in the inflated state and water displacement measured; these lungs werethen collapsed by evacuation at - 740 mmHg for 10 minutes, and then fixed withglutaraldehyde, as has been previously described (Kennedy & Little, 1974a).Thewater displacement of these fixed lungs was found to be the same as for the freshinflated lungs in each case. The effect of embedding and sectioning in our systemwas determined by cutting eight 0 9 x 0-9 cm blocks from one fixed lung and thenmeasuring the blocks after embedding and sectioning. The linear shrinkage due toembedding and sectioning in two of these blocks was measured to be 0-96 ± 0-08 (S.E.)while the remaining six blocks did not appear to have changed size between fixationand sectioning. Our observed linear shrinkage factor has not been incorporated intothe dimensions reported in this paper since it is not significantly different from unity.It appears that our plastic section technique results in better preservation of lungstructures than does conventional paraffin embedding procedures, in which shrinkagefactors for airway dimensions are significant (Dunhill, 1962; Matsuba & Thurlbeck,1971; Weibel, 1963 b).

528

Since airway diameter is one of the characteristics we used to distinguish betweenbronchi and bronchioles microscopically (Tables 7-8), the diameters of the airwaysin the casts were used to determine the numbers of bronchi and bronchioles in thevarious regions of the lung. Bronchi were defined as airways with a diameter> 0 5 mm; bronchioles as airways with a diameter < 0-5 mm and > 0-1 mm, butwith only one dimension, length or diameter, in the 0-1-0-2 mm range. If an airwayhad both dimensions in the 0-1-0-2 mm range, it was classified as a terminal bron-chiole and confirmed from the correlated histological sections.The total number and sizes (diameters and lengths) of bronchi occurring within

the various lobes were found to be approximately the same for five casts; however,they did branch off parent airways at somewhat different places and angles. Sincewe were primarily interested in frequencies and generations of the various sizedairways, a complete dissection as far as the terminal bronchioles was carried out ononly one cast.

Lengths and diameters of airways in the respiratory portion of the lungs (from therespiratory bronchioles to the alveolar sacs) were determined from histologicalsections of glycol methacrylate embedded lungs containing longitudinal sectionsfrom the terminal bronchiole to the alveolar sacs (Figs. 3-4). Measurements of agiven airway diameter in the respiratory portion of the lungs were made from theknob-like swellings at the entrance to the alveoli (Rhodin, 1974) on either side of theairway - and thus did not include the depth of the alveolar outpocketings.Two methods were used to estimate the total number of alveolar ducts and sacs

in the hamster lung: one, a method derived from cast measurements and the other,Weibel's (1963 a) method for the analysis of data from histological sections.

HistologyProcedures for preparation of lungs for glycol methacrylate embedding and stain-

ing have been described (Kennedy& Little, 1974a). The following parts of the respirat-ory system were sampled. The larynx, three sections of trachea (near the larynx,mid-way along its length, and near the bronchial bifurcation), and one section ofeach major bronchus. Within each lobe, three to five sections (from five separateanimals) were cut in a longitudinal or in a crosswise fashion and the orientation inthe lobe recorded. The cell types and frequencies present in the various airways ofthe lung were determined from both glycol methacrylate embedded lungs for lightmicroscopic analysis and lungs embedded in Epon for electron microscopic (EM)study. The procedures for preparation of lungs for EM sections involved fixation ofwhole lungs in Karnovsky's glutaraldehyde-formaldehyde fixative (Karnovsky, 1965)for 3 hours. Lungs were then cut into pieces no larger than 1 mm3, and post-fixedfor 1 hour in 1 3 % OSO4 in 0 1 M s-collidine buffer at pH 7-4. Lungs were embeddedin Epon, and 1 ,tm sections were cut and stained with toluidine blue for light micro-scopy. Silver sections were cut on a Reichert ultramicrotome, stained with leadcitrate and uranyl acetate, and examined with a Philips 300 electron microscope.

Frozen sections for histochemical studies were prepared from lungs embedded inO.C.T. compound (a mixture ofwater soluble glycols and resins obtained from FisherScientific Co.) frozen by immersion in methyl butane cooled to the viscous stage byliquid nitrogen, and cut in a microtome-cryostat (Harris Mfg. Co.) at 4 ,um. Themethod of Chayen et al. (1969) was used for toluidine blue staining, and succinicdehydrogenase activity was determined by the method of Nachlas et al. modifiedby Barka & Anderson (1963).

o'-y-rian hamster lungs 529

34 A NA125


Paraffin sections of alcoholic-zinc formalin-fixed normal lungs (two animals) wereused for the determination of mucin secretions in glands and goblet cells. The follow-ing stains were employed: the alcian blue (AB) (pH 1)-periodic acid Schiff (PAS)sequence (Luna, 1968), the high iron diamine method for sulphomucins (Spicer,1965), and Muller's modification of Mowry's colloidal iron stain for acid mucopoly-saccharides with Van Gieson's collagen fibre stain - with and without neuraminidasedigestion (Luna, 1968).

RESULTS

MorphometryGross anatomy and airway branching patternsThe respiratory system of the hamster consists of a pharynx, larynx, trachea, and

lungs made up of a single large lobe on the left, and four lobes on the right, namelyan apical, middle, diaphragmatic or caudal, and an infracardiac lobe extending bet-ween the heart and the diaphragm (Fig. 1). The anatomical arrangements of thelobar bronchi, and the ranges and average dimensions for the trachea and mainbronchi (as determined from fresh lungs) are given in Table 1.Most upper airway (trachea to large bronchioles) and some lower airway branching

in the hamster is by unequal dichotomy, in which the smaller branch forms a largerangle with the parent tube. Unequal dichotomous branching is demonstrated in thelung cast shown in Figure 2: the left main bronchus is smaller than the right, and theright lung is more of a direct extension of the trachea. After the standard branchingof the main bronchi into secondary bronchi the branching is usually dichotomous(Figs. 3-4) and occasionally trichotomous.

Airway sizesRepresentative dimensions of airways determined from fresh lungs, casts and histo-

logical sections are given in Tables 1-3 and compared with sizes of human airwaysin Table 7. Smaller diameters for the hamster trachea have been reported bySchwarze & Michel (1959-60) (1-5-1-6 mm) and a larger diameter is reported byRaabe et al. (1976a) (2-6 mm), but our results of 2 mm were quite constant. Figure 5shows the relative frequencies of large (bronchi and bronchioles) and small (terminalbronchioles) airways obtained from casts. Figure 6 demonstrates the variation ofairway diameter and length as a function of generation for the large airways. Withinany generation, the variation of length was always greater than variations in diam-eter, as seen in Figure 7 where the fourth generation is used as an example. Sixgenerations of bronchi and six genertions of bronchioles were classified in thehamster.

Terminal bronchioles were found in seven generations (4-10) of airways. The sizeof these airways was not related to the generations in which they were encountered.As can be seen in Table 3, the hamster respiratory bronchiole is often very short,

being only 2-4 alveoli in length before the alveolar duct begins. However, the lengthof the respiratory bronchioles varied, with a range of 0-1-0-5 mm (average length of0-23 mm). One (Fig. 3) or two (Fig. 4) generations of alveolar ducts appeared fre-quently in longitudinal sections. Diameter and length were approximately the samefor both generations of alveolar ducts (Table 3). In the lung casts, the usual lengthof the respiratory portion extending from a terminal bronchiole (acinus) was between0'7 and 1 mm.

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Syrian hamster lungs

b

-f4.

3< 4 , j : ,* , + s .

Fig. 1. Schematic representation of the hamster lung. a, larynx; b, trachea near fused middlerings; c, carcina; d, single lobe on left; e, right apical lobe;f, right middle lobe; g, diaphragmaticor caudal lobe; h, infracardiac lobe.Fig. 2. Silicone rubber cast of the hamster lung. Note that the right main bronchus (left of field)is more of a direct extension of the trachea than the left main bronchus. x 2.Fig. 3. Branching pattern from a terminal bronchiole showing a respiratory bronchiole, onegeneration of an alveolar duct and two alveolar sacs. x 43.Fig. 4. Branching pattern from a terminal bronchiole showing a respiratory bronchiole, twogenerations of alveolar ducts and an alveolar sac (terminating at the bronchiole from which theacinus originated). x 43.

34-2

531

I I


Table 1. Dimensions of trachea and main bronchi determinedfromfive fresh lungs

Diameter

Length Average+ S.E. Range Average+ S.E.Airway range (mm) (mm) (mm) (mm)

Trachea (to carinal 17-21 19-2±0-66 1-8-2d1 2 0+0 05cartilage), Genera-tion 1Main bronchi,Generation 2(extralobular)Left lungRight lung

5-6 564+±019 610±0-296-8 6-6+±040

194-126 125±0 03 1-7+0-081-9-2-0 2*0+0-02

Location of secondary bronchi: distance along main bronchi at which secondary bronchialbifurcations occur

LeftRight

ApicalMiddleInfracardiacDiaphragmatic

Range(mm)5-6

1-5-24-55-76-8

Average± S.E.(mm)

5*4+±*19

1*9+0-094-4±0-246-0+0446-6+0 40

1000 r-

1001-

.0

Ea)z 10p

2 4 6Airway generation

8 10

1

Fig. 5. Numbers of bronchi (0), bronchioles (A), and terminal bronchioles (O) in the hamsterrespiratory tract as a function of airway generation.

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Syrian hamster lungs100l K

101

E1-

0).Ncn

1 _

2 4 6 8 10Airway generation

Fig. 6. The variation of airway length (A) and diameter (0) versus airway generation.

2-

E

E

0D *E~~

to0

0 1 2 3 4 5 6Length (mm)

Fig. 7. The variation of airway diameter versus airway length for 25 fourth generation airwaysof the hamster lung.

533

I


Table 2. Pulmonary measurements determinedfrom a lung cast(Frequencies and generations of airways by lobe.)

Number of airways (generations in parentheses)

TerminalBronchi Bronchioles bronchioles

Lobe: Left 18 (2-7) 44 (4-9) 156 (4-9)RightApical 9 (3-7) 20 (5-8) 80 (5-9)Middle 5 (3-5) 45 (4-7) 86 (5-8)Infracardiac 4 (3-4) 22 (4-7) 48 (5-8)Diaphragmatic 13 (3-6) 77 (5-9) 170 (5-10)

Total 49 (2-7) 208 (4-9) 540 (4-10)

Range of sizes and averages for these various airways (airways classified by histologicaldefinition described in text)

Length Diameterr v A

- - - _ -

Range Average ± S.E. Average± S.E.Airway (mm) (mm) Range (mm) (mm)

Bronchi 1-05-5 2-70+0-21 0-5-2-0 0-67+0 04Bronchioles 0-1-40 1-39+0-05 0 1- <0 5 0-24+0-01Terminal bronchioles 0 1-0 2 0-15+0-003 0-1-02 0-16+0-003

Average number of respiratory units per terminal bronchiole - 2-4 ± 0 1 (100 terminal bronchioles andrespiratory units counted).

Number of alveolar ducts and sacsTwo methods were employed to estimate the total number of alveolar ducts and

sacs in the normal hamster lung. First, the average number of respiratory units perterminal bronchiole was multiplied by the total number of terminal bronchiolesfound in a cast (see Table 2). This gives the number of respiratory bronchioles.Subsequently, dichotomous branching was assumed to produce exactly 14 ducts andsacs in each unit: two first generation ducts, four second generation ducts and eightalveolar sacs. Dichotomous branching was assumed distal to the respiratory bronchi-ole, since this was observed most often in tissue sections.Thus the total number of ducts and sacs is equal to:

14 x number of respiratory units per terminal bronchiole x number ofterminal bronchioles = 18100.

Using the standard error given in Table 2, and assuming a 1 % maximum error forthe number of terminal bronchioles, the standard error of the total is 800.

Additionally, the method of Weibel (1963 a) was used to estimate the total numberof ducts and sacs. Frequency measurements were obtained by light microscopicexamination of random fields of hamster lung parenchyma. This method uses therelationship:

N= la05Vnifpibwhere N = the number of alveolar ducts and sacs, V = the parenchymal volume offixed lung, n = the number of ducts and sacs intersected per cm2 on random fields,p = the proportion of parenchymal volume consumed by ducts and sacs, b = ashape factor related to the average cross sectional area of a duct or sac.

In this measurement no distinction is made between ducts and sacs; rather the

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Table 3. Measurements of airways determinedfrom histological sections

(A). Airway dimensions (seven animals: number of units counted in parentheses)Larger airway diameters

Range (mm) Average± S.E. (mm)

TracheaBronchiBronchiolesTerminal bronchioles

1-9 -2-105 -2-0010- <050.1 -0-2

Length

2-0 +0-04(7)0 70+0 06 (25)0-23±0-02 (25)0-17+0-004 (25)

Diameterrag(m

Range (mm)Average ± R eageAverage± S.E. Range (mm) Average± S.E.

Respiratory region:Respiratory bronchioles 0-12-0-50 0-23±0-03 (23) 0-05-0-20 0-16±0-01 (23)Alveolar ducts 0-24-0-55 0-32+0-08 (23) 0-09-0 20 0-13+0-01 (23)Alveolar sacs 0-20-0-40 0-27±0-01 (18) 0 10-0 20 0-13+0-01 (18)Alveoli 0-03-0-10 0-06±0-002 (74) 0-03-0-10 0-06+0-002 (74)

Average number of alveoli per respiratory bronchiole: 4-2±0-23 (one side only) (23)Average number of alveoli per alveolar duct: 5*1 ± 0-18 (one side only) (23)Average number of alveoli per alveolar sac: 5-2± 0-27 (18)

(B). Cell type frequencies and cell numbers in airways (10 fields - two from each of five animals)

Cell typeCiliatedGobletBasal*UndeterminedTotalCiliatedGobletBasalClara*UndeterminedTotalCiliatedClara*UndeterminedTotal

Average total no.per 150,tm

epithelial length13-414-37-51.9

37*19.57.34-82*03.3

26-911-611*50-7

23-8* Undetermined category includes intermediate and brush cells.

(C). Height of epithelium (five measurements made from each of five animals)

TracheaBronchiBronchioles

Average ± S.E. (cum)20-0±0*2920-2+0-34'scalloped' appearance: Clara cells, approximately 20,tm; ciliated cells,approximately 10lm (without cilia)

(D). Alveolar regionAverage number of cells/alveolus: 11-4± 0-8 (100 alveoli counted in each of five animals - measured

on longitudinal section of alveolar ducts and sacs; does not include macrophages).Frequency of macrophages in plastic sections: for 1000 alveolar cells, counted in each of five animals,

average of 38± 0-89 macrophages (however, most probably washed out by processing - see Discussionin text).

In each of five animals studied, there were approximately one third the number of type 2 cells as com-pared to the total number of type 1 and endothelial cells.

AirwayTracheaMain bronchi

Bronchi

Bronchioles

S.E.

0-540-540-340-230-840-340-560-330-210-370-460-310-270-210-29

% of total36-138-520-25*1

35.327-117-87.4

12-3

48-748-32-9

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total number of ducts and sacs in the alveolar region is counted. Ducts and sacs maybe distinguished from respiratory bronchioles in the hamster since the latter are linedwith bronchiolar epithelial cells. Alveoli are distinguished from ducts and sacs bytheir smaller size and circumscribed appearance.A random selection of glycol methacrylate-embedded tissue specimens was

examined to determine the average number of transsections of both ducts and sacsper field. When divided by the field area, this yielded n = 426± 31 transsections percm2. These same fields were also measured in order to obtain the proportion ofparenchymal volume occupied by ducts, sacs, and alveoli. Although the methodoutlined by Weibel uses an integrating stage micrometer, the present study was per-formed by photographing microscopic fields. Enlargements were printed in fullformat and a machinist's micrometer was employed to sum the contributions ofsmall blood vessels, tissue septa and respiratory space to the lung parenchyma. Theaverage proportion of parenchymal volume devoted to alveoli, ducts and sacs wasfound to be 0-863 ± 0-023. Based on Weibel's measurements of human lungs, theparenchyma was estimated to comprise 90 % of the hamster lung by volume, and theducts and sacs were estimated to comprise 32 % of the parenchymal air exchangespace. The volume of fixed lungs was 2 5 cm3. Hence the proportion of parenchymalvolume consumed by ducts and sacs, p, is 0-863 x 0-32 = 0 275 and the parenchymalvolume is 0 90 x 2-5 = 2 25 cm3. Also from Weibel (1963 a), the shape factor, b, istaken to be 2-15 for hamster ducts and sacs (Fig. 17 in Weibel, 1963 a).

Using these values in the above equation leads to N = 18400 + 2000, where thestated error is due entirely to statistical fluctuations in n. Errors for the other para-meters were not included because these figures involve estimates based on humandata and because the error stated, a lower bound of the true error, is nevertheless ofan order which makes it possible to conclude that agreement exists between themorphometrical approach of Weibel and the cast dissection technique employedhere. Since the Weibel method does employ parameters which are more difficult toevaluate and verify, the semi-empirical results obtained directly from an intact castwere employed for all subsequent calculations.

Number of epithelial cells in the various regions of the lungUsing plastic sections, the number of epithelial cells in each region of the con-

ductive airways was estimated by converting the number of cells observed per 150 4umof airway epithelial length into a surface density. For example, Table 3 indicates that26-9 cells per 150 #rm were observed in hamster bronchi. Thus the average length ofa cell is 1 50126-9 = 5 58 ,um. If this is also taken to be the mean cell width, then atypical cell has an area of (5 58 ,um)2 = 31 -1 /ZMm2. This is equivalent to an area densityof 3-2 x 106 cells per cm2. Similar calculations were performed for the trachea, majorbronchi, bronchioles, and terminal bronchioles.The area of each airway dissected from a lung cast was then calculated and

multiplied by the appropriate cell surface density. A total for each region was thenobtained by summing the numbers of cells from all airways of that region. Resultsare given in Table 5.For the purpose of determining the number of epithelial cells in the alveolar

region, each alveolus was considered to be a sphere of radius 38 ,m. Althoughalveoli are not spherical (Weibel & Gomez, 1962), a closed sphere was consideredto be approximately equal in surface area to an alveolus of equivalent width. Thelarger surface-to-volume ratio of the alveolus compensates to a considerable extent for

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Table 4. Results of staining reactions

Staining reactions of histologic structuresStain and significance (two animals used r-for each stain) Blue Red

AB pH 2-5-PAS. All polysaccharides and Some tracheal and Most goblet cells in tracheal andmucosubstances containing hexoses or bronchial goblet cells bronchial epithelium and glandsdeoxyhexoses with vicinal glycol groups Cilia Cartilage - interlacunar matrixstain red - includes neutral mucosub- Cartilage - lacunar rims, Most glandular goblet cellsstances. Hyaluronic acid, sialomucins chondrocytes and peri-and all but the most strongly acidic chondriumsulphated mucosubstances stain blue Mast cells in tracheal and(Luna, 1968) bronchial adventitia

An occasional cell in atracheal gland

AB pH 1-PAS. PAS staining (red) A few goblet cells high in Most goblet cells in trachealsame as in above sequence. However, the trachea near the epitheliumalcinophilic (blue) substances at this larynx All bronchial goblet cellspH include only the sulphated An occasional cell in a Cartilage - interlacunar matrixmucosaccharides (Luna, 1968) tracheal gland Most glandular goblet cells

Cartilage - lacunar rims,chondrocytes and peri-chondriumMast cells in tracheal andbronchial adventitia

AB pH 0 4. Only strongly acidic Very few positive cellssulphated mucosubstances give a high in the tracheal epi-positive (blue) reaction (Luna, 1968) thelium near the larynx,

and an occasional posi-tive cell in a trachealgland

Table 5. Total number of epithelial cells in conductive or air exchangespaces as measuredfrom histological sections

Region Cells x 106

Trachea 7-3Main bronchi 4-1Bronchi 9-3Bronchioles 6-0Terminal bronchioles 1-0Alveoli 1-2 x 103

the area occupied by the alveolar opening. Since the alveolar diameter is pro-portional to the fifth root of body weight (Weibel & Gomez, 1962) in mammals, thehamster's alveolar size may be obtained from the relationship:

DR-(125)0,2 (270)-76sm(70000)0 2

where 270 ,am is the average alveolar diameter of a 70 kg human subject. This resultcompares favourably with the average measured width of 60 ,tm (Table 3). When-ever a plane circle of radius r is randomly transsected, the average chord length is(nj2) r, not 2r. As (T/2) 38 = 60, there is clearly complete agreement.The surface area of a 76 ,um diameter sphere is 1 8 x 104 jUm2. By use of Weibel's

(1972) relationship for the total surface area of a mammalian lung, a 125 g hamster

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Table 6. The hamster respiratory system (range of measurementstaken from seven animals)

Muscularis Adventitia Cartilage Mast cellsAirway thickness (mm) thickness (mm) thickness (mm) (adventitia)

Trachea Muscularis not If cartilage present, 0-02-020 xpresent 0-02-0-12; if no

Trachealis muscle, cartilage, 0 03-0060 02-0)05

Main bronchus 0-005 (in front of 0-03-012 (thickest 0 01-0410 xcartilage) - 0-02 where blood vessels

occur in all airways)Bronchus 0 02-004 0 03-0-06 - xBronchiole 0-0 01 0-005Terminal 0-0-005 0-005 - --bronchiole

Respiratory Occasional singlebronchiole muscle fibre

Alveolus Rarely a singlemuscle fibre

would have an air exchange surface of 3-2 x 1011 #am2. This implies that there are1 -8 x 107 alveoli. By comparison, Granito (1971) reported 3 0 x 107 alveoli in therat, whose alveoli are larger.The average area of an alveolar cell was estimated from the number of cells

observed along sections of alveolar wall. From Table 3, 11-4 cells occupied alveolarperimeters of mean length 7T60 1um. Hence the mean cellular area is (fT60/1114)2 =270 ,um2 as explained above. The number of alveolar cells is therefore 3-2 x 1011cIm2/lung ÷270 gMm2/cell = 1 2 x 109 cells/lung. We recognize that the assumptionsemployed in formulating these estimates preclude anything but rough accuracy.Standard errors were not calculated for these estimates because systematic sources oferror are expected to be dominant.

HistologyHistological features of the hamster lung are presented in Tables 6-8. Table 6

gives features of the muscularis and adventitia in hamster airways, Table 7 compareshistological features in hamster and human lungs, and Table 8 summarizes the clearestdistinguishing features between airways of the hamster lung.

Cartilage (data from five fresh lungs and five casts)The trachea contains 15-18 C-shaped cartilage rings and a carinal cartilage. The

carinal cartilage is composed of two semicircular segments which meet but do notfuse ventrally where the right segment gives rise to a semicircular appendage whichcourses in a sagittal plane below and behind the carina. The central tracheal ringsare often made up of two oblique C-shaped rings fused at the lateral aspect (Fig. 1).

In the left lung, C-shaped cartilage rings or large cartilaginous plates extendalong the entire extrapulmonary course of the main bronchus and thereafter about1 mm past the point at which the first secondary bronchus branches off the mainbronchus. Since the main bronchus does not appear grossly different after thisbranching, it was concluded that the main bronchus extends into the 'pulmonary'portion of the left lung. Cartilage structure in the right lung was variable. C-shapedrings or large cartilaginous plates usually extended along the entire extrapulmonary

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Table 7. Comparison ofhuman and hamster respiratory systems

RespiratoryAirway Trachea Bronchus Bronchiole bronchiole Alveoli

(A) Human respiratory system

Diameter

Epithelium

Laminapropria

Muscularis

20-25 mm (Bloom& Fawcett, 1968)

Ciliated pseudo-stratified colum-nar with gobletcells. No Claracells (Rhodin,1974)

Prominent(elastic lamina)(Sorokin, 1973)Not prominent.Only trachealismuscle (Rhodin,1974)

Submucosa Mucous and sero-mucous glands(Sorokin, 1973)

Adventitia About 20 C-shaped cartilagerings. Lymphnodes (Rhodin1974)

> 1-0 mm (Soro-kin, 1973)Same as trachea.Height, 30-50,m(Rhodin, 1974;Gastineau et al.1972)

Thin (diffusenetwork) (Soro-kin, 1973)Prominent muscu-laris. Musclefascicles: con-tinuous circularor spiral (Soro-kin, 1973)

Mucous and sero-mucous glands(Sorokin, 1973)Cartilage plates.Lymph nodes,lymphatics. Bun-dles of collagen-ous and elasticfibres. Bloodvessels, nervebundles (Rhodin,1974)

0-5-1-0 mm (Rho-din, 1974)

Ciliated simplecolumnar. Gobletcells become in-frequent and dropout; Clara cellsappear (Rhodin,1974)

Thin: looseconnective tissue(Rhodin, 1974)

Prominent muscu-laris: musclefascicles, circularand spiral. Sep-arated by con-nective tissue(Sorokin, 1973)No glands (Soro-kin, 1973)

Connective tissue,elastic fibres,blood vessels,lymphatics (Bloofn& Fawcett, 1968)

_ 0-5 mm (Bloom& Fawcett, 1968)Low columnar tocuboidal. ManyClara cells, fewciliated cells.Ciliation dropsout along thisairway (Bloom& Fawcett, 1968)

Layer of smoothmuscle cells isthin and incom-plete (Rhodin,1974)

270 /tm (Weibel &Gomez, 1962)

Types 1 and 2cells (Rhodin,1974)

Narrow bundlesof smooth musclecells encircle theentrance to eachalveolus of thealveolar duct(Rhodin, 1974)

Collagenous con-nective tissue con-taining elasticfibres (Bloom &Fawcett, 1968)

(B) Hamster respiratory system

Diameter

Epithelium

LaminapropriaMuscularis

Approximately2-0mm

Ciliated pseudo-stratifiedcolumnar inplaces to lowcolumnar. Manygoblet cells.No Clara cells.Height, 20,m

Prominent

Only trachealismuscle

Submucosa Mucous glandsAdventitia C-shaped

cartilage rings.Mast cells.Lymph nodes,nodules;lymphatics

0-5-2-0 mm

Ciliated lowcolumnar.Many gobletcells, someClara cellsHeight, 20,m

Very thin

Muscle fascicles:continuous,primarily circularNo glandsCartilage platesdrop out as theairways enterthe lungMast cells.Lymphatics;Rare lymphnodules

0-1- < 0-5 mm 0-05-0-2 mm Approximately76,cm

Ciliated low Clara cells Types 1 and 2columnar. Claraand ciliatedcells. Ciliadrop out inthe terminalbronchiole.Height: Clara cells,20,tm; ciliated cells,10,tm

Loose network: Rare singleprimarily circular muscle fibresmuscle fascicles.No glands --Connective tissue - --elastic,reticular fibres.Blood vessels;Lymphatics

539


Table 8. Major differences among airways in the hamster

Diameter Distinguishing characteristics Differences from parent branch

Approx. 2-0mm

Approx. 1-8 mm

C-shaped cartilage rings surroundairway except dorsally wheretrachea contacts oesophagus

Variation from ciliated pseudo-statified columnar to lowcolumnar epithelium withgoblet cellsMast cells in adventitiaGlands - primarily near larynxLymphatics and lymphoidtissue in adventitia

Cartilage plates and fragmentsas well as a few rings

Epithelium same as tracheaMast cells in adventitiaLymphatics and lymphoid tissuein adventitia

Bronchus 0-5-2-0 mm No cartilageLow pseudostratified ciliatedcolumnar epithelium (lowcolumnar in places) withmany goblet cells, someClara cells

Fascicles of muscle are closelypacked into a continuousmuscularisLymphatics in adventitia; rarelymphoid tissue in adventitianear bifurcationsMast cells in adventitia

Bronchiole 0 10- < 0 5 mm

Terminal 0O1-0 2 mmbronchiole

Respiratory 0 05-0 20 mmbronchiole

No cartilageEpithelium: low columnarciliated and Clara cellsMuscularis: fascicles of muscleseparated by connective tissueNo mast cells in adventitia

Epithelium: ciliated cells sparse;they drop out at approximately0<18 mm diameterEnds often flare out: 0 06 mm(minimum) to 0-12 mm (maxi-mum) diameter

Small stretches of bronchiolarepithelial cells (Clara cells)separated by alveoli

Cartilage no longer surroundsairway; plates and fragmentsare more common than ringsNo glands

Cartilage drops outMuscularis becomes prominentAppearance of Clara cells

Goblet cells drop outClara cells become prominentFascicles of muscle becomeseparated by connective tissueMast cells drop out (in adventitia)

Ciliation drops out in terminalbronchiole

Alveolar outpocketings

540

Airway

Trachea

Mainbronchus

course of the right main bronchus. However, cartilage occasionally stopped aroundthe origin of the middle lobe secondary bronchus, and sometimes extended into thepulmonary portion of the diaphragmatic lobe. Apart from the C-shaped rings orlarge cartilaginous plates, traces of cartilage could occasionally be seen in largebronchi under the light microscope (Fig. 10).

Mucous secreting apparatus: distribution and nature of mucous secretionsThe distribution of glands in the hamster is quite unlike that in the human air

passages (Table 6). Glands can be found in the hamster trachea, and are in particu-larly large numbers very high in the trachea near the larynx (Fig. 8). Glands are veryrare in the rest of the tracheobronchial tree. This paucity of glands in the hamsterlung has also been noted by Kleinerman (1972).Goblet cells are prominent in the trachea and bronchi (Figs. 9-11). The distribution

is variable (Fig. 9), with goblet cells being the predominant cell type in some areas,while being nearly absent in others. They are particularly prominent at bifurcations.Within a given airway, goblet cells decrease in number as the diameter of the airwaydecreases.The nature of the mucous secretions was determined by various staining reactions.

As can be seen in Table 4, the results of the AB pH 2-5-PAS and AB pH 1-PASsequences indicate that primarily a neutral mucopolysaccharide is produced byhamster goblet cells in the airway epithelium and glands. An occasional glandularcell, and a very few epithelial goblet cells, in the trachea near the larynx containedacidic mucosubstances (Table 4) which were shown to be strongly acidic sulphatedmucosubstances by the AB pH 0 4 stain (Table 4) and the Spicer high iron method(Spicer, 1965). Some goblet cells in the tracheal and bronchial epithelium containedsialomucins which are also acidic mucosubstances (Table 4). With prior digestion byneuraminidase, which acts on the glycoside link through which sialic acid is attachedto its substrate, and staining by Mowry's colloidal iron stain for acid mucosubstances(Luna, 1968), it was shown that the sialomucins present in tracheal goblet cells andbronchial goblet cells at bifurcations were insensitive to neuraminidase, while thosebetween bronchial branch points were sensitive to the enzyme.

Airway epitheliumCell type frequencies for various airways are shown in Table 3. For these fre-

quencies, ten randomly chosen fields were counted - two fields from five animals foreach region counted.A preliminary study of the airway epithelium was undertaken to determine whether

cell type frequencies in airways varied according to their location in the lungs. Celltype frequencies were determined by studying bronchi of diameter 0 6-0-7 mm andbronchioles of diameter 0 3-0 4 mm (Tables 2, 3) in histological sections preparedfrom the apical, middle and basal parts of all five lobes from two hamster lungs.Minor variations were found within the same animal and between animals - par-ticularly in regard to the distribution of goblet cells. However, there appeared to beno consistent pattern of variation for cell type frequencies based on location of air-ways within lungs. Thus, it was concluded that similar sized bronchi contained thesame cell types in the same proportions regardless of generation or position in thelung; the same was true for bronchioles.

In general, the cell types encountered at the electron microscopic (EM) levelappeared to be similar to those found in the human lung. However, neurosecretory

oc'-y-rian hamster lungs 541


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cells were rarely found among hamster epithelial cells. Neurosecretory cells arecommon in human bronchial, bronchiolar and tracheal epithelium (Rhodin, 1974).The cell types and frequencies are given by regions as follows:

1. Tracheobronchial epitheliumHamster tracheal and bronchial epithelium varies from low pseudostratified

(Fig. 10) to low columnar ciliated. The height of the epithelium is approximately20,um (Table 3).Two types of ciliated cells, differing primarily in lengths of their cell borders, have

been identified in tracheal and bronchial epithelium, and are illustrated in a bronchusin Figure 12. The majority of ciliated cells have a compact round nucleus whichcontains fine granular chromatin and which is located near the base of the cell; theother ciliated cells have an elongated nucleus parallel to a very long ciliated border.It is not clear from histological sections whether two ciliated cell types really exist,since a tangential section could give the appearance of long and short cell borders.Schreiber & Nettesheim (1972) have separated the two cell types by lung washings,however, and present evidence that two cell types in fact exist.

In the trachea, ciliated cells comprise about 36 % of cell types, goblet cells 39 %, andbasal cells 20 %. The remaining 5 % of cell types are intermediate or brush cells -labelled as 'undetermined' in this study. The ultrastructural morphology of the celltypes present in the normal hamster tracheal epithelium has been described in detail(Harris et al. 1971).Bronchial epithelium (Figs. 10-14) consists of the two types of ciliated cells already

described as well as goblet, basal and intermediate cells, which appear ultrastructur-ally the same as those in the tracheal epithelium. The proportion of cell types in themain bronchi is like that in the trachea. Clara cells begin to appear in the secondarybronchi of the hamster lung: they are seen in bronchial epithelium at the lightmicroscope level in Figure 11, and at the electron microscope (EM) level in Figure 14.

Fig. 8. Hamster glands (centre right) are common high in the trachea near the larynx. Theseglands are strongly PAS-positive and secrete mucus consisting of neutral mucopolysaccharide.Cartilage appears at bottom ofphotomicrograph, tracheal epithelium at top. PAS-haematoxylin.x 43.Fig. 9. Low power view of two similar bronchi showing very irregular distribution of goblet cellsin bronchial epithelium. Almost every cell is a goblet cell in the bronchus appearing at top,whereas goblet cells are more uniformly distributed in the bronchus at bottom of photomicro-graph. PAS-haematoxylin. x 43.Fig. 10. High power view of pseudostratified columnar bronchial epithelium showing gobletcells (with dark granules of PAS-positive mucus), ciliated, basal and intermediate cells. Afragment of cartilage appears at bottom of photomicrograph. PAS-haematoxylin. x 430.Fig. 11. High power view of bronchial epithelium showing Clara cells, the large dome-shapedcells having nuclei containing condensed chromatin around the edges (left and right of field) andgoblet cells, containing dark granules of PAS-positive mucus (centre and far right). Cells of thealveolar region appear at bottom of photomicrograph. PAS-haematoxylin. x 430.Fig. 12. Bronchial epithelium showing two types of ciliated cells: most ciliated cells have a com-pact, round nucleus located near the base of the cell and a short ciliated border (centre), butsome ciliated cells have an elongated nucleus parallel to the basement membrane and a longciliated border (right of field). Alveolar cells appear at bottom of photomicrograph: type 2 cellscontaining conspicuous cytosomes in the cytoplasm appear at bottom left. Haematoxylin-phloxine. x 430.Fig. 13. Bifurcation of main bronchus into secondary bronchus, showing pseudostratifiedcolumnar epithelium, muscle bundles and lymphoid tissue (centre). Alveolar region cells appearat right. Haematoxylin-phloxine. x 215.

V..oyrian hamster lungs 543


44B....._M1.Fig. 14 (A). Clara cells (C) and goblet cells (G*) can be distinguished in bronchial epitheliumusing electron microscopy. The Clara cell has a round nucleus with condensed chromatinaround the edges, extensive apical agranular endoplasmic reticulum, light secretion granules(above nucleus) and lysosomes appearing at bottom of cell. Goblet cells have dark secretiongranules containing mucus. Intermediate cells can be seen in the'-centre of field. x 5545.

(B). Higher power view of Clara cell appearing in bronchial epithelium shown in Fig. 14 (A).Clara cell light secretion granule (centre top) is distinguished from goblet cell dark secretiongranules (top right). A very dark lysosome is seen at the bottom of the Clara cell and just belowthe mucous granules of the goblet cell. x 9485.

544


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Fig. 15. Bronchiolar epithelium showing Clara cells (left and centre) with nuclei containing con-densed chromatin around the edges, light secretion granules, lysosomes and residual bodies(black) in apical centre region ofeach Clara cell. Ciliated cells appear in centre and right of field.x 3150.Fig. 16. High power view of a goblet cell showing goblet (apical part of cell) filled with dropletsof mucus, a Golgi region (upper right of cell), supranuclear granular endoplasmic reticulum,and nucleus at base of cell. Note that the secretory granules do not coalesce in hamster gobletcells. x 7950.Fig. 17. High power view of bronchiolar Clara cell showing extensive apical agranular endo-plasmic reticulum, a light secretion granule (centre top), numerous mitochondria and a myelinfigure (centre right). x 6300.

ANA 125

545

35

546 ANN R. KENNEDY AND OTHERS

As can be seen in Table 3, a large percentage of cells are in the undetermined categoryin bronchial epithelium owing to the difficulty of ascertaining whether a cell is aClara cell or a goblet cell which has discharged its PAS-positive secretion granules.At the EM level, Clara and goblet cells can be clearly differentiated. The goblet

cell, as seen in Figure 16, has stacks of rough endoplasmic reticulum (ER) in thelower part of the cell, supranuclear Golgi regions, and numerous mitochondria inthe basal and middle portions of the cell. The apical region, or goblet, is filled withdense membrane-bounded mucous droplets and some rough ER. The luminal surfacecontains microvilli. The goblet cells are generally similar to those of other species(Rhodin, 1974; Sorokin, 1970a). However, hamster goblet cells possess secretorygranules that do not coalesce as they do in many species, including man (Rhodin,1974). The Clara cell, a large dome-shaped cell, is a major cell type in both humanand hamster bronchioles (Figs. 15, 17). The basal part of the cell contains numerousmitochondria, some rough and smoothER, and free ribosomes. In many Clara cells,lysosomes can be seen at the base of the cell (Fig. 14). The nucleus is usually round,with finely dispersed chromatin centrally and clumped chromatin around the edges.In the supranuclear region, one or two Golgi complexes are usual. ER, both smoothand rough, and many mitochondria are common in the middle zone of the cell.Occasional light, membrane-bounded secretion granules are present in the middleand apical regions of the cell. The apex of the cell contains extensive agranular ER,a very characteristic feature of Clara cells, often lysosomes and residual bodies, andoccasional mitochondria and clear secretion granules. The luminal margin containsmicrovilli. These ultrastructural characteristics of Clara cells from bronchial epi-thelium are shown in Figure 14.The largest bronchi contain many goblet cells which gradually become fewer as

the airway narrows. In what we designate as a small bronchus, using criteria shownin Tables 6-8, there are occasional goblet cells and many Clara cells among theciliated, intermediate and basal cells.

2. Bronchiolar epitheliumBronchiolar epithelium consists of low columnar ciliated cells, of both the types

already described, as well as Clara cells (Fig. 15). Clara cells tower above the ciliatedcells in the bronchioles, being about twice as high (Table 3). Clara cells begin toappear in the bronchi and increase progressively in number as the size of the airwaydecreases. In medium size bronchioles (02-O4A mm diameter), the proportion ofClara to ciliated cells is about 1:1. By the distal terminal bronchiole, Clara cells arethe exclusive cell type. The ciliated cells decline markedly in numbers along theterminal bronchioles, and almost disappear when the diameter reaches about018 mm, only occasional cells being found beyond that point.

3. Alveolar epitheliumAlveolar epithelium consists of type 1 cells (also called squamous epithelial cells,

typeA cells etc.) and type 2 cells (also called great alveolar, type B cells, large alveolarcells, granular pneumonocytes, etc.). At the light microscope level it is impossible todistinguish type 1 cells from endothelial cells since they are both long and thin withsmall nuclei. At the EM level, they can be differentiated because the basementmembrane separates them. The type 2 cells are characterized by large size, largevesicular nuclei, and vacuolated cytoplasmic inclusions known as multilamellarbodies or cytosomes (Fig. 12). The characteristics of type 2 cells have been discussed

Syrian hamster lungs 547in detail elsewhere (Sorokin, 1966, 1970a, b, 1973). In hamsters there were approxi-mately one third the number of type 2 cells as compared to the sum of type 1 andendothelial cells (Table 3). In rats this also appears to be the case (Bertalanffy &Leblond, 1953). At the EM level, brush cells, originally identified in rat lungs (Mey-rick & Reid, 1968), were also seen in hamster lungs, as were connective tissue cells,which have been described in detail elsewhere (Rhodin, 1974).

Utilizing the plastic section technique, we found about 38 alveolar macrophagesfor each 1000 alveolar cells (Table 3). However, these glycol methacrylate lungsections involve liquid processing techniques which would most likely wash outmany of these free cells from the lung, as occurs with lung washing techniques(Brain, 1970).

Muscle (Table 6)The trachea does not contain a continuous muscularis, but the 0-2-45 mm thick

trachealis muscle forms a transverse layer in the dorsal trachea. The muscularis ofthe bronchi is a closely packed layer of smooth muscle fascicles of 0-02-004 mmthickness which encircle or spiral around the airway (Fig. 20). Gradually, connectivetissue increases between the fascicles and the muscle diminishes. In the medium sizebronchioles (about 0 3 mm diameter), the thickness of the muscularis decreases toa range from a maximum thickness of 10,tm to complete absence. In the terminalbronchioles, the maximum thickness is 5 ,um. In general, as the airways branch, in-creasing amounts of connective tissue separate progressively smaller, predominantlycircular, muscle fascicles (Fig. 21).

Adventitial mast cellsThe adventitia of the hamster airways consists of connective tissue and elastic

fibres; it is always thickest where blood vessels appear in the airways (maximumadventitia thickness in the trachea and main bronchi is 0 12 mm). As can be seenin Table 6, the adventitia gradually gets smaller in size as the airway diameter de-creases, and is non-existent in places in the bronchioles.The medial aspect of the trachial and bronchial (but not bronchiolar) adventitia

has a circular array of cells with strongly PAS-positive granules (Fig. 18) and a small,round nucleus. The cells were identified as mast cells by using a toluidine blue stainon a frozen lung section (Fig. 19). These mast cells in the lung appeared blue in boththe AB pH 1-PAS and AB pH 2-5-PAS staining sequences, indicating an acidicproduct. Mast cells are known to contain acidic sulphated mucopolysaccharides(Spicer, 1965).

Blood vesselsThe distribution pattern of the hamster lung vasculature is similar to that in man.

Branching of the pulmonary arterial and venous systems beyond the small venulesfollows the tracheobronchial tree (Kleinerman, 1972). The branching arterial systemprogresses from elastic to musculo-elastic to muscular arteries, ending in non-muscular precapillary vessels, and capillaries which unite into venules (Kleinerman,1972).Veins greater than 0-06 mm in diameter do not contain smooth muscle, but instead

a layer of cardiac muscle (Fig. 22), identified by the succinic dehydrogenase reactionon a frozen lung. In the smaller veins, the layer of smooth muscle is only one cellthick (Fig. 22B); the layer increases to 3-4 cells thick in the larger veins. Cardiacmuscle is often found in the veins of small mammals (Rhodin, 1974; Sorokin, 1973);but not in man.

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22BFig. 18. Section of a bronchus showing PAS-positive cells in the adventitia. PAS-haema-toxylin. x 410.Fig. 19. The PAS-positive cells shown in the bronchial adventitia in Fig. 18 are identified asmast cells by a toluidine blue stain on a frozen lung filled with O.C.T. embedding medium.A bronchus with a circular array of mast cells in the adventitia can be seen at top. A bloodvessel appears at bottom, and alveolar cells at left and right of photomicrograph. x 103.Fig. 20. Section of a bronchus showing large muscle bundles separated by only a small amountof connective tissue. Iron haematoxylin-Gomori's trichrome. x 205.

Fig. 21. There is a predominantly circular arrangement of muscle fibres, separated by connectivetissue, around bronchioles. An artery (left) appears next to a bronchiole. Haematoxylin-phloxine. x 103.Fig. 22 (A). Single cell layer of cardiac muscle in hamster veins as shown by succinic dehydro-genase reaction on a frozen lung filled with O.C.T. embedding medium. Cardiac muscle is foundin veins larger than 0*06 mm diameter. The smaller vein in the field has a diameter of 0-13 mm.x103.(B). Higher powerviewof Fig. 22 (A) showing single cell layer of cardiac muscle invein. x 410.

548

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Lymphatic systemExcept for large hilar nodes, lymphoid tissue is relatively rare in the hamster

tracheobronchial tree, contrary to the situation in human lungs where lymphoidtissue is abundant. Most hamster lungs examined contained no lymphoid tissueexcept for small nodules very high up in the tracheal region where mucous glandswere common. Some animals did have nodules of lymphoid tissue at bronchialbifurcations, however, particularly at bifurcations of a main bronchus, as shown inFigure 13. The distribution of lymphoid tissue appears to be comparable to thatreported for the mouse (Sorokin & Brain, 1975). Lymphatic vessels, on the otherhand, are common in the hamster. They were seen in the tracheal, bronchial andbronchiolar adventitia, in the connective tissue of the interlobular septa, and rarelyin the very thin pleura of the hamster lung.

DISCUSSION

MorphometryThe branching pattern of hamster airways is like that of many small mammals

(Kliment, Libich & Kaudersova, 1972). Moreover, our classifications are compatiblewith results obtained by Horsfield & Cumming (1968) and Parker et al. (1971) inhuman lungs. They found that human airways larger than 07 mm diameter exhibiteda markedly asymmetrical dichotomy when the numbering order increased towardthe carina. Smaller airways (terminal bronchioles and beyond) showed regulardichotomous branching. Sorokin (1973) in humanand caninelungsreported that totalbronchial cross sectional area increased geometrically as a function of distancefrom the carina and then declined. In the hamster, cross sectional area increasesinitially as a function of generation, then declines. It is worthy of note that the regionventilated by a terminal bronchiole is considerably shorter in the hamster lung(07-1 mm) than in the human lung (2-5 mm): this is primarily due to the presenceof more generations of respiratory bronchioles and alveolar ducts in the human lung.

Respiratory bronchioles are shaped differently in hamster and man. In humanlungs, long stretches of bronchiolar epithelial cells are interrupted by occasionalalveolar outpocketings. In hamsters, the respiratory bronchiole consists of veryshort stretches of bronchiolar epithelium, usually just a few cells, with extensivealveolar outpocketings. The few bronchiolar type cells at the entrance to an alveolusfrom a respiratory bronchiole were not appreciably greater in number than thenumber of alveolar cells in the knob-like swellings at the entrance to each alveolusin more peripheral areas of the lung (Rhodin, 1974). Since these bronchiolar typecells would be receiving similar doses as alveolar cells from exposures to irritantsand carcinogens reaching the alveolar region, we have considered them as part of thetotal number of alveolar cells for the purposes of investigating the action of carcino-gens, even though they are not histologically alveolar type cells.

Altogether, these data produce a pattern of lung morphometry for the hamsterlung which contrasts somewhat with lung models used in particle deposition esti-mates for human lungs (Nelson et al. 1969). Principally, this difference resides in thedeclining surface area of peripheral airways encountered in the hamster lung. Thedecreases in length, diameter and number of airways combines to decrease totalsurface area in regions distal to the fifth generation. Models for the human lunghave been developed and used to estimate deposition (Taulbee & Yu, 1975) and

549


clearance (Altschuler, Nelson & Kuschner, 1964; Harley & Pasternack, 1972)wherein the number and surface area always increase as a function of generation.Therefore, it is important to remember, that species differences need to be consideredwhen extrapolating data about the deposition, retention and clearance of inhaled orintubated materials from hamsters to other species, and especially to man.

HistologyIn relation to human lung carcinogenesis, it is important to note that in its hist-

ological features the hamster trachea most closely resembles the human bronchus(Kendrick, Nettesheim & Hammons, 1974), the site of most human lung cancer.The hamster trachea is made up of cartilage, glands, lymph nodes and nodules, andan epithelium containing goblet, ciliated, basal, intermediate and brush cells, all ofwhich are characteristic of the human bronchial region. Unlike the human bronchus,the hamster tracheal epithelium contains few neurosecretory cells. The height of thisepithelium is approximately 20 ,um, as compared with 30-50 ,um in man (Rhodin,1974; Gastineau, Walsh & Underwood, 1972). Tracheobronchial epithelium isnormally lower in height in small than in large mammals (Sorokin, 1970a).We have observed a slightly lower proportion of mucous cells in hamster tracheal

epithelium than that reported by Kaufman et al. (1972). Many factors influence theproportions of cell types in various regions of the tracheobronchial tree, includingage, sex, vitamin A status and environmental conditions. For example, there is adecrease in the proportion of ciliated cells and an increase in basal cells on a vitaminA-deficient diet (Boren et al. 1974). In our study young male hamsters were fed dietscontaining adequate amounts of vitamin A.

There are no published reports of cell type frequencies in the bronchial andbronchiolar regions of hamster lungs. As we have shown, Clara cells can be foundin hamster bronchial epithelium: the frequency is difficult to determine at the lightmicroscopic level due to the similarity between Clara cells and certain goblet cells.A cell with many PAS-positive mucous granules is clearly a goblet cell (Fig. 10).However, a goblet cell which has discharged its secretion granules has lost its identityand appears as just another brush cell in the epithelium (Sorokin, 1973). A cell withjust a few PAS-positive areas may be a goblet cell with some mucous granules, or aClara cell with PAS-positive lysosomes in the apical region (Fig. 15). The occasionalPAS-positive lysosomes have no doubt caused a few investigators to conclude thatClara cells produce mucus (Cutz & Conen, 1971; Fredricson, 1956).The Clara cell is of particular interest because preliminary studies suggest it may

be the cell of origin of peripheral lung tumours induced by 210Po alpha radiation(Lisco, Kennedy & Little, 1974). The Clara cell incorporates and retains a significantfraction of intratracheally administered 210po, as shown by autoradiography (Kennedy& Little, 1974b). It is also considered to be the cell of origin of human bronchiolo-alveolar carcinomas (Kuhn, 1972). We have found that the Clara cell in hamstersis present in both bronchial and bronchiolar epithelium, whereas in man it occursonly in the bronchioles. Between different species, both the distribution and thehistological characteristics of the Clara cell vary. Human (Bloom & Fawcett, 1968),but not hamster, Clara cells contain particulate glycogen. Myelin figures, frequentlyseen in hamster Clara cells, have not been described in other species. Hamster andhuman mitochondria are basically similar, but the ultrastructural morphology ofClara cell mitochondria varies among rodents as well as other mammals (Sorokin,1970a). Hamster Clara cells contain very few secretion granules (Fig. 17) and have a

550

Syrian hamster lungs 551minimal lipid content (unpublished data) as compared with human Clara cells(Cutz & Conen, 1971). These granules may contain a lipoprotein, rich in choline-based phospholipids, secretion (Azzopardi & Thurlbeck, 1969).The Clara cell secretion is the major component of the distal 'mucous ciliary

escalator' (Kilburn, 1967) involved in the removal of foreign particles and carcino-gens from the lung. Mucus of the proximal mucous ciliary escalator is produced byglands and goblet cells. In the hamster, tracheal glands produce a neutral muco-polysaccharide. The human lung contains mixed glands, which produce a neutralmucopolysaccharide and acidic mucosubstances (McCarthy & Reid, 1964b); similarglands are found in rat and mouse lungs (McCarthy & Reid, 1964a). Goblet cells inthe hamster trachea and bronchi produce most of the secretions for the proximalmucous ciliary escalator. Goblet cells in both man (McCarthy & Reid, 1964b), rats(McCarthy & Reid, 1964a) and hamsters produce neutral mucopolysaccharides, andneuraminidase sensitive and resistant sialomucins, while the goblet cells in mice(McCarthy & Reid, 1964a) only produce neuraminidase sensitive sialomucins.Sulphomucins, common in human (McCarthy & Reid, 1974b) and rat (McCarthy &Reid, 1974a) goblet cells, are essentially absent in hamster and mouse (McCarthy &Reid, 1974a) goblet cells. Such species variations in the nature of these secretionscould influence the efficacy of the mucous ciliary escalator, and in turn, alter theincidence and locations of tumours.

Species variation in cell types may also affect tumour incidence. The neuro-secretory cell, of human tracheal, bronchial and bronchiolar epithelium (Rhodin,1974; Bensch, Gordon & Miller, 1965; Gmelich, Bensch, & Liebow, 1967) has beenproposed as the cell of origin of carcinoid tumours (Gmelich et al. 1967) and oatcell tumours (Spencer, 1968). The rarity of neurosecretory cells in hamster epitheliamay explain why hamsters infrequently get carcinoid tumours, and oat cell tumoursare exceedingly rare (personal communication, Dr Curtis C. Harris).

Perhaps some of the known species differences in pulmonary susceptibility tocarcinogens and infections can be explained by differences in airway structure,distribution of lymphoid tissue and glands, mucous secretions and epithelial cells,as have been discussed.

SUMMARY

Hamster lung morphometry and histology have been studied in an attempt todetermine differences between hamster and human lungs which may have relevancefor lung carcinogenesis studies. Morphometric measurements were made on freshlungs, lung casts, and histological sections. Cell type and frequency measurementswere determined from frozen, paraffin,1 um plastic (glycol methacrylate) and electronmicroscopic sections. A standard terminology for hamster lung histology is estab-lished, and differences between hamster and human lung morphometry and histologyare discussed.

This research was supported by Contract CP-33273 and Training Grant CA-09078from the National Cancer Institute, Grant DB-37c from the American CancerSociety, and Center Grant ES-00002 from the National Institute of EnvironmentalHealth Sciences. We would like to thank Mr Frank Bettinelli for his expert assistancein the preparation of histological materials, and Dr Curtis C. Harris and Dr DavidG. Kaufman for help in the preparation of this manuscript.

552 ANN R. KENNEDY AND OTHERS

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AzZOPARDI, A., & THURLBECK, W. M. (1969). The histochemistry of the nonciliated bronchiolar epi-thelial cell. American Review of Respiratory Diseases 99, 516-525.

BARKA, T. & ANDERSON, P. J. (1963). Histochemistry Theory, Practice and Bibliography, p. 313. NewYork: Hoeber Medical Division, Harper & Row Publishers, Inc.

BENSCH, K. G., GORDON, G. B. & MILLER, R. L. (1965). Studies on the bronchial counterpart of theKultschitzky (argentaffin) cell and innervation of the bronchial glands. Journal of UltrastructureResearch 12, 668-686.

BERTALANFFY, F. D. & LEBLOND, C. P. (1953). The continuous renewal of the two types of alveolar cellsin the lung of the rat. Anatomical Record 115, 515.

BLOOM, W. & FAWCETT, S. W. (1968). A Textbook of Histology, 9th edition. Philadelphia: W. B.Saunders Co.

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