Morphometric and histological analysis of the lungs of Syrian golden hamsters
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J. Anat. (1978), 125, 3, pp. 527-553 527
With 22 figures
Printed in Great Britain
Morphometric and histological analysis of the lungs of
Syrian golden hamsters
ANN R. KENNEDY, ARTHUR DESROSIERS, MARGARET
TERZAGHI* 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 studies
since 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 the
human lung; however, there are substantial morphometric and histological differ-
ences which suggest that regional exposures and reactions to carcinogens may differ
in 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 between
small mammals. There is currently, however, no comprehensive analysis of either
the hamster lung or the lungs of other small mammals. The anatomy of the hamster
lung has been discussed by Magalhaes (1968) and Schwarze & Michel (1959-60),
and occasional specific characteristics have been described, such as the distribution
of glands (Kleinerman, 1972), the pattern of vasculature (Kleinerman, 1972), the
frequency 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). No
morphometry data on the hamster lung have been available; however, such a study
has 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 ,um
plastic (glycol methacrylate) and electron microscopic sections. These data were
correlated with the frequencies and sizes of airways in the lung, using measurements
from histological sections, fresh lungs and casts of hamster lungs. A standard
terminology for hamster lung histology is presented, and comparisons are made
between hamster and human lung morphometric and histological data.
* Current address:
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee.528 ANN R. KENNEDY AND OTHERS
MATERIALS AND METHODS
Animals
Adult male (approximately 125 g), random bred Syrian golden hamsters were ob-
tained from Dennen Animal Industries, Gloucester, Mass. They were fed Purina chow.
Morphometry
A 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 of
lungs. Lung casts (Fig. 2) were made with Medical Silastic 382 Elastomer (Dow
Corning) which, when mixed with Catalyst M, becomes a silicone rubber. The
techniques employed were described by Frank & Yoder (1966). Lungs were fixed
at approximately 90 % of maximum inflation. Peripheral airways had clearly been
completely filled with silicone rubber, because the respiratory portion of the lung
(respiratory bronchioles to the alveolar sacs) could be seen extending from the
terminal bronchioles in the casts (Fig. 2). The generation number as well as the
lengths and diameters of airways (secondary bronchi to terminal bronchioles)
within lobes of the lungs were determined from casts by dissection with the aid of a
dissecting microscope. An airway was taken to be a successive generation from a
parent airway only if it noticeably decreased in size (diameter) as compared with the
parent 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 measurements
made on successive generations in lung casts would be comparable to those of fresh
lungs at the same degree of inflation. Diameter measurements of the large airways
made on fresh lungs were also comparable to the measurements made on histological
sections of five lungs embedded in plastic (glycol methacrylate). We have carried out
two studies to determine the effects of fixation and embedding on lung structures
using our plastic section technique, since several investigators have reported that the
dimensions of lung structures alter during the preparation of lungs for paraffin
sections (Dunhill, 1962; Matsuba & Thurlbeck, 1971; Weibel, 1963b). The effect
of 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 from
hamsters in the inflated state and water displacement measured; these lungs were
then collapsed by evacuation at - 740 mmHg for 10 minutes, and then fixed with
glutaraldehyde, as has been previously described (Kennedy & Little, 1974a).The
water displacement of these fixed lungs was found to be the same as for the fresh
inflated lungs in each case. The effect of embedding and sectioning in our system
was determined by cutting eight 0 9 x 0-9 cm blocks from one fixed lung and then
measuring the blocks after embedding and sectioning. The linear shrinkage due to
embedding 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 fixation
and sectioning. Our observed linear shrinkage factor has not been incorporated into
the 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 lung
structures than does conventional paraffin embedding procedures, in which shrinkage
factors for airway dimensions are significant (Dunhill, 1962; Matsuba & Thurlbeck,
1971; Weibel, 1963 b).o'-y-rian hamster lungs 529 Since airway diameter is one of the characteristics we used to distinguish between bronchi and bronchioles microscopically (Tables 7-8), the diameters of the airways in the casts were used to determine the numbers of bronchi and bronchioles in the various 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, but with only one dimension, length or diameter, in the 0-1-0-2 mm range. If an airway had 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. Since we were primarily interested in frequencies and generations of the various sized airways, a complete dissection as far as the terminal bronchioles was carried out on only one cast. Lengths and diameters of airways in the respiratory portion of the lungs (from the respiratory bronchioles to the alveolar sacs) were determined from histological sections of glycol methacrylate embedded lungs containing longitudinal sections from the terminal bronchiole to the alveolar sacs (Figs. 3-4). Measurements of a given airway diameter in the respiratory portion of the lungs were made from the knob-like swellings at the entrance to the alveoli (Rhodin, 1974) on either side of the airway - 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. Histology Procedures 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 of each major bronchus. Within each lobe, three to five sections (from five separate animals) were cut in a longitudinal or in a crosswise fashion and the orientation in the lobe recorded. The cell types and frequencies present in the various airways of the lung were determined from both glycol methacrylate embedded lungs for light microscopic analysis and lungs embedded in Epon for electron microscopic (EM) study. The procedures for preparation of lungs for EM sections involved fixation of whole 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-fixed for 1 hour in 1 3 % OSO4 in 0 1 M s-collidine buffer at pH 7-4. Lungs were embedded in 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 lead citrate and uranyl acetate, and examined with a Philips 300 electron microscope. Frozen sections for histochemical studies were prepared from lungs embedded in O.C.T. compound (a mixture of water soluble glycols and resins obtained from Fisher Scientific Co.) frozen by immersion in methyl butane cooled to the viscous stage by liquid nitrogen, and cut in a microtome-cryostat (Harris Mfg. Co.) at 4 ,um. The method of Chayen et al. (1969) was used for toluidine blue staining, and succinic dehydrogenase activity was determined by the method of Nachlas et al. modified by Barka & Anderson (1963). 34 A NA 125
530 ANN R. KENNEDY AND OTHERS
Paraffin sections of alcoholic-zinc formalin-fixed normal lungs (two animals) were
used 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 neuraminidase
digestion (Luna, 1968).
RESULTS
Morphometry
Gross anatomy and airway branching patterns
The 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, namely
an apical, middle, diaphragmatic or caudal, and an infracardiac lobe extending bet-
ween the heart and the diaphragm (Fig. 1). The anatomical arrangements of the
lobar bronchi, and the ranges and average dimensions for the trachea and main
bronchi (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 larger
angle with the parent tube. Unequal dichotomous branching is demonstrated in the
lung cast shown in Figure 2: the left main bronchus is smaller than the right, and the
right lung is more of a direct extension of the trachea. After the standard branching
of the main bronchi into secondary bronchi the branching is usually dichotomous
(Figs. 3-4) and occasionally trichotomous.
Airway sizes
Representative dimensions of airways determined from fresh lungs, casts and histo-
logical sections are given in Tables 1-3 and compared with sizes of human airways
in Table 7. Smaller diameters for the hamster trachea have been reported by
Schwarze & Michel (1959-60) (1-5-1-6 mm) and a larger diameter is reported by
Raabe et al. (1976a) (2-6 mm), but our results of 2 mm were quite constant. Figure 5
shows the relative frequencies of large (bronchi and bronchioles) and small (terminal
bronchioles) airways obtained from casts. Figure 6 demonstrates the variation of
airway diameter and length as a function of generation for the large airways. Within
any 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. Six
generations of bronchi and six genertions of bronchioles were classified in the
hamster.
Terminal bronchioles were found in seven generations (4-10) of airways. The size
of 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 length
of the respiratory bronchioles varied, with a range of 0-1-0-5 mm (average length of
0-23 mm). One (Fig. 3) or two (Fig. 4) generations of alveolar ducts appeared fre-
quently in longitudinal sections. Diameter and length were approximately the same
for both generations of alveolar ducts (Table 3). In the lung casts, the usual length
of the respiratory portion extending from a terminal bronchiole (acinus) was between
0'7 and 1 mm.Syrian hamster lungs 531
b
I
I
3< 4 , j : ,* , + s .
-f4.
Fig. 1. Schematic representation of the hamster lung. a, larynx; b, trachea near fused middle
rings; c, carcina; d, single lobe on left; e, right apical lobe;f, right middle lobe; g, diaphragmatic
or 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, one
generation of an alveolar duct and two alveolar sacs. x 43.
Fig. 4. Branching pattern from a terminal bronchiole showing a respiratory bronchiole, two
generations of alveolar ducts and an alveolar sac (terminating at the bronchiole from which the
acinus originated). x 43.
34-2532 ANN R. KENNEDY AND OTHERS
Table 1. Dimensions of trachea and main bronchi determined from
five 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 05
cartilage), Genera-
tion 1
Main bronchi,
Generation 2
(extralobular)
Left lung 5-6 564+±019 610±0-29 194-126 125±0 03 1-7+0-08
Right lung 6-8 6-6+±040 1-9-2-0 2*0+0-02
Location of secondary bronchi: distance along main bronchi at which secondary bronchial
bifurcations occur
Range Average ± S.E.
(mm) (mm)
Left 5-6 5*4+±*19
Right
Apical 1-5-2 1*9+0-09
Middle 4-5 4-4±0-24
Infracardiac 5-7 6-0+044
Diaphragmatic 6-8 6-6+0 40
1000 r-
1001-
.0
E
a)
z 10p
1
2 4 6 8 10
Airway generation
Fig. 5. Numbers of bronchi (0), bronchioles (A), and terminal bronchioles (O) in the hamster
respiratory tract as a function of airway generation.Syrian hamster lungs 533
100l
K
101
E
1-
0)
.N
cn
1 _
I
2 4 6 8 10
Airway generation
Fig. 6. The variation of airway length (A) and diameter (0) versus airway generation.
2-
E
E
0D E~~ *
to
0
0 1 2 3 4 5 6
Length (mm)
Fig. 7. The variation of airway diameter versus airway length for 25 fourth generation airways
of the hamster lung.534 ANN R. KENNEDY AND OTHERS
Table 2. Pulmonary measurements determined from a lung cast
(Frequencies and generations of airways by lobe.)
Number of airways (generations in parentheses)
Terminal
Bronchi Bronchioles bronchioles
Lobe: Left 18 (2-7) 44 (4-9) 156 (4-9)
Right
Apical 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 histological
definition described in text)
Length Diameter
r 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 04
Bronchioles 0-1-40 1-39+0-05 0 1-Syrian hamster lungs 535
Table 3. Measurements of airways determined from histological sections
(A). Airway dimensions (seven animals: number of units counted in parentheses)
Larger airway diameters
Range (mm) Average± S.E. (mm)
Trachea 1-9 -2-1 2-0 +0-04(7)
Bronchi 05 -2-0 0 70+0 06 (25)
Bronchioles 010-536 ANN R. KENNEDY AND OTHERS total number of ducts and sacs in the alveolar region is counted. Ducts and sacs may be distinguished from respiratory bronchioles in the hamster since the latter are lined with bronchiolar epithelial cells. Alveoli are distinguished from ducts and sacs by their 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 sacs per field. When divided by the field area, this yielded n = 426 ± 31 transsections per cm2. These same fields were also measured in order to obtain the proportion of parenchymal volume occupied by ducts, sacs, and alveoli. Although the method outlined by Weibel uses an integrating stage micrometer, the present study was per- formed by photographing microscopic fields. Enlargements were printed in full format and a machinist's micrometer was employed to sum the contributions of small blood vessels, tissue septa and respiratory space to the lung parenchyma. The average proportion of parenchymal volume devoted to alveoli, ducts and sacs was found to be 0-863 ± 0-023. Based on Weibel's measurements of human lungs, the parenchyma was estimated to comprise 90 % of the hamster lung by volume, and the ducts and sacs were estimated to comprise 32 % of the parenchymal air exchange space. The volume of fixed lungs was 2 5 cm3. Hence the proportion of parenchymal volume consumed by ducts and sacs, p, is 0-863 x 0-32 = 0 275 and the parenchymal volume is 0 90 x 2-5 = 2 25 cm3. Also from Weibel (1963 a), the shape factor, b, is taken 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 the stated 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 human data and because the error stated, a lower bound of the true error, is nevertheless of an order which makes it possible to conclude that agreement exists between the morphometrical approach of Weibel and the cast dissection technique employed here. Since the Weibel method does employ parameters which are more difficult to evaluate and verify, the semi-empirical results obtained directly from an intact cast were employed for all subsequent calculations. Number of epithelial cells in the various regions of the lung Using 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 4um of airway epithelial length into a surface density. For example, Table 3 indicates that 26-9 cells per 150 #rm were observed in hamster bronchi. Thus the average length of a cell is 1 50126-9 = 5 58 ,um. If this is also taken to be the mean cell width, then a typical cell has an area of (5 58 ,um)2 = 31 -1 /ZMm2. This is equivalent to an area density of 3-2 x 106 cells per cm2. Similar calculations were performed for the trachea, major bronchi, 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 then obtained by summing the numbers of cells from all airways of that region. Results are 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. Although alveoli are not spherical (Weibel & Gomez, 1962), a closed sphere was considered to be approximately equal in surface area to an alveolus of equivalent width. The larger surface-to-volume ratio of the alveolus compensates to a considerable extent for
Syrian hamster lungs 537
Table 4. Results of staining reactions
Staining reactions of histologic structures
Stain 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 and
mucosubstances containing hexoses or bronchial goblet cells bronchial epithelium and glands
deoxyhexoses with vicinal glycol groups Cilia Cartilage - interlacunar matrix
stain red - includes neutral mucosub- Cartilage - lacunar rims, Most glandular goblet cells
stances. Hyaluronic acid, sialomucins chondrocytes and peri-
and all but the most strongly acidic chondrium
sulphated mucosubstances stain blue Mast cells in tracheal and
(Luna, 1968) bronchial adventitia
An occasional cell in a
tracheal gland
AB pH 1-PAS. PAS staining (red) A few goblet cells high in Most goblet cells in tracheal
same as in above sequence. However, the trachea near the epithelium
alcinophilic (blue) substances at this larynx All bronchial goblet cells
pH include only the sulphated An occasional cell in a Cartilage - interlacunar matrix
mucosaccharides (Luna, 1968) tracheal gland Most glandular goblet cells
Cartilage - lacunar rims,
chondrocytes and peri-
chondrium
Mast cells in tracheal and
bronchial adventitia
AB pH 0 4. Only strongly acidic Very few positive cells
sulphated 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 tracheal
gland
Table 5. Total number of epithelial cells in conductive or air exchange
spaces as measured from histological sections
Region Cells x 106
Trachea 7-3
Main bronchi 4-1
Bronchi 9-3
Bronchioles 6-0
Terminal bronchioles 1-0
Alveoli 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, the
hamster's alveolar size may be obtained from the relationship:
DR-(125)0,2 (270)-76sm
(70 000)0 2
where 270 ,am is the average alveolar diameter of a 70 kg human subject. This result
compares 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 hamster538 ANN R. KENNEDY AND OTHERS
Table 6. The hamster respiratory system (range of measurements
taken from seven animals)
Muscularis Adventitia Cartilage Mast cells
Airway thickness (mm) thickness (mm) thickness (mm) (adventitia)
Trachea Muscularis not If cartilage present, 0-02-020 x
present 0-02-0-12; if no
Trachealis muscle, cartilage, 0 03-006
0 02-0)05
Main bronchus 0-005 (in front of 0-03-012 (thickest 0 01-0410 x
cartilage) - 0-02 where blood vessels
occur in all airways)
Bronchus 0 02-004 0 03-0-06 - x
Bronchiole 0-0 01 0-005
Terminal 0-0-005 0-005 - --
bronchiole
Respiratory Occasional single
bronchiole muscle fibre
Alveolus Rarely a single
muscle fibre
would have an air exchange surface of 3-2 x 1011 #am2. This implies that there are
1 -8 x 107 alveoli. By comparison, Granito (1971) reported 3 0 x 107 alveoli in the
rat, 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 alveolar
perimeters 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 1011
cIm2/lung ÷270 gMm2/cell = 1 2 x 109 cells/lung. We recognize that the assumptions
employed in formulating these estimates preclude anything but rough accuracy.
Standard errors were not calculated for these estimates because systematic sources of
error are expected to be dominant.
Histology
Histological 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 compares
histological features in hamster and human lungs, and Table 8 summarizes the clearest
distinguishing 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 not
fuse ventrally where the right segment gives rise to a semicircular appendage which
courses in a sagittal plane below and behind the carina. The central tracheal rings
are 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 extend
along the entire extrapulmonary course of the main bronchus and thereafter about
1 mm past the point at which the first secondary bronchus branches off the main
bronchus. Since the main bronchus does not appear grossly different after this
branching, 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-shaped
rings or large cartilaginous plates usually extended along the entire extrapulmonarySyrian hamster lungs 539
Table 7. Comparison of human and hamster respiratory systems
Respiratory
Airway Trachea Bronchus Bronchiole bronchiole Alveoli
(A) Human respiratory system
Diameter 20-25 mm (Bloom > 1-0 mm (Soro- 0-5-1-0 mm (Rho- _ 0-5 mm (Bloom 270 /tm (Weibel &
& Fawcett, 1968) kin, 1973) din, 1974) & Fawcett, 1968) Gomez, 1962)
Epithelium Ciliated pseudo- Same as trachea. Ciliated simple Low columnar to Types 1 and 2
stratified colum- Height, 30-50,m columnar. Goblet cuboidal. Many cells (Rhodin,
nar with goblet (Rhodin, 1974; cells become in- Clara cells, few 1974)
cells. No Clara Gastineau et al. frequent and drop ciliated cells.
cells (Rhodin, 1972) out; Clara cells Ciliation drops
1974) appear (Rhodin, out along this
1974) airway (Bloom
& Fawcett, 1968)
Lamina Prominent Thin (diffuse Thin: loose
propria (elastic lamina) network) (Soro- connective tissue
(Sorokin, 1973) kin, 1973) (Rhodin, 1974)
Muscularis Not prominent. Prominent muscu- Prominent muscu- Layer of smooth Narrow bundles
Only trachealis laris. Muscle laris: muscle muscle cells is of smooth muscle
muscle (Rhodin, fascicles: con- fascicles, circular thin and incom- cells encircle the
1974) tinuous circular and spiral. Sep- plete (Rhodin, entrance to each
or spiral (Soro- arated by con- 1974) alveolus of the
kin, 1973) nective tissue alveolar duct
(Sorokin, 1973) (Rhodin, 1974)
Submucosa Mucous and sero- Mucous and sero- No glands (Soro-
mucous glands mucous glands kin, 1973)
(Sorokin, 1973) (Sorokin, 1973)
Adventitia About 20 C- Cartilage plates. Connective tissue, Collagenous con-
shaped cartilage Lymph nodes, elastic fibres, nective tissue con-
rings. Lymph lymphatics. Bun- blood vessels, taining elastic
nodes (Rhodin dles of collagen- lymphatics (Bloofn fibres (Bloom &
1974) ous and elastic & Fawcett, 1968) Fawcett, 1968)
fibres. Blood
vessels, nerve
bundles (Rhodin,
1974)
(B) Hamster respiratory system
Diameter Approximately 0-5-2-0 mm 0-1- < 0-5 mm 0-05-0-2 mm Approximately
2-0mm 76,cm
Epithelium Ciliated pseudo- Ciliated low Ciliated low Clara cells Types 1 and 2
stratified columnar. columnar. Clara
columnar in Many goblet and ciliated
places to low cells, some cells. Cilia
columnar. Many Clara cells drop out in
goblet cells. Height, 20,m the terminal
No Clara cells. bronchiole.
Height, 20,m Height: Clara cells,
20,tm; ciliated cells,
10,tm
Lamina Prominent Very thin
propria
Muscularis Only trachealis Muscle fascicles: Loose network: Rare single
muscle continuous, primarily circular muscle fibres
primarily circular muscle fascicles.
Submucosa Mucous glands No glands No glands --
Adventitia C-shaped Cartilage plates Connective tissue - --
cartilage rings. drop out as the elastic,
Mast cells. airways enter reticular fibres.
Lymph nodes, the lung Blood vessels;
nodules; Mast cells. Lymphatics
lymphatics Lymphatics;
Rare lymph
nodules540 ANN R. KENNEDY AND OTHERS
Table 8. Major differences among airways in the hamster
Airway Diameter Distinguishing characteristics Differences from parent branch
Trachea Approx. 2-0 mm C-shaped cartilage rings surround
airway except dorsally where
trachea contacts oesophagus
Variation from ciliated pseudo-
statified columnar to low
columnar epithelium with
goblet cells
Mast cells in adventitia
Glands primarily near larynx
-
Lymphatics and lymphoid
tissue in adventitia
Cartilage no longer surrounds
airway; plates and fragments
Main Approx. 1-8 mm Cartilage plates and fragments are more common than rings
bronchus as well as a few rings No glands
Epithelium same as trachea
Mast cells in adventitia
Lymphatics and lymphoid tissue
in adventitia
Cartilage drops out
Muscularis becomes prominent
Bronchus 0-5-2-0 mm No cartilage Appearance of Clara cells
Low pseudostratified ciliated
columnar epithelium (low
columnar in places) with
many goblet cells, some
Clara cells
Fascicles of muscle are closely
packed into a continuous
muscularis
Lymphatics in adventitia; rare
lymphoid tissue in adventitia
near bifurcations
Mast cells in adventitia
Goblet cells drop out
Clara cells become prominent
Bronchiole 0 10- < 0 5 mm No cartilage Fascicles of muscle become
Epithelium: low columnar separated by connective tissue
ciliated and Clara cells Mast cells drop out (in adventitia)
Muscularis: fascicles of muscle
separated by connective tissue
No mast cells in adventitia
Ciliation drops out in terminal
Terminal 0O1-0 2 mm Epithelium: ciliated cells sparse; bronchiole
bronchiole they drop out at approximately
0oc'-y-rian hamster lungs 541
course of the right main bronchus. However, cartilage occasionally stopped around
the origin of the middle lobe secondary bronchus, and sometimes extended into the
pulmonary portion of the diaphragmatic lobe. Apart from the C-shaped rings or
large cartilaginous plates, traces of cartilage could occasionally be seen in large
bronchi under the light microscope (Fig. 10).
Mucous secreting apparatus: distribution and nature of mucous secretions
The 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 very
rare in the rest of the tracheobronchial tree. This paucity of glands in the hamster
lung 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 airway
decreases.
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-PAS
sequences indicate that primarily a neutral mucopolysaccharide is produced by
hamster goblet cells in the airway epithelium and glands. An occasional glandular
cell, and a very few epithelial goblet cells, in the trachea near the larynx contained
acidic mucosubstances (Table 4) which were shown to be strongly acidic sulphated
mucosubstances 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 contained
sialomucins which are also acidic mucosubstances (Table 4). With prior digestion by
neuraminidase, which acts on the glycoside link through which sialic acid is attached
to 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 and
bronchial goblet cells at bifurcations were insensitive to neuraminidase, while those
between bronchial branch points were sensitive to the enzyme.
Airway epithelium
Cell 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 for
each 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. Cell
type frequencies were determined by studying bronchi of diameter 0 6-0-7 mm and
bronchioles of diameter 0 3-0 4 mm (Tables 2, 3) in histological sections prepared
from 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 be
no 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 the
same cell types in the same proportions regardless of generation or position in the
lung; the same was true for bronchioles.
In general, the cell types encountered at the electron microscopic (EM) level
appeared to be similar to those found in the human lung. However, neurosecretory542 ANN R. KENNEDY AND OTHERS
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cells were rarely found among hamster epithelial cells. Neurosecretory cells are
common in human bronchial, bronchiolar and tracheal epithelium (Rhodin, 1974).
The cell types and frequencies are given by regions as follows:
1. Tracheobronchial epithelium
Hamster tracheal and bronchial epithelium varies from low pseudostratified
(Fig. 10) to low columnar ciliated. The height of the epithelium is approximately
20,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 bronchus
in Figure 12. The majority of ciliated cells have a compact round nucleus which
contains fine granular chromatin and which is located near the base of the cell; the
other 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 %, and
basal cells 20 %. The remaining 5 % of cell types are intermediate or brush cells -
labelled as 'undetermined' in this study. The ultrastructural morphology of the cell
types 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 the
main bronchi is like that in the trachea. Clara cells begin to appear in the secondary
bronchi of the hamster lung: they are seen in bronchial epithelium at the light
microscope 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. These
glands are strongly PAS-positive and secrete mucus consisting of neutral mucopolysaccharide.
Cartilage appears at bottom of photomicrograph, tracheal epithelium at top. PAS-haematoxylin.
x 43.
Fig. 9. Low power view of two similar bronchi showing very irregular distribution of goblet cells
in 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 goblet
cells (with dark granules of PAS-positive mucus), ciliated, basal and intermediate cells. A
fragment 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-shaped
cells having nuclei containing condensed chromatin around the edges (left and right of field) and
goblet cells, containing dark granules of PAS-positive mucus (centre and far right). Cells of the
alveolar 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), but
some ciliated cells have an elongated nucleus parallel to the basement membrane and a long
ciliated border (right of field). Alveolar cells appear at bottom of photomicrograph: type 2 cells
containing conspicuous cytosomes in the cytoplasm appear at bottom left. Haematoxylin-
phloxine. x 430.
Fig. 13. Bifurcation of main bronchus into secondary bronchus, showing pseudostratified
columnar epithelium, muscle bundles and lymphoid tissue (centre). Alveolar region cells appear
at right. Haematoxylin-phloxine. x 215.544 ANN R. KENNEDY AND OTHERS
....._M1.
44
B
Fig. 14 (A). Clara cells (C) and goblet cells (G*) can be distinguished in bronchial epithelium
using electron microscopy. The Clara cell has a round nucleus with condensed chromatin
around the edges, extensive apical agranular endoplasmic reticulum, light secretion granules
(above nucleus) and lysosomes appearing at bottom of cell. Goblet cells have dark secretion
granules 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 secretion
granules (top right). A very dark lysosome is seen at the bottom of the Clara cell and just below
the mucous granules of the goblet cell. x 9485.Syrian hamster lungs 545
...
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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 of each 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 droplets
of 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 goblet
cells. 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 myelin
figure (centre right). x 6300.
35 ANA 125546 ANN R. KENNEDY AND OTHERS As can be seen in Table 3, a large percentage of cells are in the undetermined category in bronchial epithelium owing to the difficulty of ascertaining whether a cell is a Clara 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 the lower part of the cell, supranuclear Golgi regions, and numerous mitochondria in the basal and middle portions of the cell. The apical region, or goblet, is filled with dense membrane-bounded mucous droplets and some rough ER. The luminal surface contains microvilli. The goblet cells are generally similar to those of other species (Rhodin, 1974; Sorokin, 1970a). However, hamster goblet cells possess secretory granules 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 human and hamster bronchioles (Figs. 15, 17). The basal part of the cell contains numerous mitochondria, 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 smooth and rough, and many mitochondria are common in the middle zone of the cell. Occasional light, membrane-bounded secretion granules are present in the middle and 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, and occasional mitochondria and clear secretion granules. The luminal margin contains microvilli. 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 shown in Tables 6-8, there are occasional goblet cells and many Clara cells among the ciliated, intermediate and basal cells. 2. Bronchiolar epithelium Bronchiolar 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 ciliated cells in the bronchioles, being about twice as high (Table 3). Clara cells begin to appear in the bronchi and increase progressively in number as the size of the airway decreases. In medium size bronchioles (02-O4A mm diameter), the proportion of Clara to ciliated cells is about 1:1. By the distal terminal bronchiole, Clara cells are the exclusive cell type. The ciliated cells decline markedly in numbers along the terminal bronchioles, and almost disappear when the diameter reaches about 018 mm, only occasional cells being found beyond that point. 3. Alveolar epithelium Alveolar epithelium consists of type 1 cells (also called squamous epithelial cells, type A cells etc.) and type 2 cells (also called great alveolar, type B cells, large alveolar cells, granular pneumonocytes, etc.). At the light microscope level it is impossible to distinguish type 1 cells from endothelial cells since they are both long and thin with small nuclei. At the EM level, they can be differentiated because the basement membrane separates them. The type 2 cells are characterized by large size, large vesicular nuclei, and vacuolated cytoplasmic inclusions known as multilamellar bodies or cytosomes (Fig. 12). The characteristics of type 2 cells have been discussed
Syrian hamster lungs 547
in 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 and
endothelial 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 macrophages
for each 1000 alveolar cells (Table 3). However, these glycol methacrylate lung
sections involve liquid processing techniques which would most likely wash out
many 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 of
the bronchi is a closely packed layer of smooth muscle fascicles of 0-02-004 mm
thickness which encircle or spiral around the airway (Fig. 20). Gradually, connective
tissue increases between the fascicles and the muscle diminishes. In the medium size
bronchioles (about 0 3 mm diameter), the thickness of the muscularis decreases to
a range from a maximum thickness of 10,tm to complete absence. In the terminal
bronchioles, the maximum thickness is 5 ,um. In general, as the airways branch, in-
creasing amounts of connective tissue separate progressively smaller, predominantly
circular, muscle fascicles (Fig. 21).
Adventitial mast cells
The adventitia of the hamster airways consists of connective tissue and elastic
fibres; it is always thickest where blood vessels appear in the airways (maximum
adventitia thickness in the trachea and main bronchi is 0 12 mm). As can be seen
in 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 stain
on a frozen lung section (Fig. 19). These mast cells in the lung appeared blue in both
the AB pH 1-PAS and AB pH 2-5-PAS staining sequences, indicating an acidic
product. Mast cells are known to contain acidic sulphated mucopolysaccharides
(Spicer, 1965).
Blood vessels
The distribution pattern of the hamster lung vasculature is similar to that in man.
Branching of the pulmonary arterial and venous systems beyond the small venules
follows the tracheobronchial tree (Kleinerman, 1972). The branching arterial system
progresses 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 reaction
on a frozen lung. In the smaller veins, the layer of smooth muscle is only one cell
thick (Fig. 22B); the layer increases to 3-4 cells thick in the larger veins. Cardiac
muscle is often found in the veins of small mammals (Rhodin, 1974; Sorokin, 1973);
but not in man.
35-2'*f:itJ"os. > -*,'8:S.
548 ANN R. KENNEDY AND OTHERS
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22B
Fig. 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 as
mast 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 blood
vessel 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 amount
of connective tissue. Iron haematoxylin-Gomori's trichrome. x 205.
Fig. 21. There is a predominantly circular arrangement of muscle fibres, separated by connective
tissue, 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 found
in 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.Syrian hamster lungs 549
Lymphatic system
Except for large hilar nodes, lymphoid tissue is relatively rare in the hamster
tracheobronchial tree, contrary to the situation in human lungs where lymphoid
tissue is abundant. Most hamster lungs examined contained no lymphoid tissue
except for small nodules very high up in the tracheal region where mucous glands
were common. Some animals did have nodules of lymphoid tissue at bronchial
bifurcations, however, particularly at bifurcations of a main bronchus, as shown in
Figure 13. The distribution of lymphoid tissue appears to be comparable to that
reported for the mouse (Sorokin & Brain, 1975). Lymphatic vessels, on the other
hand, are common in the hamster. They were seen in the tracheal, bronchial and
bronchiolar adventitia, in the connective tissue of the interlobular septa, and rarely
in the very thin pleura of the hamster lung.
DISCUSSION
Morphometry
The branching pattern of hamster airways is like that of many small mammals
(Kliment, Libich & Kaudersova, 1972). Moreover, our classifications are compatible
with results obtained by Horsfield & Cumming (1968) and Parker et al. (1971) in
human lungs. They found that human airways larger than 07 mm diameter exhibited
a markedly asymmetrical dichotomy when the numbering order increased toward
the carina. Smaller airways (terminal bronchioles and beyond) showed regular
dichotomous branching. Sorokin (1973) in human and canine lungs reported that total
bronchial cross sectional area increased geometrically as a function of distance
from the carina and then declined. In the hamster, cross sectional area increases
initially as a function of generation, then declines. It is worthy of note that the region
ventilated 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 presence
of more generations of respiratory bronchioles and alveolar ducts in the human lung.
Respiratory bronchioles are shaped differently in hamster and man. In human
lungs, long stretches of bronchiolar epithelial cells are interrupted by occasional
alveolar outpocketings. In hamsters, the respiratory bronchiole consists of very
short stretches of bronchiolar epithelium, usually just a few cells, with extensive
alveolar outpocketings. The few bronchiolar type cells at the entrance to an alveolus
from a respiratory bronchiole were not appreciably greater in number than the
number of alveolar cells in the knob-like swellings at the entrance to each alveolus
in more peripheral areas of the lung (Rhodin, 1974). Since these bronchiolar type
cells would be receiving similar doses as alveolar cells from exposures to irritants
and carcinogens reaching the alveolar region, we have considered them as part of the
total 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 hamster
lung which contrasts somewhat with lung models used in particle deposition esti-
mates for human lungs (Nelson et al. 1969). Principally, this difference resides in the
declining surface area of peripheral airways encountered in the hamster lung. The
decreases in length, diameter and number of airways combines to decrease total
surface area in regions distal to the fifth generation. Models for the human lung
have been developed and used to estimate deposition (Taulbee & Yu, 1975) and550 ANN R. KENNEDY AND OTHERS
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 considered
when extrapolating data about the deposition, retention and clearance of inhaled or
intubated materials from hamsters to other species, and especially to man.
Histology
In 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, and
an epithelium containing goblet, ciliated, basal, intermediate and brush cells, all of
which are characteristic of the human bronchial region. Unlike the human bronchus,
the hamster tracheal epithelium contains few neurosecretory cells. The height of this
epithelium is approximately 20 ,um, as compared with 30-50 ,um in man (Rhodin,
1974; Gastineau, Walsh & Underwood, 1972). Tracheobronchial epithelium is
normally 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 the
proportions of cell types in various regions of the tracheobronchial tree, including
age, sex, vitamin A status and environmental conditions. For example, there is a
decrease in the proportion of ciliated cells and an increase in basal cells on a vitamin
A-deficient diet (Boren et al. 1974). In our study young male hamsters were fed diets
containing adequate amounts of vitamin A.
There are no published reports of cell type frequencies in the bronchial and
bronchiolar regions of hamster lungs. As we have shown, Clara cells can be found
in hamster bronchial epithelium: the frequency is difficult to determine at the light
microscopic 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 identity
and appears as just another brush cell in the epithelium (Sorokin, 1973). A cell with
just a few PAS-positive areas may be a goblet cell with some mucous granules, or a
Clara cell with PAS-positive lysosomes in the apical region (Fig. 15). The occasional
PAS-positive lysosomes have no doubt caused a few investigators to conclude that
Clara 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 significant
fraction 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 hamsters
is present in both bronchial and bronchiolar epithelium, whereas in man it occurs
only in the bronchioles. Between different species, both the distribution and the
histological characteristics of the Clara cell vary. Human (Bloom & Fawcett, 1968),
but not hamster, Clara cells contain particulate glycogen. Myelin figures, frequently
seen in hamster Clara cells, have not been described in other species. Hamster and
human mitochondria are basically similar, but the ultrastructural morphology of
Clara cell mitochondria varies among rodents as well as other mammals (Sorokin,
1970a). Hamster Clara cells contain very few secretion granules (Fig. 17) and have aSyrian hamster lungs 551
minimal 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 by
glands and goblet cells. In the hamster, tracheal glands produce a neutral muco-
polysaccharide. The human lung contains mixed glands, which produce a neutral
mucopolysaccharide and acidic mucosubstances (McCarthy & Reid, 1964b); similar
glands are found in rat and mouse lungs (McCarthy & Reid, 1964a). Goblet cells in
the hamster trachea and bronchi produce most of the secretions for the proximal
mucous ciliary escalator. Goblet cells in both man (McCarthy & Reid, 1964b), rats
(McCarthy & Reid, 1964a) and hamsters produce neutral mucopolysaccharides, and
neuraminidase 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 secretions
could influence the efficacy of the mucous ciliary escalator, and in turn, alter the
incidence 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 been
proposed as the cell of origin of carcinoid tumours (Gmelich et al. 1967) and oat
cell tumours (Spencer, 1968). The rarity of neurosecretory cells in hamster epithelia
may explain why hamsters infrequently get carcinoid tumours, and oat cell tumours
are exceedingly rare (personal communication, Dr Curtis C. Harris).
Perhaps some of the known species differences in pulmonary susceptibility to
carcinogens 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 to
determine differences between hamster and human lungs which may have relevance
for lung carcinogenesis studies. Morphometric measurements were made on fresh
lungs, lung casts, and histological sections. Cell type and frequency measurements
were determined from frozen, paraffin,1 um plastic (glycol methacrylate) and electron
microscopic sections. A standard terminology for hamster lung histology is estab-
lished, and differences between hamster and human lung morphometry and histology
are discussed.
This research was supported by Contract CP-33273 and Training Grant CA-09078
from the National Cancer Institute, Grant DB-37c from the American Cancer
Society, and Center Grant ES-00002 from the National Institute of Environmental
Health Sciences. We would like to thank Mr Frank Bettinelli for his expert assistance
in the preparation of histological materials, and Dr Curtis C. Harris and Dr David
G. Kaufman for help in the preparation of this manuscript.You can also read
