Epithelial surfaces of the trachea andprincipal ... - Thorax · Departments of Anatomy and Medicine, Monash University, Baker Institute, Alfred Hospital and School of Biological Sciences,

Thorax (1975), 30, 171.

Epithelial surfaces of the trachea and principalbronchi in the rat

IVAN ALEXANDER, B. C. RITCHIE,J. E. MALONEY, and C. R. HUNTER

Departments of Anatomy and Medicine, Monash University, Baker Institute, Alfred Hospitaland School of Biological Sciences, La Trobe University, Melbourne, Victoria, Australia

Alexander, I., Ritchie, B. C., Maloney, J. E., and Hunter, C. R. (1975). Thorax, 30,171-177. Epithelial surfaces of the trachea and principal bronchi in the rat. Theepithelial surfaces in the trachea and principal bronchi of healthy rats were examinedby scanning electron microscopy. A system of four cell types, ciliated, microvillous,brush, and goblet cells, in this order of frequency, were found and intermediate typecells were not seen. An extensive area of the surface examined was covered by denselyciliated epithelium. The presence of other cell types beneath the cilia was confirmed bytransmission electron microscopy.

Areas up to 1 mm in diameter and randomly distributed were observed wheremicrovillous cells predominated and only occasional ciliated cells were found. Mostciliated cells in these areas were adjacent to glandular openings or goblet cells.The larger microvilli of the brush cells were arranged in a coronal configuration

elucidated by the scanning electron microscope.Preparatory techniques recently introduced for the examination of soft tissue in the

scanning electron microscope facilitated the confirmation of cell types present and themicroarchitecture of the epithelial surface.

Scanning and transmission electron microscopywas used to demonstrate the structure of thetracheal and principal bronchial epithelialsurfaces in the rat. Cell types present, theirrelationship to each other, and their pattern ofdistribution were studied.

Scanning electron microscopy, due to theversatility in its range of magnification and greatdepth of focus, was particularly useful in demon-strating the microarchitecture of epithelialsurfaces. Recently introduced techniques of softtissue preparation for the vacuum of the scan-ning electron microscope were instrumental indepicting the tissues examined.

Tissue surfaces consisted of four distinctvarieties of cell types, ciliated cells outnumberingthe other three, in decreasing frequency, micro-villous, brush, and goblet cells. A number of areasof up to 1 mm in diameter were found wheresheets of microvillous cells formed the epithelialsurface, relieved only occasionally by one of theother three cell types. The same surface patternwas found throughout the entire tracheobronchialarea examined.

MATERIAL AND METHODS

Healthy 5-month-old male and female rats of theSprague Dawley strain, 270-370 g in weight, keptunder standard laboratory conditions, fed on a dietof Gro-Chick Ration (Clark King and Co. Pty. Ltd.,Melbourne) containing approximately 1800 IU ofvitamin A per kg (Harris et al., 1972) were injectedintraperitoneally with heparin (Heparin injection BP,5000 IU/kg body weight). They were anaesthetized onehour later with intraperitoneal sodium pentobarbi-tone (50 mg/kg body weight), their chests wereopened, and the aorta was cannulated through theleft ventricle; 5% phosphate buffered glutaraldehyde(Sjostrand, 1967) was injected into the aorta bygravity at approximately the mean arterial pressure(120 cm fixative). Fixative was kept at 4°C and in-jected at the rate of about 500 ml/kg body weightper minute for 20 minutes. The trachea and mainbronchi were removed from each animal in toto.Specimens were then divided longitudinally and theresultant pieces were separated transversely into fiveportions. Fixation was continued in the same bufferedglutaraldehyde overnight at 4°C. Washing in bufferand post-fixation in 2% osmium tetroxide for onehour at 4°C and dehydration in graded alcoholsolutions followed.

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I. Alexander, B. C. Ritchie, J. E. Maloney, and C. R. Hunter

Tissues were then transferred into an epoxypropane-Araldite mixture and finally embedded inAraldite (Glauert and Glauert, 1958; Alexanderet al., 1973). After two hours at room temperaturethey were polymerized for 30 minutes at 60°C,soaked for 10 minutes in acetone, and scoured withit until all surfaces presented a mat appearance, andfinally polymerized at 60°C for 48 hours. Specimenswere then mounted on metal stubs with a plasticizednitrocellulose solution (Colorless Cutex SpillproofPolish, Cheseborough-Ponds International Ltd.,Clayton, Victoria). The stubs bearing the specimenswere positioned at an angle of 450 to an evaporationsource and rotated in vacuo while a conducting layerof gold was deposited on all exposed surfaces. Thethickness of the layer was controlled by matchingthe colour of a similarly exposed tile to a standard.Transmission electron microscopic examination ofsimilarly treated material, embedded and sectionedappropriately, showed the thickness of the gold layerto be 20 nm. The bases of the specimens were paintedwith a colloidal silver solution (DAG, 915-AchesonColloids Company, Prince Rock, Plymouth,England).Specimens were examined in various scanning

electron microscopes at an accelerating voltage of20 keV. Micrographs were taken on Polaroid series42 and 55 films (Polaroid Land Co.).For control purposes selected tissues were handled

separately from the stage of Araldite embedding,polymerized in blocks, and thin sectioned. Sectionswere mounted on uncoated grids, stained with uranylacetate (Huxley and Zubay, 1961) and lead citrate(Reynolds, 1963), and examined in various trans-mission electron microscopes at 75 and 80 keV.

RESULTS

Four cell types and four cell types only, i.e.,ciliated, microvillous, brush, and goblet cells,make up the epithelial surfaces of the tracheaand bronchi in that order of frequency.The largest area of the tissue surfaces showed

nothing but a regular array of cilia obscuring cellsurfaces (Fig. 1). Cilia were relatively largecylindrical structures (6 [km long and 0 2 jtm indiameter) and they were interposed between thescanning beam and the cell surfaces.The scanning electron microscopic appearance

of the ciliated cells differed in no way from thedescription given by Barber and Boyde (1968).The transmission electron microscopic image ofthis part of the cell was first described two decadesago in the classic paper of Fawcett and Porter(1954). The extracellular parts of the ciliapossessed nine doublets of peripheral micro-tubules. These surrounded a central pair of fila-ments miaking up the well-known axoneme. Thispattern is almost invariable (Fig. 6).

Areas were found in the tissues examined whereciliated cells were almost completely absent. Inthese tracts microvillous cells outnumbered allothers (Fig. 5). Here, as in all other parts of thetissues examined, every cell type was representedbut in different proportions. The microvillous cellsoccasionally formed sheets of cell surfaces exclud-ing all others (Fig. 2). The transmission electronmicrograph (Fig. 11) shows microvillous cells anda brush cell intermingled with ciliated cells.

All the cell apices in the tracheobronchial tractexamined were bordered by straight lines ofmicrovilli (Figs 3 and 6). Microvilli of the respira-tory surface resembled the brush border of theintestinal epithelium (Ito, 1965; Revel and Ito,1967). Microvilli are covered with a cell mem-brane and have a core of cytoplasm; this is con-tiguous with the cytoplasm of the cell itself. Inthe cores the cytoplasm is dense and filamentous(Fig. 12). Filaments extend into the terminal webof the cell and outside the cell membrane into afilamentous halo, the glycocalyx. Microvilli are03 ,um in length and approximately 01 jum inwidth on all types of cells (Fig. 6). In scanningmicrographs the microvilli are about 0 25 km in

FIG. 1. Array of cilia covering the largest area ofthe tissues examined. Their dense mass prevents thescanning beam from depicting all underlying struc-tures (approx. X 1000).

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FIG. 3. SEM of microvillous cells in the trachea. AFIG. 2. SEM of microvillous cells from one of the double row of microvilli separates the cellular apicesmajor bronchi. Central elevations bear most of the (approx. X7000).microvilli. Apices of the cells are clearly demarcated(approx. X5000).-s 4 44L A_Ll __s____~~~~~~~~~~~~~~~~~~~~~~

FIG. 5. SEM of microvillous area near the carina. TheFIG. 4. SEM of microvillous area in the lower part goblet cell on the lower left is bordered by cilia. Aof a principal bronchus relieved by two brush cells small brush cell is visible in the upper right part ofand a ciliated cell (approx. X800). the micrograph (approx. X1000).



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1 ,um in diameter with alternating darker andlighter demilunes (Fig. 6).Brush cells were seen with equal frequency in

the ciliated and microvillous areas. Their apiceswere commonly pentagonal and each sidemeasured some 4 ym in length (Fig. 9). Approxi-mately 100 very large microvilli, nearly 2 tsmlong and 0 2 tsm in diameter (Figs 9 and 10),occupied the centre of these cells' apices and weregathered into a corona surrounding a centralopening of 1 ,um in diameter. Their transmissionviews (Fig. 10) were in accordance with similar

P'',¢ vcells described by Silva (1966), Meyrick and Reid(1968), and Luciano, Reale, and Ruska (1969).

Goblet cells were sparse. Each goblet cell seenin the microvillous areas was marked by a singleadjacent ciliated cell (Fig. 5). Transmission viewsdid not differ from those of Rhodin and Dalhamn(1956), Rhodin (1959), and Cireli (1966).There were few glandular openings or goblet

- ~~~~~~~~~cells(Fig. 8). The one referred to and depictedshowed its funnel-like opening and its entrancelined by microvillous and ciliated cells.

FIG. 6. TEM view of microvillous and ciliated cellapices. The detached part is due to skewed sectioningand emphasizes the central elevations of the micro- W';villous cell. Granules show characteristic demilunes. ...Microvilli are seen at the junction of all cells. Ciliatedpatterns are apparent on the left side of the micro- 'graph (approx. X 15,000).

width (Fig. 9) probably due to the inclusion of the weglycocalyces in the sample (Ceccarelli, Clementi,and De Giuli, 1969). Microvillous cells found 4i W O.

throughout the tissues examined had an apical A

diameter of approximately 10 ,um and were colum-nar, being approximately 20 /im in length and10 ,am in diameter. A'

Tracts of up to 1 mm in diameter were foundalmost completely covered by these cells. Tractsof this description were relieved by relatively fewof the other types of cells. The transmission elec-tron microscopic appearance of microvillous cells t :1twas similar to that of the 'mucous' cell of Rhodin(1959).Membrane-bound granules of varying appear-

ance in accordance with their location in the cellwere numerous. Near the cell centre they were .40 5 ,um in diameter, containing small round dark FIG. 7. TEM of supranuclear region of microvillousbodies of approximately 30 nm in diameter, super- cell. A mitachrondrion is seen between two character-imposed on a granular lighter background (Fig. 7). istically speckled granules in the immediate vicinityNearer the apex the granules were approximately of the Golgi apparatus (X 16,000).

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FIG. 8. SEM of glandular opening funnelling outinto the tissue near the first ring of the trachea. It isin a microvillous area. A ciliated cell is visible andcilia reach up from the glandular orifice (approx.X 1000).

DISCUSSION

Rhodin and Dalhamn (1956) have describedciliated, goblet, brush, and 'basal' cells. Moreadvanced techniques utilized by Rhodin (1963)established that the epithelial covering is made upof four cell types. Intermediate cells were des-cribed similar to, but not identical with, the abovefour, and it was stated that the tracheal andprincipal bronchial surfaces were uniform.Blenkinsopp (1967), using light microscopy only,identified four clearly recognizable superficial celltypes.

Microvillous cells discussed above were referredto by Alexander, Ritchie, and Maloney (1971) as

'collicular'. A similar cell type was termed 'non-ciliated' by Hansell and Moretti (1969) in theirtransmission electron microscopic description ofthe mouse trachea.Vitamin A deficiency, possibly responsible for

areas lacking cilia, was prevented by adequateamounts of this substance being incorporated in

the pellets fed to the experimental animals (Harriset al., 1972).The plastic infusion and scouring techniques

employed here and described previously (Alex-ander et al., 1971 and 1973) had their origin in anidea of Barber and Boyde (1968), who suggested,in a scanning electron microscopic study of cilia,that tissues might be fixed and embedded as fortransmission electron microscopy and then, usinga solvent, the excessive embeddent eroded away soleaving just the surface. The sucessful applicationof this idea demonstrated the large microvillousareas and the configuration of the brush cell apex.The few glandular openings found could per-

haps be explained by the large number of secre-tory granules of variable electron densities seenin every cell of the tissues examined (Fig. 11). Itis interesting in this regard to compare the similarcomposition of the apical granules of the micro-villous cells found in the tracheobronchial sur-faces in the rat (Fig. 6) and the ones seen byMeyrick and Reid (1970) in human bronchialglandular tissue, which may account for thescarcity of these glands in the rat, similar granulesbeing discharged from surface cells.

Large tracts of nonciliated areas seem not tointerfere with the flow of mucus in healthyanimals-this may be due to the microvilli beingable to induce movement in the deeper, relativelynon-viscous layer of the mucus (Friedmann andBird, 1971). It is interesting to note that bothgoblet cells and glandular openings were associ-ated with at least one adjacent ciliated cell, evenin the microvillous areas (Figs 5 and 8).Scanning electron microscopy confirmed by

transmission electron microscopy has shown thatthe highly sophisticated and structured micro-architecture of the rats' tracheobronchial surface.consists of a mixture of four cell types. These areall distinct, and it is now possible to state thatintermediate or 'basal' cells are not ordinarilyfound to exist on the surface of healthy animals.The regularity of configuration has shown theimportance of electron optical investigation ofepithelial surfaces.

This work was supported by the Australian TobaccoResearch Foundation.

The help and advice of Professors G. C. Schofieldand A. B. Wardrop and the technical help of Mrs. V.Case, Miss M. L. Exelby, Mrs. M. Rose, and MissM. L. Coyne are gratefully acknowledged.

Professor I. W. B. Thornton kindly read themanuscript.

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FIG. 9. SEM of microvillous cells surrounding abrush cell. The long microvilli are seen in their distinc-tive position with the central opening. Cell bordersare marked by straight lines of microvilli (approx.X1000).

FIG. 10. TEM of apical region, brush cell. The longmicrovilli have a central core of fine filaments extend-ing into the cells web in individual bundles (X21,000).

lk~~~~~~9

FIG. 11. Longitudinal section of principal bronchialepithelium shown by TEM. Microvillous and ciliatedcells alternate on the surface. A brush cell is seen inthe upper right-hand portion of the micrograph(approx. X4000).

FIG. 12. TEM of surface of microvillous cells. Micro-villi are seen in longitudinal section. Their filamentouscore is connected with the terminal web of the cellsbelow and the glycocalyces surrounding the micro-villi (X30,000).



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REFERENCES

Alexander, I. G. S., Capicchiano, P. M., Ritchie,B. C., and Maloney, J. E. (1973). Plastic embed-ding and surface erosion techniques in soft tissuescanning electron microscopy. Journal of Micro-scopy, 99, 69.

- , Ritchie, B. C., and Maloney, J. E. (1971).Scanning electron microscopy of respiratory tis-sues in the rat. Journal of Anatomy, 110, 491.

Barber, V. C. and Boyde, A. (1968). Scanning electronmicroscopic studies of cilia. Zeitschrift fur Zell-forschung und mikroskopische Anatomie, 84,269.

Blenkinsopp, W. K. (1967). Proliferation of respiratorytract epithelium in the rat. Experimental CellResearch, 46, 144.

Ceccarelli, B., Clementi, F., and DeGiuli, C. (1969).Aspetto di alcune mucose dell'apparato digerenteal microscopio elettronico a scansione. Bollettinodella Societa' Italiano di Biologia Sperimentale,45, 646.

Cireli, E. (1966). Elektronenmikroskopische Analyseder pra- und postnatalen Differenzierung desEpithels der oberen Luftwege der Ratte. Zeit-schrift fuir mikroskopische-anatomische For-schung, 74, 132.

Fawcett, D. W. and Porter K. R. (1954). Study of thefine structure of ciliated epithelia. Journal ofMorphology, 94, 221.

Friedmann, I. and Bird, E. S. (1971). Ciliary structure,ciliogenesis, microvilli. (Electron microscopy ofthe upper respiratory tract). Laryngoscope, 81,1852.

Glauert, A. M. and Glauert, R. H. (1958). Araldite asan embedding medium for electron microscopy.Journal of Biophysical and Biochemical Cytology,4, 191.

Hansell, M. M. and Moretti, R. L. (1969). Ultrastruc-ture of the mouse tracheal epithelium. Journal ofMorphology, 128, 159.

Harris, C. C., Sporn, M. B., Kaufman, D. G., Smith,J. M., Jackson, F. E., and Saffloti, U. (1972).Histogenesis of squamous metaplasia in the ham-ster tracheal epithelium caused by vitamin Adeficiency or benzo(a)pyrene-ferric oxide. Journalof the National Cancer Institute, 48, 743.

Huxley, H. E. and Zubay, G. (1961). Preferentialstaining of nucleic acid-containing structures forelectron microscopy. Journal of Biophysical andBiochemical Cytology, 11, 273.

Ito, S. (1965). The enteric surface coat on cat intes-tinal microvilli. Journal of Cell Biology (supple-ment of Journal of Biophysical and BiochemicalCytology) 27, 475.

Luciano, L., Reale, E., and Ruska, H. (1969). Bursten-zellen im Alveolarepithel der Rattenlunge. Zeit-schrift fir Zellforschung und mikroskopischeAnatomie, 95, 198.

Meyrick, B. and Reid, L. (1968). The alveolar brushcell in rat lung-a third pneumonocyte. Journalof Ultrastructure Research, 23, 71.and . (1970). Ultrastructure of cells in thehuman bronchial submucosal glands. Journal ofAnatomy, 107, 281.

Revel, J. P. and Ito, S. (1967). Electron microscopicstudies of the surface components of animalcells. In The Specificity of Cell Surfaces, p. 211.Prentice-Hall, New Jersey.

Reynolds, E. S. (1963). The use of lead citrate athigh pH as an electron-opaque stain in electronmicroscopy. Journal of Cell Biology, 17, 208.

Rhodin, J. A. (1959). Ultrastructure of the trachealciliated mucosa in rat and man. A nnals ofOtology, Rhinology and Laryngology, 68, 964.. (1963). An Atlas of Ultrastructure. Saunders,Philadelphia.and Dalhamn, T. (1956). Electron microscopy ofthe tracheal ciliated mucosa in the rat. Zeitschriftfiur Zellforschung und mikroskopische A natomie,44, 325.

Silva, D. G. (1966). The fine structure of multivesi-cular cells with large microvilli in the epitheliumof the mouse colon. Journal of UltrastructureResearch, 16, 693.

Sj6strand, F. S. (1967). Electron Microscopy of Cellsand Tissues, Volume 1. Academic Press, NewYork and London.

Requests for reprints to: Dr. I. G. S. Alexander,School of Biological Sciences, La Trobe University,Bundaora, 3083 Victoria, Australia.

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Epithelial surfaces of the trachea andprincipal ... - Thorax · Departments of Anatomy and Medicine, Monash University, Baker Institute, Alfred Hospital and School of Biological Sciences,