MICROSCOPIC PROPERTIES OF T H E BASEMENT MEMBRANE AND ELASTIC FIBERS OF TRACHEA

AND BRONCHUS OF SMOKERS AND NONSMOKERS Y . HAYASHI, M.D., E. V. COWDRY, PH.D., AND V. SUNTZEFF, M.D.

E HAVE undertaken a histological study of the basement membrane and elastic

fibers in the trachea and bronchus of smokers and nonsmokers.

At the boundary between the epithelial membrane and its supporting connective tis- sue there is generally a thin layer of a special kind of intercellular substance (ground sub- stance) that is called the basement mem- brane.12 I t is optically homogeneous and may vary from a fairly fluid to a gel-like con- sistency.9 MutoZ3 has classified basement mem- branes into 3 morphological types: (1) argeno- fibril nets with liquid-like or sol-like ground substance (glands of the uterus); (2) argeno- fibril nets with gel-like ground substance (epi- dermis): (3) a combination of types 1 and 2 (seminiferous tubule). According to this classification, the basement membrane of the trachea and bronchus is of type 2.

The ground substance is composed of a polymerized, presumably organized fluid, or gel, containing oriented fibrils. It may be polymerized to a different degree in the same organ at different times and in different or- gans at the same time.9

The elastic tissue of the respiratory system is an extremely important functional element and is found in a definite morphological pat-

as will be shown later. During de- flation, the coils of elastic tissue contract in unison with the contraction of the longitud- inal elastic fibers effecting shortening of the bronchus and trachea. T h e Iatter is shortened 20 to 30% of its The present study is restricted to the alteration of the longitudinal elastic layer of the trachea and the bronchus.

MATERIAL AND METHODS

The data that are presented in this report

From the TYernse Cancer Research Laboratory, Washington University School of Medicine, St. Louis, hfo.

This work was supported by a grant (L-8B(t)) from the American Cancer Society, Inc.

Received for publication Dec. 20, 1960.

are gathered from specimens of the human trachea and right bronchus of white men 40 to 77 years of age. Most of this material in- cludes cases previously studied in our labora- tory by Ide and co-workers.16 Only fatal cases, with histories of myocardial infarctions andl 2 cases of cerebral hemorrhage, were used in this study of the basement membrane since it appears that other diseases, such as diabetes mellitus, rheumatism, and glomerulonephritis, may cause changes in the ground substance, as will also such local conditions as malignant tumors, pulmonary edema, and pneumonia. Autopsy material was obtained less than 5 hours after death. The classification of smok- ing habits by Watson and C ~ n t e , ~ ~ which .we used, describes a minimal smoker as one who smokes less than 20 cigarettes a day; a mod- erate smoker, one who smokes up to 20 cig- arettes a day for 20 or more years; and a heavy smoker, one who smokes 20 to 60 cigarettes a day for 20 or more years with inhalation.

Tissues routinely fixed in Bouin’s solution were prepared for general histological observa- tions, but some tissues were fixed in Carnoy’s fluid. Longitudinal sections were routinely used, stained by Lillie’s periodic acid Schiff (PAS) leucofuchsin technique,l’ Weigert’s stain for elastic fibers, and Van Gieson’s picro- acid fuchsin stain. In order to color with Mallory’s phosphotungstic acid hematoxylin it was necessary to place the sections in a mordant of mercuric chloride solution for 12 hours. No difference was found between thlose sections fixed with Bouin’s and Carnoy’s fluids after staining with PAS. All observations of pas-stained specimens refer to Bouin’s-fixed ones. Material fixed in Carnoy’s fixing fluid was stained with alcian blue and periodic acid-Schiff stain for carbohydrates22 and Rine- hart and Abul-Haj’s26 modification of Hale’s method. We also used several enzymic di- gestion techniques, by hyaluronidase, col- lagenase, and trypsin for 72 hours at 37” C . with a concentration of I mg. per cu. cm. of phosphate buffer (pH 7.0). Before digestion, 8-v sections were denatured in absolute alco-

I175

1176 CANCER Nouem ber-December 1961 Vol. 14

TABLE 1 THICKNESS OF BASEMENT MEMBRANE IN SMOKERS AND NONSMOKERS

Thickness membrane, p

Pt. Trachea Bronchus Smoking Case age, habits no. Yr. Range Average Range Average

Nonsmoker 71 68 5.1-2.1 3 .6 4.6-2.2 3 .4 111 42 5.2-2.7 4 . 0 4.9-2.1 3.5

3 . 3 5.8 3 .8 4 .1 4 . 0

138 51 4.9-2.6 3 .8 144 60 7.6-2.6 5.1 148 75 5.8-2.3 4.1 153 75 5.4-2.3 3.9

5.7-2.4 4 .1 AVERAGE

light smoker Minimal or

AVERAGE Moderate smoker

103 69 5.4-2.0 3.7 117 47 9.8-4.6 7.2 139 40 6.5-2.8 4.7 142 67 6.8-2.2 4.5 207 63 5.9-2.6 4 .3 188 76 7.2-3.1 5.2

9s .~

127 132 137 212

17.890 AVERAGE

Heavy smoker 29 77* 99

110 116 201

AVERAGE

66 79 63 62 61 43

48 60 63 52 64 69

6.9-2.9 4 . 9 11.0-4.0 7 .5 8.3-2.8 5.6 6.3-2.6 4 .5 9.3-3.0 6.2

11.7-4.1 7.9 8.4-2.9 5.7 9.2-3.2 6 . 4 6.5-2.3 4 .4 1.8-1.0 1 .4 8.4-2.4 5 .4 6.7-2.8 4 . 8 5.5-2.1 3 .8

14.7-3.5 9.1 8.4-2.6 4.8

4.4-2.1 8.0-3.6 5 .O-2.5 5.6-2.6 5.4-2.5

3.3-1.8 5.1-2.2

10.4-3.7 3.2-1.6

6.2-2.6 5.5-2.4

4.7-2.2

6.9-3.0 7.4-2.1 6.3-2.2 6.4-2.9

7.2-3.7 7.2-2.8 7.1-2.4 1 .2-1 .o 0.1-4.4 7.1-1.9 5.0-1.8 7.4-5.1 9.3-3.1

9.1-3.0

2.6 3.7 7.1 2 .4 3 .5 4 .4 4 . 0 5.0 4 . 8 4 .3 4.7 6.2 5 .5 5.1 4 . 8 1 . 1 7.3 4.5 3 .4 1 .3 5.4

*This patient had cancer, and the case is not included in the average.

hol for 24 hours. Controls were run in buffer alone.

Measurements of the thickness or distribu- tion of the basement membrane stained with PAS were made with an ocular micrometer. Five sections were selected from each slide, then each section was divided into 5 fields of vision under the microscope at a magnifica- tion of ~ 1 0 0 . Measurements were taken of areas that were cut longitudinally. Because some areas of the inner part of the basement membrane were diffuse and ill defined and therefore difficult to measure, we only meas- ured compact homogeneous-appearing areas.

RESULTS

Basement Membrane. Morphological Classi- fication. The ground substance may be po- lymerized to different degrees in the same organ at different times, thereby making it possible to distinguish several types of base- ment membranes. These are listed in Figs. 1 to 5 as types A to E. There are transitional states in each type. The characteristics of each type are:

Type A: Thin and relatively straight (or slightly wavy) and homogeneous basement membrane (Fig. 1).

Type B: The basement membrane is con- siderably thicker than type A and distinctly homogeneous. Sometimes PAS-positive sub- stance spreads diffusely into the submucosa (Fig. 2).

Type C : Some areas are like type B, but in general the adjacent connective tissue contains fine fibrils (“feather-bed” structure) or edema, with or without round cell infiltration. In these places the PAS-positive substance is very scanty and similar to type D (Fig. 3).

Type D: Thin, straight basement mem- brane is very distinct. The homogeneous struc- ture of the basement membrane is difficuIt to see because the membrane is extremely thin. The adjacent connective tissue is similar to type C (Fig. 4).

Type E: A very thin basement membrane that is difficult to find in places. In these areas the connective tissue containing many capil- laries comes in close contact with the meta- plastic epithelium.

Similar changes in the connective tissue as

No. 6 CHANCES IN TRACHEA & BRONCHI IN SMOKERS & NONSMOKERS * Hayashi et d. 1177

found in types C and D are also found here but to a lesser degree (Fig. 5) .

Based on these findings, it seems that edema in the connective tissue adjacent to the base- ment membrane is an important factor in the thinning process of the basement membrane.

Staining Properties. The basement mem- brane of the trachea and bronchus appears as a brilliant red sheet when stained only with PAS; however, wherever ground substance ex- tends toward the subepithelial connective tis- sue these areas appear less red than does the upper zone or membrane proper. Lillie’s method, using PAS with picric acid, only mod- erately stains the basement membrane so that it appears red with increasing paleness toward the underlying connective tissue.

Bouin-fixed specimens stained with alcian blue and PAS and also with Rinehart’s method have the same brilliant red color, but speci- mens fixed with Carnoy’s fixing fluid appear reddish-purple.

The basement membrane was found not to stain with PAS after the enzymic action of col- lagenase, while hyaluronidase had no signifi- cant effect, and the action of trypsin is not yet clear.

Measurements. The measurements of the thickness of the basement membranes in 2!4 cases, grouped according to the patients’ smok- ing habits, are recorded in Table 1. These measurements included the maximum and minimum thicknesses. As is indicated in Table 1, the basement membrane of smokers is gen-

FIG. 1. Type A. Thin and relatively straight, homogeneous basement membrane. ( ~ 4 4 0 . ) FIG. 2. Type B. The basement membrane is considerably thicker than type A and distinctly homogeneous.

FIG. 3. Type C . Some areas are like type B, but in general the adjacent connective tissue contains fine fibrils.

FIG. 4. Type D. The basement membrane is thin and straight. The homogeneous structure is difficult to see

(X440.)

(X440.)

because the membrane is extremely thin. (X440.)

1178 CANCER Nouem ber-December 1961 Vol. 14

FIG. 5. Type E. The basement membrane is difficult to find in places (carcinoma in situ?). (~440.)

erally thicker than that of nonsmokers, but there are some exceptions:

Nonsmokers’ (NS) average, 4.1 p for trachea (TR); 4.0 p for bronchus (BR). Minimal or light smokers’ (LS) average, 4.9 p (TR); 4.0 p (BR). Moderate smokers’ (MS) average, 6.4 p (TR); 5.1 p (BR). Heavy smokers’ (HS) average, 4.8 p (TR); 5.4 p (BR).

The average thickness of the basement mem- brane in the trachea and bronchus of non- smokers is almost identical (4.1 p and 4.0 p). Smoking appears to be associated with a greater thickening of the basement membrane in the trachea than in the bronchus among minimal and moderate smokers.

The average maximum thickness of base- ment membrane of the trachea seems to in- crease with the amount of smoking. However, with the small sizes of the samples, the differ- ences among the means are not significant (P slightly greater than 0.05).

The same is true for the bronchus, with the P greater than for the trachea.

Observations concerning the relationship between the changes in the epithelium, in- cluding changes in the height of epithelium, and smoking habits have been made by oth- ers.4,16 Included in this report is the relation- ship between the changes of the epithelium

and the thickness of the basement membrane. In Table 2, the thickness of the basement

membrane is less in cases in which there is simple columnar epithelium (TR 4.8 p; BR 3.9 p) than in those in which there are some small areas of stratified epithelium (TR 5.5 p, BR 6.6 p). In the trachea, the thinnest base- ment membranes appear with stratified flat- tened or squamous epithelium (2.7 p). In the bronchus, the thinnest basement membrane appears with carcinoma in situ (1.1 p). In gen- eral, the thickness of the bronchus basement membrane varies much more than that of the basement membrane of the trachea.

Since expanding tumors create increased localized pressures in tissues and are associated with unusual thickenings of the basement membrane, it was thought that perhaps the pressure from thickened epithelium might also have a similar effect on the basement membrane.29

We attempted to analyze the relationship between the height of the epithelium and the thickness of the basement membrane. I t was observed that some smokers whose epithelia average 50. p to 60. p in thickness have very thick basement membranes. But a few non- smokers, whose epithelia also average between 50. p and 60. have thin basement mem- branes. Therefore it seems that an increase in the height of epithelium is not accompanied by a significant increase in the thickness of the basement membrane in the trachea and bronchus.

Measurements of varying thickness of the basement membrane according to age show that age does not appear to influence the thickness of the basement membrane within the 40 to 77 year age range studied.

Type of Epithelium Basement Membrane and Smoking Habits. The relationship be- tween the type of epithelium and the type of basement membrane and smoking habits is indicated in Table 3. I n general, nonsmokers have type A basement membrane, and smokers have type B or type C basement membrane. Cases of carcinoma in situ present type E. If the epithelium of nonsmokers and smokers is of the same type, i.e., pseudostratified or sim- ple columnar, then the nonsmoker’s basement membrane belongs to type A but the smoker exhibits type I3 basement membrane.

Elastic Fibers of Trachea and Bronchus. In cross sections, which are those usually studied, the elastic tissues of the trachea and bronchus are composed of 4 distinct layers: (1) sub-

No. 6 CHANGES IN TRACHEA & BRONCHI IN SMOKERS & NONSMOKERS * Hayashi et al. 1179

basement membrane layer; (2) longitudinal layer; (3) muscular layer; and (4) a layer en- closing cartilage nodules.21

Between the longitudinal layer and the ligamenta annularia is found an elastic bun- dle.14 The sub-basement membrane layer and the longitudinal layer appear very distinctly in longitudinal sections.

The present paper is restricted to a study of the longitudinal layer, which, according to Miller,21 is the most distinct layer in the walls of the larger air tubes. Longitudinal sections were stained in Weigert’s resorcin fuchsin, and some were stained in Mallory’s hema- toxylin.

The elastic fibers of trachea and bronchus in most cases of nonsmokers, when stained with resorcin fuchsin, are relatively straight and regular, deeply colored, and generally show an even thickness and stainability (Fig. 6). In most cases of nonsmokers and minimal smokers, Mallory’s hematoxylin gives definite deep or pale-blue staining of elastic fibers. No significant difference between minimal smok- ers and nonsmokers is demonstrable. Half of the moderate smoker cases present irregularly wavy elastic fibers. When the fibers appear irregularly wavy or are unusually coiled, there are usually more of irregular thickness and stainability. In these cases the fibers appear closer to one another than in those in which the fibers are slightly wavy, and most of the fibers do not stain with Mallory’s phospho- tungstic acid hematoxylin. These fibers have been called “elastically degenerated,” and “al- terative process” of elastic fibers.10

Considerable changes in the elastic tissue were found among heavy smokers (Fig. 7).

TABLE 2 TYPE OF EPITHELIUM AND AVERAGE

THICKNESS OF BASEMENT MEMBRANE Trachea Bronchus

Av. Av. Type No. thick., No. thick., epithelium cases cases

Simple columnar Simple columnar+

pseudostratified Pseudostratified Pseudostratified +

stratified Stratified Sm. area squamous

stratified Squamous strati-

fied or squamous Carcinoma in situ

2 4 .8 6 3 .9

8 5 . 1 7 4 . 2 3 4 . 8 3 5 . 5

7 5 . 8 6 5 . 5 2 4 . 6 2 4 .7

3 5 .5 2 6 . 6

3 2 . 7 1 4.7 . . . . . . 1 1.1

Most of these cases were distinguished by an increase in the amount of elastic fibers and irregular waviness, thickness, and stainability of these fibers. I t appears that heavy or me- dium smoking may cause these alterations of the elastic fibers in the trachea and bronchus.

Relationships between changes of elastic tissue in trachea and bronchus with age were studied in 11 cases of nonsmokers and light smokers. From the 11 cases studied, 5 showed slight changes of elastic tissue. In another group consisting of 12 cases of medium and heavy smokers, 10 cases showed, also, slight changes of elastic tissue, but the difference between these 2 groups was not striking.

DISCU5SION

A recent electron microscopic study of the trachea showed that the term “basement mem- brane” should be reserved for a continuous, structureless, medium-opaque membrane. For- merly the collagen fibers and elastin, together with the fibroblasts that are located close to and below the basement membrane, were interpreted as components of the basement membrane.25

McManus,20 and Lillie,l7 employed PAS

techniques. Hotchkiss,l5 has stated that the basement membrane is fairly specific for water and/or alcohol-insoluble polysaccharides or polysaccharide-containing complexes. Gersh and Catchpole9 found that the ground sub- stance and basement membrane are composed of a polymerized, presumably organized fluid or gel containing oriented fibrils, and the basement membrane is ground substance con- centrated to form a denser region between the connective tissue and epithelium. von Ebner has reported the thickness of the basement membrane in the trachea to be about 11 y.”

According to Wagner,33 it has become ob- vious that the ground substance is a compli- cated structure and differs in chemical com- position in various organs; several different mucopolysaccharides have been isolated.3

In this study the presence of acid- and neu- tral-mucopolysaccharides in the basement membrane was demonstrated with alcian blue- PAS staining after fixation with Carnoy’s fixing fluid. T h e homogeneous component of the basement membrane was removed by the ac- tion of a proteolytic enzyme, namely, col- lagenase. Treatment with hyaluronidase for 72 hours did not affect the affinity of the base- ment membrane for PAS. Conflicting results

1180 CANCER November-December 1961 Vol. 14

have been reported concerning the effect of hyaluronidase on the basement membrane. Gersh and Catchpoleg observed that after treatment with hyaluronidase no traces of a homogeneous component could be detected. But, in another report, the intensity of the red stain of PAS was described as not weakened by previous treatment with hyaluronidase.30 In the gingiva it was not feasible to determine the effect of hyaluronidase on the basement membrane.8

Evidently many factors bring about altera- tions in the ground substance, “collagen dis- eases.” Modifications are also caused by hor- mones and by hyaluronidase.5 Local chronic edema, as in bronchial asthma, is accom- panied by a thickening of the basement mem- brane of medium-sized bronchi.11

The foregoing observations would appear to indicate that smoking produces thickening of the basement membrane, by accumulation of neutral and acid mucopolysaccharides. Epi- thelial metaplasia seems to have some relation- ship to thickening of the basement membrane.

Factors that cause degeneration of the elastic fibers have been discussed by some au- thors.13~35 The effects of some chemical sub- stances have been studied by Bierich.2 He in- dicated that a small dose of arsenic acid produced slight hyperplasia of the epithelium

and also a remarkable increase in the quantity of elastic fibers. A marked increase in the amount of elastic fibers and hyperplasia of the epithelium occurred after treatment with arsenic acid plus tar, and with roentgen radi- ation. In our cases, heavy smoking produced changes in the elastic fibers, i.e., irregular waviness, increased amounts, and uneven thickness and stainability. Also, these fibers lost their stainability with Mallory’s phos- photungstic acid hematoxylin. However, some fibers of nonsmokers and smokers, which stained in the usual way with Weigert’s stain, were not colored with Mallory’s stain. Why this should be so is not clear.

Among bronchial asthma cases, the only sig- nificant alteration is an increase in the num- ber of elastic fibers in the walls of medium- sized bronchi.24 Clinically, chronic cough is often attributed to heavy smoking. Swineford and Ochota31 found 19 cases of bronchial asthma among 52 smokers who complained of chronic cough.

An acute exudative process usually is char- acterized by early disappearance of the stain- ing capacity of elastic fibers, with thinning of the fibers.32 Sawada28 did not, however, note these changes in the elastic fibers in his cases of bronchopneumonia.

We found irregularly wavy and sometimes

FIG. 6. The elastic fibers of trachea and bronchus of nonsmokers are relatively straight and regular. (~440.) FIG. 7. Increase in the thickness and in the amount of elastic fibers and irregular waviness in heavy smokers

at a lower magnification to show how thick the layer of elastic tissue is. (x215.)

No. 6 CHANGES IN TRACHEA & BRONCHI IN SMOKERS & NONSMOKERS - Hayashi et d. 1181 TABLE 3

TYPE OF EPITHELIUM, BASEMENT MEMBRANE, AND SMOKING HABITS - Type basement membrane, no. pt.*

A B C D E -

T R BR T R BR T R BR T R BR T R BR Type epithelium - Simple columnar 2 NS 2 NS 1 MS 2 MS ... 1 LS . . . 1 LS . . . . I .

1 NS Simple columnar+

. . . pseudostratified 4KS 5 N S I N S 1 N S . . . 1 LS 1 LS 2 L S 2 MS

1 LS

2 MS

1 MS 2 AIS Pseudostratified 1 NS . . . 1 LS 1 LS . . . 1 MS . . . . . . . . . . . . Pseudostratified +

stratified . . . . . . 1 N S 1 N S l N S 1 M S I L S 1LS . . . . . . 2 L S 1 L S 1 HS 2 H S 3 M S 1 H S 1 HS

Stratified . . . . . . 2 H S 2 H S . . . . . . . . . . . . . . . . . . Squamous stratified f

stratified . . . . . . 1 L S 1 N S 1 H S . . . . . . . . . . . . . . .

Squamous stratified . . . . . . 1 HS . . . . . . 1 MS 1 HS . . . 1 MS . . . 2 M S 1 H S

*See text for explanation of different types of basement membrane; the symbols used here indicate: TR, trachea; BR, bronchus; NS, nonsmoker; LS, light or minimal smoker; MS, niediurn smoker; HS, heavy smoker.

scattered elastic fibers among bronchopneu- monia cases; however, these changes are more distinct in some cases of heavy smokers.

Many physiological studies concerned with disorders of the respiratory tract due to smok- ing have been made.l,7218,36 However, these studies are primarily concerned with acute changes in the bronchial or pulmonary func- tion before and after smoking.

Our study shows that smoking, especially for long periods of time, causes alterations in the elastic fibers. These changes are responsi- ble for some loss of elasticity in the trachea and bronchus.

SUMMARY

1. The effects of smoking on the basement membrane and elastic fibers of the trachea and bronchus have been studied in human autopsy material. We also studied the process of changing thickness of the basement mem- brane.

2. Smoking was correlated with thickening of the basement membrane associated with the accumulation of neutral and acid mucopoly- saccharides. The average thicknesses of base-

ment membranes were: in nonsmokers, 4.1 p for trachea and 4.0 p for bronchus; in light smokers, 4.9 p for trachea and 4.0 p for bron- chus; in medium smokers, 6.4 p for trachea and 5.1 p for bronchus; and in heavy smokers, 4.8 p for trachea and 5.4 p for bronchus.

Epithelial metaplasia also seems to have some relationship to thickening of the base- ment membrane. In general, cases in which there were some small areas of stratified squa- mous epithelium also had very thick basement membranes.

In the trachea the thinnest basement mem- branes underlay transitional or squamous epi- thelium. T h e thinnest basement membranes in the bronchus were found with carcinoma in situ. T h e thickness of basement mem- branes varies much more in the bronchus than in the trachea.

3. Heavy smoking, especially 2 packages a day for a long period of time, produced more changes of the elastic fibers than those seen in light smokers. These changes included ir- regular waviness, increased thickness and stainability, and increased amounts of elastic fibers. They are responsible for some loss of elasticity in the trachea and bronchus.

REFERENCES

1. ATTINGER, E. 0.: GOLDSTEIN, M. M., and SEGAL, 3. CALKIUS, E.; SOODAK, M., and BAUER, W.: Metabo- M. S.: ERects of smoking upon mechanics of breathing; lism and clinical significance of carbohydrate com- I, in normal subjects. Am. Rev. Tuberc. 77: 1-9, 1958. ponents of connective tissue. New England J . M e d . 253:

2. BIFRICH, R.: Ober die Beteiligung des Bindege- 865-872, 1955. webes bei der experimentellen Krebsbildung. Virclzows 4. CHANG, S. C.: Microscopic properties of whole Arch. Path. Anat . 239: 1-19, 1922. mounts and sections of human bronchial epithelium of

1182 CANCER hrovember-December 1961 Vol. 14

smokers and nonsmokers. Cancer 10: 1246-1262, 1957. 5. DURAN-REYNALS, F., Chairman: Ground substance

of mesenchyme and hvaluronidase. Ann. New York Acad. Sc. 52: 943-1195, i950.

6. VON EBNER, V., [Ed.]: A. Koelliker's Handbuch der Gewebelehre des Menschen, 6. Aufl., Bd. 3. Leipzig, Germany. Verlag von Wilhelm Engelmann. 1902; pp. 293-297.

7. EICH, R. H.; GILBERT, R., and AUCHINCLOSS, J. H., JR.: Effects of smoking on respiratory mechanics in chronic pulmonary emphysema. Clin. Res. Proc. 4:

8. ENGEL, M. B.: Water-soluble mucoproteins of gingiva. J. Dent. Res. 32: 779-784, 1953.

9. GERSH, I., and CATCHPOLE, H. R.: Organization of ground substance and basement membrane and its significance in tissue injury, disease and growth. Am. J. Anat. 85: 457-521, 1949.

10. GILLMAN, T.; PENN, J.; BRONKS, D., and Roux, M.: Abnormal elastic fibers; appearance in cutaneous carcinoma, irradiation injuries, and arterial and other degenerative connective tissue lesions in man. A . M . A . Arch. Path. 5 9 733-749, 1955.

11. GUNN, F. D.: Lung. I?z ANDERSON, W. A. D., Ed.: Pathology, 3d ed. St. Louis, Mo. The C. V. Mosby Company. 1957; pp. 635-682; 644-645.

12. HAM, A. W.: Histology, 2d ed. Philadelphia, Pa. J. B. Lippincott Company. 1953; pp. 129-130.

13. HASS, G. M.: Elastic tissue. Arch. Path. 27: 334- 365, 583-613, 1939.

14. IIEIss, R.: Der Atmungsapparat. In VON MOEL- LENDORF, W., Ed.: Handbuch der mikroskopischen Anatomie des Menschen, Bd. 5, TI. 3. Berlin, Germany. Verlag von Julius Springer. 1936; pp. 709-798; 742-750.

15. HOTCHKISS, R. D.: Microchemical reaction re- sulting in staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16: 131-141, 1948.

16. IDE, G.; SUNTZEFF, V., and COWDRY, E. V.: Com- parison of histopathology of tracheal and bronchial epithelium of smokers and nonsmokers. Cancer 12: 473- 484, 1959.

17. LILLIE, R. D.: Histopathologic Technic. Phila- delphia, Pa. The Blakiston Company. 1948.

18. LOOMIS, T. A.: Broncho-constrictor factor in cigarette smoke. Proc. SOC. Exper. Biol. & Med. 92: 337- 340, 1956.

19. MACKLIN, C. C.: Note on elastic membrane of bronchial tree of mammals, with interpretation of its functional significance. Anat. Rec. 24: 119-135, 1923.

20. MCMANUS, J. F. A.: Histological demonstration of mucin after periodic acid. [Letter to the Editor.]

151-152, 1956.

Nature, London 158: 202, 1946. 21. MILLER, J.: Arrangement of elastic fibres in bron-

chi and lung. J. Anat. 5 Physiol. 40: 162-170, 1905- 1906.

22. MOWRY, R. W.: Alcian blue and alcian blue- periodic acid Schiff stains for carbohydrates. In Manual of Histologic and Special Staining Technics. Washin ton, D.C. Armed Forces Institute of Pathology. 195'5;

23. MUTO, K.: Zur Kenntnis der Basalmembran. Virchows Arch. path. Anat. 300: 652-669, 1937.

24. PAGEL, W.: Zur Pathologie des Asthma bron- chale. Virchows Arch. path. Anat. 286: 580-590, 1932.

25. RHODIN, J., and DALHAMN, T.: Electron micros- copy of tracheal ciliated mucosa in rat. Zschr. Zell- forsch. u. mikr. Anat. 44: 345-412, 1956.

method for histologic demonstration of acid muco- polysaccharides in tissues. A . M . A . Arch. Path. 52: 189-194, 1951.

27. SAUSER, G.: Paries elastico-muscularis tracheae. Acta anat. 30: 705-712, 1957.

28. SAWADA, K.: Uber Zerstorung und Neubildung des elastischen Gewebes in der Lunge bei verschie-

pp. 140-142.

26. RINEHART, J. F., and ABUL-HAJ, s. K.: Improved

denen Erkrankungen. Virchows Arch. 3ath. Anat. 169: 263-278, 1902.

29. SOMMERS, S. C.: Basement membranes, ground substance, and lymphocytic aggregates in aging organs. J. Gerontol. 11: 251-260, 1956.

30. STOUGHTON, R., and WELLS, G.: Histochemical study on polysaccharides in normal and diseased skin. J. Invest. Dermat. 14: 37-51, 1950.

31. SWINEFORD, O., JR., and OCHOTA, L.: Smoking and chronic respiratory disorders; results of abstinence. Ann. Allergy 16: 455-458, 1958.

32. UNNA, P. G.: The Histopathology of Diseases of the Skin. (Transl. by N. Walker.) New York, N.Y. Macmillan & Co. 1896; pp. 981-982.

33. WAGNER, B. M.: Hypersensitivity, role of con- nective tissue. In MIILLORS, R . C., Ed.: Analytical Pathology; Treatises in the Perspective of Biology, Chemistry, and Physics. New York, N.Y. McGraw-Hill Book Company, Inc. 1957; pp. 429-470.

34. WATSON, W. I,., and CONTL, A. J.: Smoking and lung cancer. Cancer 7: 245-249, 1954.

35. WEIDMAN, F. D.: Ageing of skin. In COWDRY, E. V., Ed.: Problems of the Ageing; Biological and Medical Aspects, 2d ed. Baltimore, Md. The Williams & Wilkins Company. 1942; pp. 391-411; 400-401.

36. WHITFIELD, A. G. W.; MELVILLE, A. W., and WATERHOUSE, J. A. H.: Effect of tobacco on lung- volume. Quart. J. Med. 20: 141-147, 1951.