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Title Tracheobronchial airway structure as revealed by casting techniques.

Permalink https://escholarship.org/uc/item/461306bx

Journal The American review of , 128(2 Pt 2)

ISSN 0003-0805

Authors Phalen, RF Oldham, MJ

Publication Date 1983-08-01

DOI 10.1164/arrd.1983.128.2p2.s1

License https://creativecommons.org/licenses/by/4.0/ 4.0

Peer reviewed

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Airway Structures

Tracheobronchial Airway Structure as Revealed by Casting Techniques13

ROBERT F. PHALEN and MICHAEL J. OLDHAM

Introduction The mammalian tracheobronchial airways SUMMARY Research quality tracheobronchial airway casts, prepared in the intact thorax, have participate in several processes that impact proved to be useful in comparative mammalian anatomic studies. Grossly, casts trimmed free on the overall health of an individual spe­ of alveoli are quite different in appearance for different species. Overall organ shape, tracheal cies. These processes are heavily influenced length/diameter ratio, presence or absence of a tracheal , and degree of branching sym­ by the structural characteristics of the air­ metry constitute the major gross characteristics. Detailed morphometric measurements per­ ways. The airways serve as a tubular system formed on such casts reveal important species differences in branch shape, number of divisions that conducts air into and out of the al­ in the tree, and variations in such structure as a function of airway generation number. Of the veolar portions of the . Additionally, mammalian tracheobronchial casts examined to date, those of humans have several distinctive tracheobronchial airways participate in hu- characteristics. Their overall shape is the most nearly spherical; most other mammals have midification and temperature adjustment that are significantly longer along the tracheal axis in relation to their width or thickness. of inspired air and are involved in the cap­ Human branches are typically relatively symmetric with respect to both daughter tube diameter ture, absorption, and elimination of in­ ratio and daughter branch angle ratio. In short, of all of the studied mammalian lungs those of haled airborne pollutants. Tracheobronch­ humans appear to be the least heterogeneous. AM REV RESPIR DIS 1983; 128:S1-S4 ial airways can also become involved in dis­ ease processes such as bronchitis, , vary with respect to state of the organ will typically have with ovoid (not and carcinoma (1). (fresh versus fixed), the choice of casting circular) cross sections, impressions of Proper selection of animal models is an compound (wax, metal, latex, acrylic, sili­ tracheal cartilagenous rings, smooth or important aspect of the scientific study of cone rubber, etc.), and the location of the faintly striated airways, clearly defined bi­ the above phenomena. An understanding organ during infusion (in situ versus ex­ furcations, and straight to gently curved of normal tracheobronchial anatomy not cised). Although most combinations of the airways. Features associated with artifacts, only aids in the selection of these animal above have been used at one time or an­ in addition to those listed in table 1, include models but also provides baseline informa­ other, most recent morphometric studies a circular cross section of a , a high tion that is useful for recognizing abnormal have used a technique first described by proportion (greater than approximately anatomic states. Furthermore, quantitative Phalen and associates (7) in which casts are 0,5%) of bent or twisted airways, grossly descriptions of tracheobronchial airways made by tracheal infusion of room temper­ lumpy airways, thin, flat or "stretched" air­ have been used in mathematical models for ature vulcanizing silicone rubber (RTV) ways, and irregularly shaped carinas. predicting capture efficiencies for inhaled mold-making compounds into fresh lung particulate pollutants (2). prior to removal of the organ from the Morphometric Measurements Given the many uses for comparative thorax (5, 6, 8, 9). The information that Unambiguous, repeatable morphometric tracheobronchial information, very few di­ follows will focus on mammalian tracheo­ measurements on airway casts require two rect comparative anatomic studies have bronchial casts in which infusion of the things, an idealized geometric model and a been performed using more than one spe­ casting compound took place in situ within unique address system that allows each cies. Klimet and coworkers (3) and Klimet the relatively intact thorax. measured airway to be repeatedly located. (4) published considerable data on the When morphometric data are to be tak­ A model and numbering system that has guinea pig and rabbit. Phalen and cowork­ en, an airway cast should be as free of been successfully used (5) is shown in figure ers (5) performed morphometric studies on artifacts as is practically achievable. Several 1A and B. This particular model was de­ replica tracheobronchial casts of humans, factors that can be controlled contribute to vised to provide proper morphometric in­ dogs, rats, and hamsters. Schlesinger and the major artifacts. These factors, their re­ formation for use in mathematical models McFadden (6) published additional com­ lated artifacts and methods for minimiza­ predicting deposition probabilities for in­ parative information on humans and on the tion of artifacts, are given in table 1. When haled particles. Such information includes donkey, rabbit, dog, rat, and hamster. Each adequate precautions are taken, casts can airway lengths, diameters, branch angles, of the above investigators used replica casts be produced in which airway size and and inclinations to gravity. The numbering in their morphometric studies. branch angle artifacts are negligible. An system begins with the trachea (Generation In the sections that follow, the methods analysis of such artifacts has been pub­ 1) labeled 1. The 2 branches off of the for preparing, evaluating, and measuring lished by Yeh and coworkers (10). replica casts of the tracheobronchial air­ Further validation of the fidelity of air­ ways are described. Additionally, new com­ way replica casts can be achieved by com­ 1 From the Air Pollution Health Effects Lab­ parative anatomic data are presented for parison of parameters obtained from mor­ oratory, Community and Environmental Medi­ several mammals. cine, University of California, Irvine, California. phometric or other measurements on the 2 cast with corresponding pulmonary func­ Supported by Contract No. AO-128-32 from Preparation of Research Quality Casts the California Air Resources Board and by Con­ tion or radiographic data obtained prior to tract No. RO1962-1 from the Electric Power Re­ An airway cast is a replica of the air spaces; death of the subject. Once the cast tech­ search Institute. it is typically obtained by the controlled in­ nique has been sufficiently validated and all 3 Requests for reprints should be addressed to fusion of a liquid mold-making medium steps in the procedure standardized, re­ Robert Phalen, Community & Environmental followed by curing and digestion of the sur­ search quality casts can be routinely pro­ Medicine, University of California, Irvine, CA rounding tissue. Cast-making techniques duced. Research quality mammalian casts 92717.

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S2 PHALEN AND OLDHAM

TABLE 1 CONSIDERATIONS FOR CONTROL OF ARTIFACTS FOR IN SITU SALINE REPLACEMENT METHOD

Factors Artifacts on Cast Remedy

Animal quality Atypical anatomy, missing and Use of top-quality animals, good distorted airways husbandry practices Casting compound Shrinkage artifacts, failure to Selection of material with low coefficient cure, fragility, thermally induced of shrinkage, elimination of curing distortion inhibitor contaminants, use of RTV* material Tissue condition Missing, thin, or otherwise Cast within hours of death and distorted airways refrigerate organ if delayed; lavage with physiologic saline prior to casting Air in lung Bubbles in cast or missing Replace air with water soluble gas and portions infuse degassed saline prior to cast material injection Infusion rate If too rapid, overdistention of Infuse at proper rate, 15- to 60-min large airways. If too slow, filling time for most silicone rubber incompletely cast small airways materials Filling volume Over- or underdistended airways Fill to predetermined endpoint; for example, till cast material visible through pleura and lung just fills thorax Curing Improper major branch angles, Cover thorax to prevent drying of lung distorted overall cast shape tissue and leave in thorax until cured cast has full memory

* Room temperature vulcanizing silicone rubber. trachea retain the tracheal identification size, shapes of bifurcations, and organiza­ number followed by either a 1 or a 2 de­ tion and shapes of large airways. Subgross pending on whether the daughter is the characteristics, obtained through hand mor­ larger (in diameter) or smaller of the two, phometry, include branching angles, air­ respectively. For equal diameter daughters, way lengths and airway diameters as a func­ another feature, say length, is used for this tion of generation number, and average selection. This numbering system has sev­ number of generations in the nonalveolar eral important features. Each airway is portion of the tracheobronchial tree. Some uniquely identified and assignable to one of of these characteristics for several mam­ 2 populations, "major" if the identity num­ malian species are summarized in table 2. ber ends in 1, and "minor" if the identity In overall shape, the human lung tends to number ends in 2. The numbering system is be more spherical than the lungs of the continuous in that it contains the complete other species (figure 2); the ferret lung, for Fig. 1. A. Idealized model defining parameters for linkage of each airway back to the trachea. example, is relatively long (in the tracheal morphometric measurement. B. Binary identifi­ Also, the number of digits is equal to the midline direction) in relation to its width. cation system used for locating individual generation number of an airway. Another The significance of overall shape is seen in airways. useful ordering system devised by Horsfield the relatively symmetric airway branching and associates (11) sequentially numbers scheme at essentially all levels in the human airways from smaller to larger. This sys­ compared with the long tapering mono- successive bifurcations also show species tem, however, does not assign unique num­ podial airways with small lateral branches differences. bers to each airway. characteristic of all of the other species in The difference between the more sym­ Hand measurements (morphometry) on table 2. metric branching in the human and the casts are usually facilitated by trimming Tracheal length-to-diameter (L/D) ratios monopodial nature of most mammals is away small airways. For tracheobronchial differ considerably among mammals. This seen in the typical airway L/D ratio and the morphometry, severing the most proximal ratio is near 20 in the goat and ferret. The typical branch angles for major and minor respiratory in each will guinea pig and rabbit also have relatively daughter airway tubes. Missing data in leave a complete conducting airway. On a long tracheas with L/D ratios of about 14 table 2 for these and other characteristics dog lung cast this procedure can require and 12, respectively. Dog, monkey, rat, result from a lack of morphometric data on more than 200 h. Direct measurements on and hamster are intermediate, with this casts. casts are performed using dial or vernier- ratio varying between 10.6 and 8.8. The hu­ With respect to average number of type micrometer calipers, magnifying eye­ man is again an extreme at 6. branchings (tracheal carina = number 1) pieces with calibrated reticles, and trans­ Additional species variation is seen in the down to the terminal , the hu­ parent protractor scales. Such measure­ shape of the main tracheal bifurcation (fig­ man has the narrowest range. Again this is ment of an entire dog tracheobronchial tree ure 3). The dog has a blunt bifurcation fol­ a reflection of the overall symmetry of the takes about 1 person-year (figure 1). lowed in order of increasing sharpness by human lung. the goat, pig, human, rhesus monkey, fer­ Respiratory bronchioles, as defined on ret, guinea pig, rabbit, hamster, and rat. replica casts, are airway tubes with walls Comparative Lung Structure The rat has an exceptionally tall, thin, flap­ that contain alveoli. Such alveoli, which are Airway casts can be used to examine gross like tracheal carina. Further, the main clearly seen under low-power magnifica­ and subgross morphologic characteristics bronchi of the rat and hamster become no­ tion, can be very few in number or can al­ in mammals. Gross characteristics include ticeably wider after branching from the most completely cover an airway tube. overall shape of the lung, relative tracheal trachea. As seen in table 2, the shapes of When a tube is completely alveolarized it is

TRACHEOBRONCHIAL CASTS S3

TABLE 2 COMPARATIVE AIRWAY STRUCTURE AS REVEALED ON REPLICA CASTS

Typical Structure (GEN 6) Typical Gross Structure Number of Branch Angles Branches Trachea Airway (Major Daughter/ to Mammal Airway L/D Tracheal Major Airway L/D Minor Daughter) Terminal Respiratory Body Mass Branching (cm) Bronchus Bifurcations (ratio) (degrees) Bronchiole Bronchioles

Human, 80 kg Relatively 12/2 Extremely Sharp for about the 2.2 11/33 14-17 About 3-5 orders symmetric rare first 10 generations, relatively blunt thereafter Beagle dog, Strongly 17/1.6 Absent Blunt tracheal 1.3 8/62 15-22 About 3-5 orders 10 kg monopodial bifurcation, others sharp Laboratory Rat, Strongly 2.3/0.26 Rare Very sharp and very 1.5 13/60 12-20 Essentially absent 0.3 kg monopodial high throughout lung Ferret, 0.61 kg Strongly 10/0.5 Absent Sharp 2.0 16/57 About 3-4 orders monopodial Guinea pig, 1 kg Monopodial 5.7/0.4 Absent Very sharp and high 1.7 7/76 About 1 order Rabbit, 4.5 kg Strongly 6/0.5 Absent Sharp 1.9 15/75 About 1-2 orders monopodial Goat, about 30 kg Monopodial 30/1.5 Present Mixed blunt and sharp 2.1 12/70 About 5-6 orders Golden hamster, Strongly 2.4/0.26 Absent Very sharp 1.2 15/63 10-18 About 1 order 0.14 kg monopodial Rhesus monkey, Monopodial 3/0.3 Absent Mixed blunt and sharp 2.6 20/62 About 4 orders about 2 kg

Definition of abbreviations: L/D = length/diameter ratio. usually referred to as an or an airways within a single generation that replica casts of lungs of the human, dog, alveolar sac. The rat, hamster, and guinea could be classified as producing one order goat, ferret, and rhesus monkey. pig lungs are essentially free of respiratory of respiratory bronchioles. Rabbit lungs The significance of these highly variable bronchioles, although the hamster and appear to have some scattered respiratory structural characteristics is relatively un­ guinea pig have a transition to alveolarized bronchioles, and several orders are seen on known. One postulates that air-flow char-

Fig. 2. Trimmed in situ tracheobronchial replica casts for adult male human (left) and adult male beagle dog (right).

S4 PHALEN AND OLDHAM

acteristics, inhaled particle deposition pat­ 4. Klimet V. Dichotomical model of respiratory terns, and the distribution of dead space airways of the rabbit and its significance for the will all be significantly influenced. construction of deposition models. Folia Mor- phol (Praha) 1974; 22:286-90. A cknowledgment 5. Phalen RF, Yeh HC, Schum GM, Raabe OG. Application of an idealized model to mor­ The writers thank Dianne Wilner and Mike phometry of the mammalian tracheobronchial Numamoto for ferret casts and Sonia Usdansky tree. Anat Rec 1978; 190:167-76. for help in preparation of the manuscript. 6. Schlesinger RB, McFadden LA. Compara­ tive morphometry of the upper bronchial tree in References six mammalian species. Anat Rec 1981; 199:99- 1. Phalen RF, Faeder EJ, Cavender FL, Reischl 108. P. Response of the to inhaled 7. Phalen RF, Yeh HC, Raabe OG, Velasquez pollutants. In: Willeke K, ed, Generation of FERRET DJ. Casting the lungs in-situ. Anat Rec 1973; aerosols. Ann Arbor: Ann Arbor Science, 1980: 177:255-63. 125-38. 8. Schreider JP, Raabe OG. Replica casts of the 2. Morrow PE, Bates DV, Fish BR, Hatch TF, entire respiratory airways of experimental ani­ Mercer TT. Deposition and retention models for mals. J Environ Pathol Toxicol 1980; 4:427-35. internal dosimetry of the human respiratory tract. Health Phys 1966; 12:173-207. 9. Schlesinger RB. Particle deposition in model systems of human and experimental animal air­ 3. Klimet V, Libich J, Kaudersova V. Geometry ways. In: Willeke K, ed, Generation of aerosols. of guinea pig respiratory tract and application of Ann Arbor: Ann Arbor Science, 1980:553-75. Landahl's model of deposition of aerosol parti­ cles. J Hyg Epidemiol Microbiol Immunol 1972; 10. Yeh HC, Hulbert AJ, Phalen RF, Velas­ 16:107-14. quez DJ, Harris TD. A stereoradiographic tech­ nique and its application to the evaluation of RHESUS MONKEY lung casts. Invest Radiol 1975; 10:351-7. Fig. 3. Variations in the mammalian tracheal cari­ 11. Horsfield K, Dart G, Olson DE, Filley GF, na. Each tracing was made from either a photo­ Cumming G. Models of the human bronchial graph or a projection of an in situ replica cast. tree. J Appl Physiol 1971; 31:207-17.

Structure as Revealed by Airway Dissection

A Comparison of Mammalian Lungs1-3

CHARLES G. PLOPPER, ANDREW T. MARIASSY, and LANCE O. LOLLINI

Introduction The study of corrosion casts has provided SUMMARY Microdissection of mammalian pulmonary airways demonstrates branching pat­ a great deal of new information concerning terns and provides precisely defined tissue samples for morphologic study. The dissections are intrapulmonary airway morphologic and performed on lung fixed by airway infusion at standard pressures. Using fine scissors and a high toxicologic features (1). Measurements of resolution dual-viewing dissecting microscope, extrapulmonary and intrapulmonary airways are casts have demonstrated differences in air­ dissected down their axial pathways. The plane of dissection is chosen to include as many minor way length, diameter, and angle of branch­ daughter (side) branches as possible. Lungs from 5 species: sheep, goat, cat, rabbit, and bonnet ing, and the numbers of generations of monkey have been dissected, photographed, successive generations numbered, and pieces of branching among mammalian species (2). tissue processed for LM, TEM, and SEM. Branching patterns differ between lobes (cranial versus While this type of information is extremely caudal) of the same species and between the same lobe in different species. Marked differences useful for developing models to estimate ef­ in epithelial population distribution within the airway tree are found between the same lobe of fects of particulate and noxious gas distri­ different species (i.e., cranial lobes of rabbit and sheep) and between different lobes in the same butions in airways, including amounts, species (i.e., cranial and caudal lobes of the sheep). The dissection approach to pulmonary air­ rates, and sites of deposition within the air­ way morphologic studies provides specimens of precisely defined branching history, generation way tree, the nature of the cellular response number, and anatomic position within regions of the lung and within specific segments. This al­ lows studies that compare: {1) different airway generations in the same pathway, (2) bifurcation 1 From the Departments of Anatomy and Vet­ points and the airway segments between them, (3) terminal airways of differing pathway lengths erinary Pathology and the California Primate and numbers of branching, (4) terminal airways of different regions of same lobe, (5) same airway Research Center, School of Veterinary Medicine, generations in different lobes, and (6) same airway generations from animal to animal and spe­ University of California, Davis, California. cies to species. AM REV RESPIR DIS 1983; 128:S4-S7 2 Supported in part by Grants No. HL-28978, No. DRR-00169, and No. 222Y01-ES-0056 from the National Institutes of Health, and by Grant cannot be assessed because of the tissue thelium in mammalian lungs. This report is No. R808533010 from the U.S. Environmental Protection Agency. destruction necessary to produce the cor­ a summary of one approach to the problem 3 Requests for reprints should be addressed to rosion casts. The data derived from these of precision in the selection of airway tis­ Charles G. Plopper, Department of Anatomy, models suggest the need for more specifici­ sue. Airway microdissection is followed by School of Veterinary Medicine, University of ty in the selection of tissue samples for ana­ processing of large blocks of tissue for California, Davis, CA 95616. tomic and pathologic studies of airway epi­ light, transmission, and scanning electron