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inary isters zone- Res. :kjen, rogen ,uinea , AND v plehysiol. -1110. \ oneiduced Respir A. L ibrosis mycin. iriables 0. L, J. J id lung Appl 48(6). , n E. L lU c-.U S Physiol method irmance >igs and ric acid col. 63. , M. F lmonar> products FUNDAMENTAL AND APPLIED TOXICOLOGY 5, 435-450 ( 1985) Clearance from the Respiratory Tract1 Richard B. Schlesinger Institute o f Environmental Medicine. New York University Medical Center. 550 First Avenue. New York. New York 10016 Clearance from the Respiratory Tract. Sc h l e s in g e r , R. B. (1985). Fundam Appl Toxicol. 5, 435-450. Clearance is an essential component of the defense system of the respiratory tract. Alterations in the clearance of insoluble particles have been shown to provide a sensitive indicator of response due to exposure to various inhaled materials. This paper reviews clearance mechanisms, the significance o f clearance changes, and the methodology used to assess them. 1985 Society of Toxicology. Clearance is the physical removal out of the respiratory tract of material which deposits on airway surfaces. It is a major component of a defense arsenal which in total serves to prevent or reduce both local damage by and systemic absorption of inhaled toxicants. Examination of the effects of inhalants upon lung defense systems should be a part of overall toxicological assessments, since regardless of the specific protocols o f any inhalation study, determination of the fate of these materials is essential for interpretation of results obtained from the exposures. The rates and routes of clearance relate to the residence time of deposited agents within the respiratory tract and it is ultimately this time which, when coupled with aspects of metab olism and physicochemical properties o f the inhalant, relates to the degree of local and/ or systemic toxic response. This paper describes the use of techniques designed to measure changes in the clearance f insoluble, nonviable particles from the respiratory tract o f experimental animals and humans as an endpoint in inhalation toxicoiogical studies, i.e., as an indicator o f toxic response and/or as a predictor of respiratory tract disease. Presented at the Symposium on New Approaches fof the Evaluation of Pulmonary Toxicology, 23rd Annual Meeting 0f the Society o f Toxicology, Atlanta, Ga., M,r<th 12-16, 1984. OVERVIEW OF CLEARANCE MECHANISMS AND PATHWAYS Clearance comprises various physiological functions, and is not a uniform process throughout the entire respiratory tract. There are regional differences, both mechanistically and temporally. Clearance of insoluble material from the conducting airways occurs via the mucociliary system. Except for the vestibular region of the nasal cavity and the posterior nasophar ynx, the nasal passages and the tracheobron chial tree through the terminal bronchioles are lined with a ciliated epithelium overlaid by a fluid, commonly called mucus. In the upper respiratory tract and the bronchi, the fluid blanket has two distinct layers (Lucas and Douglas, 1934), a lowviscosity hypophase, which surrounds the cilia and within which they move, and a high-viscosity epiphase lying on top of the cilia. In bronchioles, the fluid layer lacks the epiphase and is thinner (Luchtel, 1976); its composition and structure resemble that of surfactant (Macklem et al.. 1970; Gil and Weibel, 1971). Deposited material is cleared from most conducting airways by movement of the epiphase due to the coordinated beating o f the cilia; movement of mucus from the nasal passages occurs distally toward the na sopharynx, and from the bronchial tree prox imally toward the oropharynx. In the bron- 435 0272-0590/85 $3.00 Copyright (.c) 1985 by the Society of Toxicology All rights of reproduction in any form reserved. 4 3 6 RICHARD B. SCHLESINGER chioles, fluid movement is likely due both to ciliary beating as well as to surface tension differences between bronchioles and alveoli. The nasal vestibular region is cleared via mechanical means, i.e., blowing, wiping, or sneezing. Clearance from the nonciliated respiratory region of the lung occurs via a number of mechanisms and pathways, but the relative importance of each is not always certain and may depend to some extent upon the physi cochemical properties and amount of material deposited. The first line o f defense is the alveolar macrophage. These large cells move on the alveolar epithelium via amoeboid motion, are phagocytes, and contain an array of proteolytic enzymes that allow them to digest a wide variety of materials. Contact with deposited particles may occur by chance, or be due to directed motion resulting from the release of chemotactic factors. Although most deposited particles are ingested, which helps prevent their penetration through al veolar epithelium and translocation to other sites, the efficiency of phagocytosis may de pend on specific properties o f the particles, e.g., size, shape, and composition (Camner, 1980). Particle-laden macrophages, as well as free particles, may be cleared from the respiratory region via a number of pathways, one being the mucociliary system. However, the route(s) by which macrophages reach the distal end of the mucous blanket is uncertain. Suggested pathways are along the alveolar surface, due to surface fluid flux or directed locomotion (Kilbum, 1968; Scarpelli and Condorelli, 1975; Sorokin and Brain, 1975; Ferin, 1976), or via the pulmonary interstitium (Brundelet, 1965; Green, 1973; Kilbum, 1974; Tucker el al., 1973), as cells sieve through the alveolar epithelium into interalveolar areas, from which they then penetrate into the bronchiolar lumen. Although it is not certain whether macro phages can actually reenter the interstitium from alveolar spaces, free particles may be directly translocated through the alveolar ep ithelium (Lauweryns and Baert, 1974; Rob ertson, 1980; Brody el al., 1981). Once there, these particles may be engulfed by resident macrophages which, in turn, may migrate to a nearby lymphatic channel or be carried in the flow of interstitial fluid toward the lym phatic system, bronchial tree, or to perivenous or subpleural sites. Uningested particles in the interstitium may traverse the capillary endothelium (Robertson, 1980), while extremely small particles, i.e., those with diameters < 10 nm, may enter the blood directly from alveoli via diffusion through the air-blood barrier (Gross and Westrick, 1954; Raabe, 1982); however, entry into the lymphatic system is more likely. Clearance via the lymphatic system is relatively slow (Ferin, 1976; Sorokin and Brain, 1975), and particles may be trapped in bronchial lymph nodes, which often be come major reservoirs of retained materials. Soluble particles can dissolve in the alveolar fluid, then diffuse through the epithelium and interstitium into the lymph or blood (Chinard, 1966; Morrow, 1973). Although dissolution competes with other clearance mechanisms for temporal advantage in all respiratory tract regions, because the me chanical clearance processes in the alveolar region are slow, materials considered to be relatively "insoluble" may have high rates of dissolution in vivo, especially if the particle size is very small (Raabe, 1982). Unfortu nately, the factors affecting solubility are poorly understood, although it is known to be influenced by surface-volume ratio and other surface properties o f deposited particles (Morrow. 1973). In terms of kinetics, clearance from the respiratory tract occurs at different temporal scales in different regions and, to some extent, even within the same region. For example, the velocity of the mucus blanket increases from distal to proximal airways in the bron chial tree. Insoluble material that deposits and remains on ciliated airways will normally be cleared from the respiratory tract within 24 hr; the exact time depends upon the depth of penetration. However, the completeness of bronchial clearance within this time has there, sident ate to ied in lymenous titium elium small 0 nm, oli via (Gross wever, more tem is a and apped en be:erials. lveolar elium K xl T ,n arance in all e meIveolar to be ates ol ^article nfortuity are 3\vn to io and articles :>m the mporal extent Lamp!1-'icreasi"e bronleposHv ormai1' withm e dep11' letenc" RESPIRATORY TRACT CLEARANCE 437 occasionally been challenged, and it has been suggested that slower clearance from periph eral bronchi may extend beyond this 24-hr period (Clarke and Pavia, 1980) and that a small fraction of deposited material may actually be retained within the bronchial tree for days (Patrick and Stirling, 1977a). Particles depositing in respiratory airways generally remain longer than those deposited in conducting airways. Casarett (1972) has related a series o f three temporal phases for normal alveolar clearance to specific physio logical processes: an initial fast phase, having a half-time o f ~ 2 - 6 weeks, representing rel atively rapid clearance by macrophages via the bronchial tree; an intermediate phase of slower macrophage-mediated clearance, in volving translocation via the lymphatic chan nels, and having a half-time on the order of months; and a phase o f still slower clearance, with a half-time o f months to years, repre senting removal by dissolution. SIGNIFICANCE OF CLEARANCE AS A TOXICOLOGICAL ENDPOINT A valid toxicological endpoint should meet certain basic criteria. It should be reproducible (in controls), it should be fairly sensitive to change following exposure to appropriate agents, and it should have some relationship lo respiratory tract disease. Both alveolar and mucociliary clearance rates are well-defined functional characteris tics of an individual when tests are performed under the same conditions. There is a close correspondence in repeated analyses in the same individual for alveolar clearance rates In- for example, humans (Bohning et al., 1982), dogs (Gibb and Morrow, 1962), and donkeys (Halpem et al., 1981), and for mucociliary clearance rates in humans Lippmann et al., 1980), donkeys (Schlesinger a al , 1978, 1979), and rabbits (Chen, 1983). Work performed in this laboratory has *hown bronchial mucociliary clearance altera,ln to be a sensitive physiologic indicator f irritant response, based upon acute expo sures to one particular material, sulfuric acid (H2S 0 4) mist, a common ambient air pollut ant. Transient alterations in bronchial mu cociliary rate have been produced at low exposure levels in humans (0.1 mg/m3) (Lei kauf et al., 1981), donkeys (0.2 mg/m3) (Schlesinger et al., 1978), and rabbits (0.1 mg/m3) (Schlesinger el al., 1984). Although such a response to inhaled irritants may be adaptive, helping to maintain organ homeo stasis, these changes are more likely a patho physiological response of the airways and, although temporary, they may foreshadow more permanent alterations or progressive changes which would follow continued ex posures (Schlesinger et al., 1978, 1979). Interpretation of mucociliary clearance al terations in terms of potential health problems is somewhat speculative. Dysfunction o f mu cous transport may be involved in the patho genesis of both acute and chronic respiratory disease, but the clinical relevance of muco ciliary change is only recently being elucidated and its importance to pulmonary disease development is only beginning to be experi mentally established (e.g., Schlesinger et al., 1983). Clinical studies of individuals with a disease syndrome characterized by impaired clearance, i.e., primary ciliary dyskinesia (PCD), may be used to assess the importance o f mucociliary transport and the effect o f its dysfunction upon respiratory disease, and to provide information on the role o f mucoci liary clearance in maintaining the integrity o f the lung. The lack of mucociliary function in PCD is directly responsible for the early development o f recurrent respiratory tract infections and, eventually, chronic bronchitis and bronchiectasis (Wanner, 1980; Rossman el al., 1984). It is, however, not certain whether partial impairment of the mucocili ary system will increase the risk of lung disease. In this regard, the rate o f mucociliary clearance may be a factor affecting the de velopment o f infectious disease. Pathogenic organisms depositing in the conducting air ways are confronted with the mucociliary barrier, and the extent of penetration of vectors through the mucus to the underlying 4 3 8 RICHARD B. SCHLESINGER cells may be important in disease develop ment. This rate, relative to the rate o f mu cociliary transport out of the respiratory tract, could determine the effectiveness of inhaled pathogens in initiating disease (Proctor, 1979; Niederman et al., 1983). It is known that in conditions characterized by retarded clearance from conducting airways, e.g., chronic bron chitis, there is a predisposition to respiratory infection (Cumming and Semple, 1980). In addition, destruction of the functional integ rity o f the ciliated epithelium can result in impaired defense against bacteria (Laurenzi and Guameri, 1966), and impaired transport has been observed in viral respiratory infec tions (Bang and Foard, 1964; Lourenco et al., 1971b). Retardation o f mucociliary clearance may also be a factor in the pathogenesis of bron chial cancer. Inhaled aerosols tend to selec tively deposit at airway branching sites in the bronchial tree (Schlesinger and Lippmann, 1978). Histological studies (Kotin and Falk, 1959; Auerbach et al., 1961) have indicated that neoplastic and preneoplastic lesions pre dominate at these bifurcation regions. Macklin (1956) and Hilding (1957) have suggested that bifurcations are areas o f normally slower mucus movement; this would add to the residence time o f particulates which deposit within these areas, or are being carried through them on the mucociliary escalator. Thus, the selective distribution o f lesions at bifurcations within the upper bronchial tree may be the result o f both selective deposition and slowed clearance, resulting in prolonged retention of high local concentrations of de posited carcinogens. Although there is no direct evidence that ineffectual clearance is a contributory factor to the development of bronchogenic carcinoma, a causal relation has been suggested between adenocarcinoma and sites of local particle retention and in adequate clearance in the nasal passages of furniture workers (Morgan et al.. 1974; Hadfield and Macbeth, 1971). There is accumulating evidence that dys function o f bronchial clearance plays a role in the pathogenesis o f chronic bronchitis; it is known that mucus transport is impaired in individuals who have this disease (Wanner, 1977) . The rate at which individual healthy humans clear deposited particles from the lungs varies widely. In cigarette smokers and persons with chronic obstructive pulmonary disease, there is a somewhat wider variation (Albert et al,, 1973; Gongora et al., 1981). In such groups, the within-subject variation is also greatly increased, suggesting that loss of control o f mucociliary transport could cause and/or result from such disease. Schle singer et al. (1983) showed, in an experimen tal animal, that modest changes in mucoci liary clearance rates may be associated with secretory epithelial changes in small bronchi and bronchioles, changes which, if continued, could lead to clinical manifestations of bron chitis. Other studies suggest that mucociliary dysfunction is an early indication o f disease changes in the lungs. For example, retarded clearance has been demonstrated in bronchitics who showed no sign o f airway obstruction (Mossberg and Camner, 1980), while young smokers having various degrees of impair ment of tracheal mucus transport rates had no overt bronchitic symptoms and had nor mal pulmonary function (Goodman et al.. 1978) . The pathogenic implications of alterations in clearance from the alveolar region have not been examined to the extent that changes in mucociliary clearance have. Alveolar clearance rates appear to be reduced in people with chronic obstructive lung disease (Bohning et al., 1982) and in cigarette smokers (Cohen et al., 1979; Bohning et al., 1982). suggesting some relation between altered de fense and disease development. Clearance dysfunction has also been shown in expenmental animals having viral infections (Cresia e t a i . 1973). The adequate performance of alveolar macrophages is critical in determining the effectiveness o f pulmonary region defense in minimizing the residence time o f deposited toxicants. For example, phagocytosis plays an important role in the prevention of particle entry into fixed tissues of the lung, a region , impaired : (Wanner, al healthy from the lokers and >ulmonary - variation al., 1981). variation g that loss >ort could ase. Schlexperimenn mucociiated with ill bronchi continued, is o f brontucociliary of disease retarded n bronchi>bstruction ii' 'oung j. ipair- rates had l had norlan et al- alterations gion have at changes Alveolar 1in people ise (Bohni smokers al., 1982). altered de Clearance in expenms (Cresia f alveolar lining the defense m deposited osis pla>^ of particle a regie'1 RESPIRATORY TRACT CLEARANCE 439 from which clearance is very slow; accumu lations of several types of dust have been directly linked to development o f lung disease. Damage to macrophages has also been im plicated in the pathogenesis o f chronic lung diseases involving proteolysis (e.g., emphy sema) and fibrogenesis (e.g., silicosis, asbestosis) (Brain. 1980; Warheit et al.. 1984), as well as in an increased risk o f viral and bacterial infections (Hocking and Golde, 1979). Furthermore, the viability and func tional activity of macrophages is impaired in people with asthma (Godard et al.. 1982). species may not necessarily be more suitable than another as a model for clearance studies. Although there are anatomical and physio logical differences, these may be secondary to a project whose endpoint is alteration of clearance due to an inhaled toxicant. Specific animals, thus, may be chosen for use based upon other criteria. For example, accurate measurements over extended periods o f time favor the use of unanesthetized animals, and are facilitated by the use of species which are relatively docile. TECHNIQUES OF CLEARANCE MEASUREMENT Choice of Animal Model The species of choice in clearance studies is humans, but certain experimental protocols preclude their use. Fortunately, the broad mechanisms of clearance, i.e., mucociliary in conducting airways and cellular and disso lution in respiratory airways, are similar in humans and other mammals commonly used in inhalation toxicology studies. Interspecies differences do occur, for example, in secretory cell morphology and distribution, macro phage function, the relative role of each clearance mechanism, or rates and efficiency of clearance from various regions, differences which could affect retention and dose (Brain and Mensah, 1983). Short-term, i.e., muco ciliary, times especially may show a substan tial difference between species, even for sim ilar particles deposited under comparable ex posure conditions; much greater interspecies similarity occurs in patterns of clearance from aerosols deposited distally to ciliated airways (Thomas, 1972; Phalen, 1984). Although rates of mucociliary clearance may be species specific, the response to nu merous toxicants, e.g., H2S 0 4, S 0 2, and cig arette smoke, is qualitatively similar between humans and many experimental animals (Lippmann et al., 1982; Schlesinger et al, '984; Phalen, 1984). Thus, one particular Specific Techniques Clearance studies with humans or experi mental animals are generally performed by examining the rate of removal or transport of markers from the lungs as a whole, or from specific individual airways. When using experimental animals, serial sacrifice or fecal analysis techniques may be employed. The actual measurements of rates or times are strongly influenced by the specific method ology. Measurements o f Local Mucus Velocity Mucus transport velocities in the nasal passages, trachea, and main bronchi may be directly measured by monitoring markers on the epithelium, or by measurement of the movement of boli of radioactive particles selectively deposited within these airways or moving through them from more distal areas. Actual rates of movement are obtained by determining the time needed for the bolus or marker to traverse a calibrated distance or to move between two anatomically defined areas. The markers used have included cel lular debris, or material specifically intro duced into the airways, e.g., india ink drop lets, Teflon disks, radiolabeled resin beads, powders, colored solutions and dyes, or pollen grains. Various measurement techniques have been used. Proctor et al. (1977) placed sac 4 4 0 RICHARD B. SCHLESINGER charin particles in the nose o f humans and measured the time until the subject reported the first taste of sweetness. Some nasal mark ers have been viewed directly, or indirectly by looking, with mirrors, for their appearance in the nasopharynx after placing them on the anterior nasal mucosa (van Ree and van Dishoeck, 1962; Bang et al., 1967). Radio active or radioopaque markers in the upper respiratory tract or central airways may be viewed using external monitoring techniques, including fluoroscopy (Friedman et al., 1977; Goodman et al., 1978; Mezey et al., 1978), cinebronchofiberoscopy (Sackner et al., 1973; Santa Cruz et al., 1974; Landa et al., 1975; Toomes et al., 1981), scintillation detection (Proctor et al., 1977; Patrick and Stirling, 1977b; Man et al., 1980; Wolff et al., 1982b), or gamma-camera imaging (Quinlan et al., 1969; Chopra et al.. 1977; Puchelle et al., 1982; Wolff et al., 1982b). Transport velocities in the trachea and main bronchi have also been assessed by monitoring the movement o f inhaled boli of radiolabeled particles. Yeates el al. (1975) selectively deposited a bolus o f large, tagged albumin microspheres in human central air ways. Movement along the trachea was mon itored by a gamma camera. Later techniques employed a multidetector probe consisting of a series o f three or six small collimated scintillation detectors aligned over the trachea, and boli o f tagged ferric oxide microspheres (Yeates et al., 1981 a,b). Foster et al. (1978; 1982), using a gamma camera, assessed the transport velocity in the major bronchi of labeled ferric oxide. Wolff et al. (1982b) measured the leading edge o f instilled, tagged albumin in the trachea o f rats using scintil lation detectors, and in dogs with a gamma camera. Tracheal mucus velocities have also been measured in donkeys by assessing the time required for a bolus o f tagged particles to traverse a fixed distance between two tracheal fields as these particles moved up from distal airways (Schlesinger et a!., 1978). The advantage of local velocity techniques is that they allow measurement in anatomi cally defined, albeit limited, airways. In ad dition, because of the use of a specific site, there is no question as to whether altered clearance rates due to toxicant exposure re sulted from actual alterations in the mucous system, or to a change in deposition pattern; the latter is a possibility when using wholelung clearance assays, as discussed below. However, there are a number o f disadvan tages. The marker techniques are invasive, in that particles are usually selectively intro duced into the airway of interest. The nec essary use o f anesthetics may affect the ob served transport rates (Landa et al., 1975; Patrick and Stirling, 1977b), and the actual method of introduction may result in some trauma to the airway. In addition, if only one or two marker particles are used, place ment is critical, since zones o f slower and faster transport may occur in the airways (Phipps, 1981). It is also possible that the physicochemical properties and, perhaps, size o f the marker may determine the extent of penetration into the mucus and, therefore, the manner in which it is transported (Lee et al., 1979; Wolff and Muggenburg, 1979). However, a few studies have shown that the rate of tracheal transport of one material in various animals or o f various materials in one animal was not influenced by the char acter of the marker, i.e., its size, mass, or composition (Antweiler, 1958; Man et al.. 1980). Ahmed et al. (1979) suggested that radiation from the tracer particles may affect transport rate, but a later study by Wolff et al. (1982a) did not support this. Because it is easier to measure than is whole-lung clearance, alteration in tracheal transport rate has been used as the sole endpoint in a number o f studies aimed at assessing the effects o f inhaled toxicants upon the mucociliary clearance system. However, such transport velocities may not be adequate indices to imply an overall effect. For ex ample, studies performed in this laboratory in both humans (Leikauf et al., 1981) and donkeys (Schlesinger et al., 1978, 1979; Albert et al.. 1974) demonstrated irritant aerosol- RESPIRATORY TRACT CLEARANCE 441 a adsite, Itered re reucous ttem; /hole>elow. dvanasive, intro ; necre ob- 1975; actual some f only place;r and irways at the js, size tent of refore. c -e 1>>9). hat the ;rial in ials in e charass, or et al. 2d that y affect Volff el than is racheal he sole med at ts upon owever. dequate For ex,0 ratoo 81) and ); Albert aerosol- induced changes in bronchial clearance that were not associated with any alteration in tracheal transport rate. The reason for the lack of effect upon tracheal transport in these studies may be that the trachea is more resistant to irritant effects or, more likely, the small size of the irritant aerosols used precluded significant tracheal deposition. This latter is supported by Wolff el al. (1981), who reported a depres sion in tracheal transport rate in anesthetized dogs exposed to a 0.9-/im H2S 0 4 mist while no effect was observed at identical exposures to a 0.3-jim aerosol. With exposures o f sheep to even higher mass concentrations, Sackner et al. (1978) found no change in tracheal 1 mucus velocity when using a 0.1-^m H2S 0 4 aerosol. Thus, the use of alterations in clear ance in specific airways as a monitor of mucociliary function is valid only if enough of the agent o f interest deposits within the region measured. Therefore, care must be exerted in selecting regions o f interest based upon the properties of the toxicant employed in the study. Whole-Lung Clearance Radioaerosol technique. The most com monly used technique to measure whole-lung clearance involves inhalation of a radiolabeled tracer aerosol. The total amount o f radioac tivity remaining in the lung at selected inter vals is then measured by external detector systems. The decline in emission rate, cor rected for radioactive decay, represents clear ance. Tracer materials used have included Teflon, polystyrene latex, hematite, magne tite, clay, and albumin. Various types and configurations of scin tillation detectors have been used to monitor clearance; these may be divided into profile in n in g (mobile) or stationary (fixed) sys tems. In the former arrangement, either (1) he detectors move relative to the thorax of he subject who has inhaled the tracer aerosol ILaBelle et al., 1964; Holma, 1967a,b; Camner et al., 1971; Camner and Philipson, 1971, 1978), or (2) the subject is moved relative to stationary detectorfs) (Albert et al., 1968). In either case, measurements are obtained in discrete preselected positions, or during continuous scanning, i.e., movement at a constant rate. Scanning systems are relatively indepen dent o f the apex to base distribution of deposited tracer particles in the lung, since they provide a longitudinal activity distribu tion map. They also reduce counting varia tions due to slight differences in positioning the subject or due to subject movements, since they are not as dependent upon the exact equivalent geometry o f the subject's location in relation to the detectors as are fixed systems. In fixed systems, collimated detectors are placed in one or more positions relative to the subject's thorax. Configurations used in clude single, central anteriorly placed detec tors (Albert and Arnett, 1955; Toigo et al., 1963; Thomson and Short, 1969); two detec tors, one placed centrally and one laterally (Luchsinger et al., 1968); twin axially opposed detectors placed anterioposteriorly (Booker et al., 1967; Thomas el al., 1974; Leikauf et al., 1981) or laterally (Schlesinger et al., 1982b); and multiple detector arrays (Albert et al., 1969; Stahlhofen et al., 1981; Bailey et al., 1982). Additionally, two other systems have been used. These are the gamma camera (Klimek et al., 1969; Lourenco et al., 1971a; Sanchis et al., 1972; Wilkey et al., 1980; Puchelle et al., 1982) and the whole-body counter (Cresia et a l, 1973; Bohning el al, 1982; Snipes et a l, 1983). Most fixed systems provide for measurements in only one subject at a time. A system developed by Schlesinger et al. (1982b) for rabbits allows simultaneous measurement in up to 15 animals. Unilateral detector systems require exact positioning for reproducible results, while dual or multidetector systems (where the signal output is combined) are less sensitive to changes in position of the subject in the measurement plane or to effects o f redistri bution of retained tracer particles in the 4 4 2 RICHARD B. SCHLESINGER lungs. The gamma camera provides an as sessment o f total clearance and also allows actual visualization of the distribution of retained particles at various times postexpo sure. The imaging field is usually divided into central and peripheral areas, although more recent techniques subdivide the lung further. The airways within which the test aerosol deposits exist in a three-dimensional array in the lungs and are, therefore, at various depths relative to the detectors. Thus, the efficiency at which retained activity within each airway is measured will vary. It therefore follows that the detector configuration will affect the ultimate shape of the clearance curve. In addition, because of sensitivity differences, the amount of activity needed for a successful study varies, depending upon the type of detectors used. The gamma camera offers the greatest advantage in terms of spatial reso lution but it has low sensitivity and requires large amounts of activity. Scanning systems have poorer spatial resolution but better sen sitivity, while multiple stationary detectors, which offer little information on intrathoracic particle distribution, are the most sensitive. The most responsive fixed system is the whole-body counter. However, this technique is not suitable for use during the first few days after tracer exposure since, because it is not collimated, it cannot effectively distin guish between activity in the lungs from that cleared into the stomach during the initial, rapid tracheobronchial clearance phase. It can, however, be used to monitor long-term clearance once the activity in the rest of the body is low compared to that in the lungs. Normal clearance patterns which are car ried out beyond 1 day have two distinct broad temporal phases: an initial rapid phase, and a subsequent slower phase. The former is presumed to represent mucociliary clear ance of aerosol initially deposited on the bronchial tree, while the latter is controlled largely by nonmucociliary mechanisms, and presumably represents material deposited be low the level o f the mucous escalator. Since regional deposition is not directly measured, these apportionments are based on the as sumption that material cleared within 24 hr after inhalation resided initially on the bron chial tree. Therefore, radioactivity remaining after this time represents material in the alveolar region. Although the assumption is a useful one, it has occasionally been chal lenged, as discussed previously. Quantitation is necessary to describe the effects of toxicants, and clearance curves have been analyzed in a number of ways. They have been fitted with single exponen tials, or a series of such functions. However, because all clearance mechanisms occur si multaneously, the terms in such functions may not necessarily be related to specific physiologic mechanisms. Curves have also been described in terms o f the fraction or percentage of initial lung burden which re mains at various times postexposure or by the time required for a certain percentage of this initial activity to be cleared. Total clear ance may also be assessed by measuring the area under the clearance curve. Gamma camera images are analyzed in terms of changes in the distribution of particles in the various lung zones. One of the major problems associated with external monitoring techniques is the depen dence o f the observed mucociliary clearance pattern upon the pattern o f initial deposition of the tracer aerosol. This is because the techniques are indirect, and clearance pa rameters are proportional to transit pathways. Thus, for example, an apparent increase in clearance rate after toxicant exposure could be due to a proximal shift in deposition of the tracer aerosol rather than to an effect on the clearance system itself. This may be a special problem when comparing different groups, e.g., healthy subjects and those with chronic obstructive lung disease, since the latter may tend to have greater central de position than the former for the same size tracer aerosol (Lippmann et al.. 1980). The influence of aerosol size upon the pattern of bronchial clearance of a tracer RESPIRATORY TRACT CLEARANCE 443 rred, aerosol after irritant exposures was shown by a as- Lippmann el al. (1981). This study indicated '.4 hr that measurement of the clearance rate of a >ron- single monodispersed tracer aerosol may not ining be adequate for simultaneously assessing ef i the fects o f a toxicant on all conducting airways, on is and that the deposition pattern of the tracer chal aerosol can make an important difference in the apparent effect of the toxicant on mucus e the transport. This type of problem may be urves avoided by exposure to two different sized ways, tracer aerosols incorporating different tags to jnen- allow monitoring o f clearance from both the /ever, upper and lower bronchial tree, simulta- ur si;tions ecific I neously. This technique would also provide a more precise localization of the site of action of a toxicant on the lungs. : also Another potential problem in analyzing on or bronchial clearance is the effect o f coughing. :h re Coughs may produce a stepwise decrease in or by the amount of tracer particles left in the age of lung, and must be taken into account when dear assessing clearance times due solely to mu ie *he cociliary transport (Camner et al., 1973; San- chis et al., 1973). ns of The shapes of mucociliary clearance curves in the are highly dependent upon tracer particle deposition. In studies of alveolar clearance, d with however, different clearance rates may also iepenarance asition occur when using different sized particles (Bailey ei al., 1982), since there may be sizedependent differences in macrophage phago se the cytosis, in vivo solubility, etc. Differences in ce pahways. ;ase in could ion of feet on y be a iffereni with ice the ral de ne size the long-term clearance of equivalent-sized particles but consisting o f different materials have been noted by Stahlhofen et al. (1981) >n humans, and by Schlesinger et al. (1982a) in donkeys. Because o f wide differences in techniques, comparisons between studies of different in stigators is difficult, and lung-clearance Percentages and times may not be compara ble. Nevertheless, clearance studies with ra diolabeled tracers have been successfully used lo demonstrate dysfunction following expo ) on the tracer SUre to numerous inhaled agents or due to d|sease (Schlesinger et al., 1984; Last, 1982; banner, 1977). These techniques allow clearance to be measured repeatedly in the same animals so fewer numbers are needed, and the use o f each animal as its own control in many studies markedly reduces the vari ability inherent in comparisons between in dividuals. Magnetopneumography. The assessment of alveolar clearance requires measurements to be performed over perhaps several months. When using radioactively tagged tracer aero sols a nuclide having a relatively long half life is required. In addition, since the total dose to the subject should be minimized, especially if humans are used, long counting times may be required to obtain statistically reliable data. Thus, very-long-term clearance studies using humans may preclude use of radioisotopic tracers due to potential health risks. A relatively new technique which avoids these problems is magnetopneumography (MPG). The measurement of magnetic fields due to contaminants in the lung was first reported by Cohen (1973). Based upon this work, controlled exposures to an inert, mag netic dust (magnetite, Fe30 4) have been per formed in humans and experimental animals to assess clearance (Cohen et al., 1979; Val berg and Brain, 1979; Halpem et al., 1981; Freedman and Robinson, 1981). The subject is exposed to the aerosol and, at various times, a magnetic field is applied externally to the thorax; the field must be of sufficient strength and be applied for an appropriate length o f time to magnetize the bulk o f the deposited particles. Two magnetization tech niques have been employed. In uniform-field MPG, the whole lung is magnetized. In localized-field MPG, one region is magnetized at a time and the resultant field is measured and then erased prior to application of the magnetic field to the next region. The latter technique helps avoid internal cancellation of fields which may occur when the entire thorax is magnetized at once (Robinson and Freedman, 1979). After the magnetizing field is removed, a remanent field remains. The subject is moved 4 4 4 RICHARD B. SCHLESINGER toward and then away from a magnetic sen sor, which is positioned at a specific location relative to the chest; the change in field strength as measured by the sensor is a reflection of retained particles in the measured region o f interest. This method o f differential measurement is necessary because strong, low-frequency variations o f the local field often make it impossible to perform contin uous measurements of the remanent chest field. Because the lung remanent field is much weaker than the earth's magnetic field or field variations due to environmental sources, measurements must be performed in a magnetically shielded chamber or with a gradiometer-type field sensor, which reduces sensitivity to both uniform background fields and local fluctuating fields. Specific sensors range from large, superconducting devices (SQUID) to table-top fluxgate magnetome ters. Magnetopneumographic techniques have some advantages over radioaerosol techniques in terms o f both temporal resolution and spatial resolution in the measurement plane. However, there are a number of significant problems. All sources of external magnetic contamination on the subject must be re moved. The mobile units used to move the subject toward the sensor must be constructed o f nonmagnetic material. In addition, there are difficulties in deducing actual particle distribution in the lungs from the data, since critical positioning is required; depending upon field size, a slight misalignment can result in a dramatic change in the results. This is because the magnetic-field measure ments are much more sensitive to tracer particles in the lung periphery, which is closer to the probe, than to dust in other areas. Thus, material translocated toward the pleural surface of the lung as part o f the clearance process would result in an increase in the observed magnetic field, while trans location from this area would result in a field decrease. Because o f the sensitivity o f the remanent magnetic-field measurements to the actual distribution of retained particles within the effective viewing window, considerable caution must be exercised in directly inter preting field data in terms of lung clearance. Use of localized field MPG with partial era sure may provide information on particle depth, and permit a distinction between in ternal translocation and clearance (Freedman et al., 1982). Magnetic techniques do have potential ad vantages in that certain information may be obtained using them which is not obtainable by other whole-animal in vivo techniques. When the external field is applied, the re sulting magnetic moment increases rapidly at first, then more slowly (the moment can be deduced from the initial remanent field measurement). This time dependence is af fected by the viscosity o f the medium in which the deposited particles reside (Wil liamson and Kaufman, 1981). Therefore, as free particles are translocated from alveolar surfaces or engulfed by macrophages, the response to an externally applied field may change. Thus, when magnetic measurements are used to assess clearance over a long time period, it is possible that some or all of any observed decline in remanent moment is due to immobilization of particles. In addition, the hysteresis curves o f magnetization as well as relaxation (loss o f magnetic alignment) may provide information on the amount of fibrosis in lung tissue (Cohen, 1975). The validity of magnetopneumographic techniques as a monitor of clearance was shown in this laboratory by Halpem et al. (1981). In this study, donkeys inhaled mag netite which had been neutron activated to 5,Fe30 4, so that concurrent measurements using a proven, radiologic technique could be performed. The paired radiologic and magnetic curves displayed definite changes in slope at approximately the same times postexposure, as well as similar rates of de crease with time. This confirmed that the reduction of the initial remanent field over several months represented particle clearance and was not due simply to a chemical change o f the iron compound to a nonmagnetic form or to dissolution, with loss of magnetic properties. s u-- ition. ; well nent) nt of RESPIRATORY TRACT CLEARANCE 445 Bronchographic technique. One technique used for whole-lung clearance in both humans and experimental animals allows visualization of tracer particle distribution without the need for radiolabeled aerosols. This involves insufflation of radioopaque tantalum powder through a tracheal catheter. Serial chest X rays are then performed over a period of months to provide a visualization of clear ance, which is quantitated based upon visual scoring o f film intensity (Gamsu et a i, 1973; Wood et al., 1973). Although the technique provides some measure of whole lung and regional clearance patterns, and allows mea surement in anatomically defined airways, it is invasive, only semiquantitative, and re quires high radiation doses, making it un suitable for routine use in humans. In addi tion, it requires several grams of tantalum, which may overload clearance systems. This technique, therefore, has not found wide spread use. Fecal analysis technique. A technique for indirect monitoring of clearance which in volves radioaerosols but not external moni toring is fecal analysis (LaBelle and Brieger, 1959; Kenoyer et al., 1981; Mannix et al., 1983). This procedure involves collection of feces at fixed intervals after exposure to a radiolabeled aerosol. The fecal excretion ac tivity curve presumably represents material cleared via the mucociliary system into the gastrointestinal tract and can thus be used to provide an index of tracheobronchial clear ance. It makes the assumption that all ma terial cleared from the lung is transported to he gastrointestinal tract and excreted in the feces. This technique is very sensitive to feeding behavior of the animals; those that do not eat or do not excrete for a particular fraction of the sampling intervals cannot be included in the analysis. Spritzer et al. (1971), and Spritzer and Batson, (1964) employed an esophageal coi t i o n method in rats to monitor bronchial c|earance. At various times postexposure, a Election tube placed in the esophagus and which exited through the abdominal wall was ^shed, and the activity counted in the washes to provide clearance rates, i.e., parti cles cleared per collection interval. The rela tion between clearance rates as measured in such surgically altered animals and normal clearance rates is not known. Serial sacrifice technique. Clearance of de posited particles may be monitored in exper imental animals by serial sacrifice at various intervals after exposure, followed by analysis of material in various parts of the respiratory tract or in the lungs as a whole. The lung burden plotted as a function of time provides an assessment of clearance. The method of analysis is dependent upon the nature o f the material used. Techniques employed have included gamma-ray spec trometry for neutron-activated or radiolabeled particles (LaBelle and Brieger, 1959; Reznick and Borgmeyer, 1980; Wehner and Wilkerson, 1981; Sweeney et al., 1983); autoradiog raphy (Sweeney et al., 1983); light and/or electron microscopy (Sorokin and Brain, 1975); and chemical analyses (Ferin et al., 1983). Sacrifice techniques have the advantage of being very sensitive, i.e., they have the poten tial to detect very small amounts of material retained in the lungs, using appropriate an alytical techniques. Although microscopic techniques generally provide only qualitative assessments of particle distribution and clear ance from various regions, other techniques may allow quantitative determination of the amounts of material retained in different regions of the respiratory tract, and without interference from material in adjacent areas. The major disadvantages are that a large number of animals are needed for statistical reliability, intraindividual variability in clear ance cannot be examined, and it is not possible to examine the effects of toxicants upon the course of clearance in the same individual on different occasions. ACKNOWLEDGMENTS The author is a recipient o f a Research Career Devel opment Award from the National Institute of Environ mental Health Sciences (ES 00108). 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Chest 80(Suppl ), 870-873. lUNDAMES I Inh A nev pulmo tential bronci ical ar medic variou This \ et al., not be not O! and si exami ' Pre for the Meetin; March