INTRODUCTION

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RAT AND MONKEY ARE BOTH SPECIES that are used as models of human airway hyperresponsiveness. However, the wall structures of rat and monkey airways are very different from each other, with that of the monkey more closely resembling that of humans. In particular, the most distal airways in the rats are unalveolarized bronchioles, whereas those in monkeys and humans are extensively alveolarized or respiratory bronchioles. The most common methods used to evaluate airway responsiveness are to measure total pulmonary resistance or airway resistance in the whole animal or to measure tension in isolated airway segments. Airway strips or rings measure changes in smooth muscle tension, which only approximates airway narrowing, because the mechanical interactions with other components of the airway wall are not evaluated. None of these methods allows for measurement of the most distal airways. One method used to study distal airways is to determine the tissue resistance (Rti) component of total pulmonary resistance or airway resistance. However, only some of the potential contributors to Rti involve the small airways. Methacholine-induced increases in Rti have been ascribed to airway-tissue interdependence, small-airway closure, interstitial contractile elements, airway inhomogeneities, and/or airway wall shunting (13). Another method to study distal airways is to measure changes in tension in parenchymal strips. However, this technique measures the behavior of multiple small airways and may measure the activity of myofibroblasts in the parenchyma. These measures of airway function are inadequate to evaluate 1) the functional differences due to the distinctive airway wall structure of different airway generations or 2) the toxicological problems that arise from generation-specific or focal injury to airways. Finally, these methods do not allow an evaluation of airway histology with physiology in the same small-airway segment.

Videomicrometry of airways in lung slices holds several advantages. First, individual airways can be evaluated by airway location, including the smallest of airways. Videomicrometry evaluates the airway in situ with all the influences of tethering and of an intact and functional wall, although without such in vivo effects as tidal volume oscillations (9). Local immune and nerve responses are functional, but proximal nervous system and systemic circulation influences are absent. Airway heterogeneity in sensitivity and closure to spasmogens can be studied. A number of investigators have used lung-slice videomicrometry to study rat (4, 5, 15) and guinea pig (8) airways. Problems experienced include an inability to identify airway location, variability in the data, and poor resolution of images. In this study, our aim was to address these technical issues and to use videomicrometry to study airway responsiveness in two species, rat and monkey, with markedly different airway morphology. We were particularly interested in evaluating whether the respiratory bronchioles of the monkey would show significant narrowing and how they would compare with the distal bronchioles of the rat. We hypothesized that the known differences in wall structure of these airways would help explain differences in proximal and distal airway responsiveness in the two species.

	MATERIALS AND METHODS

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Animals. Sprague-Dawley female rats (n = 7) were obtained from Zivic-Miller (Zelienople, PA) and utilized in these experiments at 45-55 days of age. Rats were received at 4-5 wk of age and housed in filtered-air rooms for at least 1 wk before necropsy. The rhesus monkeys (Macaca mulatta; n = 28) used in this study were born at the California Regional Primate Center colony and represented animals from three different ongoing projects at California Regional Primate Center. The ages of young monkeys were 30 days (n = 3), 70 days (n = 8), 90 days (n = 3), and 6 mo (n = 8). Six 3-yr-old monkeys were also used. With the exception of four female 70-day-old monkeys, all monkeys were males. The young monkeys were housed from birth indoors in filtered-air rooms, with the exception of two 6-mo-old monkeys that were housed out of doors. Seventeen monkeys' skin tested negatively for dust mite. Three of the 6-mo-old and eight of the 70-day-old monkeys were not skin tested. All 3-yr-old monkeys were field controls, housed in outdoor field corrals. Care and housing of animals complied with the provisions of the Institute of Laboratory Animal Resources and conformed to practices established by the American Association for Accreditation of Laboratory Animal Care.

Tissue preparation. Rats were anesthetized with pentobarbital sodium (1 mg/kg ip). A ventral incision in the neck was made to cannulate the trachea, the chest was opened, and heparin (100 U) was injected into the right ventricle. The pulmonary artery was cannulated via the right ventricle, the left ventricle was incised, and 50 ml of warm (37°C) Krebs buffer with 2% bovine serum albumin were perfused through the lungs. The lungs and heart were removed en bloc from the thoracic cavity, and warm (37°C) agarose (1.25%) in Waymouth medium was slowly instilled into the lungs. The filling progressed evenly, with the tip of the infracardiac lobe filling last. At this point, the filling was stopped and the agarose-filled lungs were chilled in ice-cold (4°C) Waymouth medium for at least 30 min. The pleura of the infracardiac lobe was translucent and showed no signs of uneven filling either at the time of agarose inflation or on inspection of the slices after the lobe was cut. The slicing procedure was performed between 60 and 150 min after death. The infracardiac lobe was separated from the rest of the lung and cut in 10-mm blocks in a plane perpendicular to the long axis of the lobe. To prepare lung slices, the lung lobe block was glued to the cutting platform of the vibratome and submerged in ice-cold Waymouth medium. Lung slices, cut perpendicular to the axial airway of the lobe, were 650 µm thick for proximal airways and 600 µm thick for distal airways. Each slice was tacked to a coverslip with small, discrete spots of Nexaband glue near the pleural edges and placed in 35-mm tissue culture dishes (Corning, NY) in 3 ml ice-cold Waymouth medium. Slices were inspected, and only those airways with edges completely in focus and with no airway branching were chosen for methacholine challenge. The agarose plug from each airway was removed before study by gently probing it with the use of a 32-gauge needle.

Image capture. Lung slices in culture dishes were placed under an Olympus BH2 light microscope with water-immersion lenses connected to a Power Macintosh 7300/180 computer running National Institutes of Health (NIH) Image software via a DAGE MTI video camera and observed on the monitor. Image magnification on the video screen was ×75 or ×150 depending on the size of the airway. Images of the cross-sectional area of the main axial airway path were captured by using NIH Image software.

Experimental protocol for determining reactivity of airways. In the rat studies, the lung slices were kept in ice-cold Waymouth medium until studied 4-8 h after death. Throughout the airway responsiveness studies, the slices were placed in Krebs buffer (37°C) with or without methacholine and were continually aerated with 95% O₂ and 5% CO₂ (carbogen), at pH 7.35 ± 0.03. Solutions were aerated with carbogen for at least 5 min, and pH was measured before application to slices. Preliminary studies showed that pH was constant after 5 min of aeration with carbogen. After a minimum of 40 min, with Krebs buffer changes every 5 min, airways with a luminal area changing <10% between washes and pulsating <10% by visual inspection were challenged with methacholine. Drug concentrations started at 10⁸ M (rat) or 10⁹ M (monkey), with each subsequent concentration increasing by at least one-half log increments to a maximum 10⁴ M. For each concentration, the old solution was removed and the new one added. An image was captured 5 min after each buffer change and after 5 min of incubation in each concentration of drug. Preliminary studies with rats and monkey slices found the responses to drug to be stable between 2 and 8 min of incubation time. Preliminary experiments showed that, when this procedure was performed with Krebs buffer without methacholine, the luminal area was stable in both rats and monkeys. Distal and proximal airways were paired in the image-capture protocol, to avoid any time delay effects in comparing proximal vs. distal responsiveness.

Histological evaluation of the airways. After measurement of airway response, all lung slices were fixed in 1% paraformaldehyde for a minimum of 24 h before being embedded in paraffin (Paraplast-20, Oxford Labware, St. Louis, MO). Serial sections (5 µm thick) were cut by using a microtome (Carl Zeiss, Thornwood, NY) and stained with hematoxylin and eosin. The airway within each lung slice was examined by light microscopy. The alveoli appeared to be properly inflated and of uniform size. The airway epithelium showed no evidence of damage from removal of the agarose plug. Airways were identified by structural features. A bronchus was identified by the presence of cartilage in the wall, a bronchiole by the lack of cartilage or alveolar outpocketings within the wall, and a respiratory bronchiole by alveolar outpocketings along the wall.

Reagents and solutions. SeaPlaque agarose was obtained from FMC Bioproducts (Rockland, ME). Waymouth mouth MB 752/1 medium was purchased from GIBCO BRL, Life Sciences (Grand Island, NY). Sodium bicarbonate (2.24 mg/l) was added to the Waymouth solution, and pH was adjusted to 7.4 at 4°C. The Krebs buffer (119 mM NaCl, 4.7 mM KCl, 3.2 mM CaCl₂, 21 mM NaHCO₃, 1.17 mM MgSO₄ · 7H₂O, 1.18 mM KH₂PO₄, and 0.1% D-glucose; Mallinkrodt, Paris, Kentucky, and Sigma Chemical, St. Louis, MO) was made fresh weekly and heat sterilized. NaHCO₃ and CaCl₂ were added on the day of the experiment; the buffer was aerated with carbogen and adjusted to a pH of 7.4 at 37°C. Final concentrations of methacholine (acetyl--methylcholine chloride; Sigma Chemical) were prepared fresh on the day of each experiment in Krebs buffer from 10² M stock. Nexaband was purchased from Veterinary Products Laboratories, and bovine serum albumin was purchased from Sigma Chemical.

Data analysis and statistical evaluation. The epithelial luminal border of the airway was traced, and the luminal area was calculated by using NIH Image software. The data were expressed as a percentage of the lumen measured in the last buffer wash. Concentration-response curves were compared by using a repeated-measures analysis of variance. When appropriate, post hoc analyses consisted of a series of Scheffe contrast tests among the treatment groups (SAS/Stat, SAS Institute).

	RESULTS

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Videomicrometry technique. Water-immersion lenses provided excellent visualization of the epithelial luminal border as airway smooth muscle constricted, as shown in Fig. 1. In addition, some of the airways in the monkeys were noted to pulse by spontaneously opening and partially closing in an ~3-s cycle, with ~0.5 s for constriction and 2.5 s for relaxation. This pulsation was not affected by addition of indomethacin (10³ M). If the pulsation was significant, the slice was not used for data acquisition. If the pulsation was minor (<10% by visual inspection), the image was captured in the most open configuration.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   Example of the captured images (top; nos. represent log molar concentrations of methacholine) and corresponding concentration response curve (graph) from an axial airway in a proximal lung slice from the accessory lobe of a monkey. Increasing concentrations of methacholine were applied to the airway, and the luminal area was determined by using National Institutes of Health (NIH) Image software.

View larger version (81K): [in this window] [in a new window]   Fig. 2.   Effect of cutting an airway obliquely on airway responsiveness to methacholine. A: example of an obliquely cut distal airway from a rat. An airway was defined as obliquely cut if 20% or more of the long diameter (line a + line b) of the airway lumen included exposed inner airway wall (line b). B: example of a cross-sectionally cut distal airway from a rat. C: comparison of responsiveness to methacholine in obliquely vs. cross-sectionally cut distal airways in the rat. Increasing concentrations of methacholine were applied to the airways, and the luminal area was determined by using NIH Image software. Values are means ± SE. Ctl, control. The obliquely cut airways appeared to be less reactive to methacholine and closed less completely (P = 0.0001; n = 6 oblique, n = 13 cross section).

Histological evaluation of the airways. By histological evaluation of the monkey lung slices, it was determined that 95% of the proximal airways were bronchi, with the presence of cartilage in the wall and intact epithelium lining the lumen. Ninety-five percent of distal airways in monkey lung slices were found to have alveolar outpocketings in the walls and were defined as respiratory bronchioles. The remaining airways were labeled as bronchioles and were typically within one to two generations of a respiratory bronchiole. In contrast, rat lung slices contained only bronchioles, with large bronchioles identified in proximal slices and small bronchioles identified in distal slices. In all instances for both species, the epithelial lining of all airways was found to be intact.

Comparison of 7-wk-old rat and 6-mo-old monkey proximal and distal airways. Histological evaluation of the proximal airways (Fig. 3, Table 1) from the lung slices showed the expected morphology for the rat and monkey airways. Specifically, the monkey proximal airway had pseudostratified columnar epithelium, cartilage, goblet cells, and submucosal glands, whereas the rat had simple columnar epithelium and no cartilage, goblet cells, or submucosal glands. In the distal airways, the monkey had alveolar outpocketings indicative of respiratory bronchioles, whereas the rat had bronchioles without alveolar outpocketings. In airway slices used in the comparison, the rat proximal airways were approximately the same diameter as those from the monkey, whereas the rat distal airways were bigger than those from monkey. All airways, independent of size and type, demonstrated the presence of interrupted, circumferential bands of smooth muscle segments. Proximal airways for both monkeys and rats possessed prominent bands of smooth muscle, whereas distal airways contained fewer. Physiological evaluation (Fig. 4) showed that the rat and monkey proximal airways were equally responsive to methacholine. However, rat distal airways were more responsive to methacholine than monkey distal airways. In the rat, the distal airways were more responsive to methacholine than the proximal airways, whereas, in the monkey, the reverse was observed: distal airways were less responsive to methacholine than proximal airways.

View larger version (126K): [in this window] [in a new window]   Fig. 3.   Examples of rat proximal airway (A), rat distal airway (B), monkey proximal airway (C), and monkey distal airway (D). The expected histological differences were observed (see text).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Methacholine responsiveness of rat and monkey proximal and distal airways. Increasing concentrations of methacholine were applied to proximal and distal airways of 7-wk-old rats and 6-mo-old monkeys, and the luminal area was determined by using NIH Image software. Values are means ± SE. Rat (n = 8 slices, 6 rats) and monkey (n = 17 slices, 8 monkeys) proximal airways were equally responsive (P = 0.40), whereas rat distal airways (n = 13 slices, 4 rats) were more responsive (P = 0.0003) than monkey distal airways (n = 9 slices, 5 monkeys). Within each animal species, rat distal airways were more responsive than their proximal airways (P = 0.04), whereas monkey distal airways were less responsive than their proximal airways (P = 0.01).

Comparison of monkey proximal and distal airways at different ages. Airway responsiveness to methacholine was evaluated in monkeys at various ages. Because airway responsiveness in monkeys 6 mo of age and younger did not differ, the data were combined and compared with data from airways of 3-yr-old monkeys. The proximal and distal airways of young monkeys were more responsive than those of older monkeys (Fig. 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of age on airway responsiveness in monkeys. Increasing concentrations of methacholine were applied to proximal and distal airways from monkeys 6-mo-old and younger and from monkeys 3-yr-old. The luminal area was determined by using NIH Image software. Values are means ± SE. The proximal airways (A) from the 6-mo-old and younger monkeys (n = 39 slices, 22 monkeys) were more responsive to methacholine than the proximal airways of the 3-yr-old monkeys (n = 11 slices, 6 monkeys; P = 0.0005, interaction, 2-way ANOVA). The distal airways (B) from the 6-mo-old and younger monkeys (n = 29 slices, 18 monkeys) were also more responsive to methacholine than the distal airways of the 3-yr-old monkeys (n = 12 slices, 6 monkeys; P = 0.004, interaction, 2-way ANOVA).

Intra-animal heterogeneity in airways from young rhesus monkeys. Intra-animal heterogeneity of airway responsiveness to methacholine is shown in Fig. 6. The mean of the individual animal variances for the EC₅₀ in the proximal airways (0.24 ± 0.13; n = 13) did not differ statistically from that in the distal airways (0.28 ± 0.12; n = 11; P = 0.42). However, for the maximal response, the mean of the individual animal variances was much less in the proximal airways (31 ± 12) than in the distal airways (401 ± 259; P = 0.02).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Intra-animal heterogeneity of airway responsiveness to methacholine in young monkeys. If at least 2 airways from the same region (proximal or distal) in the same animal were available, the concentration in log molar units of methacholine that produced 50% of the maximal response (EC50) and the maximal response were determined. , data from one airway slice. A and C: frequency distribution of EC50 in proximal (n = 13 animals) and distal airways (n = 11 animals). B and D: frequency distribution of the maximum response in the proximal and distal airways. One hundred represents the original luminal area, and zero represents an airway that is completely closed. Whereas the variances of the EC50 within each animal did not differ between proximal and distal airways, the variances of the maximal response were less in the proximal than in the distal airways (P = 0.02).

	DISCUSSION

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By using videomicrometry in conjunction with lung airway slices, a method that allows measurement of airway contractility by individual generations with intact peribronchial constituents, we found variability by airway generation, age, and species. This study is the first to compare the responsiveness of proximal and distal airways of monkeys and rats. In fact, to the best of our knowledge, this represents the first time the responsiveness of the respiratory bronchiole, as defined by its anatomic structure, has been measured in any species. We demonstrated that the rat and monkey proximal airways used in this study were remarkably similar in their response to methacholine. In contrast, rat distal bronchioles were more responsive than proximal airways to methacholine, whereas, in the monkey, respiratory bronchioles (i.e., distal airways) were less responsive than bronchi (i.e., proximal airways). In addition, both proximal and distal airways from young monkeys were more responsive to methacholine than those from 3-yr-old monkeys.

An extensive database exists to identify differences between the rat and primate airway wall structure (23, 26). Proximal airways in the rat differ from those of the monkey by the presence of a simple cuboidal or columnar epithelium, rather than psueudostratified epithelium. In addition, proximal airways of the rat lack goblet cells, submucosal glands, and cartilage. The presence of cartilage in the proximal airways of the monkey might be expected to limit the narrowing of the airway lumen. Jiang and Stephens (10) showed that bronchial smooth muscle without cartilage attached demonstrated greater maximum shortening capacity and greater maximum velocity of isotonic shortening than bronchial smooth muscle with cartilage intact. In the present studies, virtually all of the proximal airways studied from monkeys contained cartilage. However, the presence of cartilage did not appear to inhibit proximal airway responsiveness.

Distal airways in the rat differ from those in the monkey to an even greater degree than do the proximal airways (Table 1). In the rat, up to 16 airway generations are present before reaching the terminal bronchiole, immediately giving rise to the alveolar duct (26). In the monkey, there are up to 16 generations of airways (personal communication, Michelle Fanucchi, University of California, Davis), which subsequently give rise to three to six generations of respiratory bronchioles before reaching the level of the alveolar duct (23). In this study, virtually all distal airways in monkeys were identified anatomically as respiratory bronchioles. Despite the interruption of the wall with outpocketings of alveoli, the monkey respiratory bronchioles were responsive to methacholine. However, as shown statistically in the younger monkeys, the respiratory bronchioles were the least responsive and most variable airway studied. There was no obvious histological explanation for the hyperresponsiveness of rat distal airway compared with the proximal airway. Possible mechanisms include fewer infolds (internal tethers); thinner lamina propria and submucosa (21); thick epithelial basal lamina (24); different smooth muscle area, configuration, or cell biology; less diffusion distance (18); and increased epithelial permeability (18). Recently, Wohlsen et al. (25), using videomicrometry techniques, showed that smaller airways of Wistar rats were more responsive to allergen and ketanserin, a serotonin antagonist, than were larger airways. Similarly, Mitchell and Sparrow (18) demonstrated, in pig airways in vitro, that small-lumen (3 mm²) bronchial tubes were more sensitive to methacholine and closed more completely than did larger lumen bronchial tubes (18 mm²). On the other hand, Minshall et al. (17), using videomicrometry techniques similar to those used in the present study to examine human airways from lung tissue resected for cancer, did not see a difference in responsiveness between large and small airways. However, differences between proximal and distal airways may have been obscured, because the airways came from different lobes and parts of lobes and because the airways were inflamed as a result of cigarette smoke exposure. Thus it may be that, in general, airway responsiveness increases in the distal airways compared with the proximal airways but that, once the airways become respiratory bronchioles as they do in the human and monkey, airway responsiveness is less.

A number of studies in rats (12), rabbits (22), and humans (3, 7, 14) have suggested that airway responsiveness is greater in younger than in older animals. We previously showed that the isolated perfused lungs from 8-wk-old rats were two- to threefold more responsive to methacholine at the highest dose given than were those from 15-wk-old rats (12). This is the first study to compare the developmental aspects of airway reactivity of proximal and distal airways in the monkey. We showed that both proximal and distal airways were more responsive to methacholine in younger than in older primates.

The observation of pulsing airways in monkeys (rarely found in rats) was an unexpected and interesting phenomenon. This pulsation was not due to cyclooxygenase products, because indomethacin did not inhibit this unique feature. In vivo airway physiology in primates may be even more dynamic than had been previously appreciated.

It is generally thought that airways within a person or animal show heterogeneity in responsiveness. In fact, airway heterogeneity is one of the potential explanations for the increase in Rti with methacholine administration (13). The study of human airways by Minshall et al. (17) reported marked heterogeneity in human airways by using videomicrometry techniques. Although we also saw some heterogeneity in responses in the young monkeys, it was much less than they reported. Our intra-animal variances for EC₅₀ in both proximal and distal airways, and for maximal effect in the proximal airways, were fivefold less than that reported by Minshall et al. However, we did see marked variability in maximal effect in the distal airways. The variance of these airways was 12-fold that in the proximal airways. Thus, although heterogeneity exists, we believe it is mostly confined to the degree of closure of respiratory bronchioles.

We believe that improvement in technique is the reason we showed less heterogeneity than has been reported by others. We have made a number of adjustments in our technique to reduce variability. We always studied generations within the axial airway of the lobe. We cut our slices with a vibratome to a consistent thickness (600-650 µm) that would allow for clearly defined airway lumen and also be thick enough for histology procedures. We glued the edges of the lung slice to a coverslip to optimize the intrinsic tethering forces of the surrounding parenchyma. This is important because in vivo studies have shown that tethering of the airways affects airway responsiveness to spasmogens (1, 2) and Mitchell et al. (19) showed in vitro in lung slices that bronchoconstriction will stretch adjacent lung parenchyma, imposing a load on airway smooth muscle. Although the concentration of agarose can affect the closure of airways (5), we used the same concentration (1.25%) in all our studies. Inflation volume with the agarose can also affect airway closure (5). We used a standard method to fill the lungs with agarose, which was done by the same person for every experiment. Histological examination showed the alveoli to be equally distended. A water-immersion lens, rather than an inverted microscope (5, 15), allowed for a more precise image of the airway. We also extended our prewashes until the luminal area of the airway was changing by no more than 10%, and we rejected airways that failed to stop pulsating. Finally, as shown in Fig. 2, eliminating obliquely cut slices provided much more consistent data, especially for distal airways.

In order for comparisons to be made within and between species, there must be consistency in the method for airway selection. This was accomplished by 1) studying airways from a lobe with a single primary axial airway, 2) localizing the distance along the lobe (proximal or distal), and 3) describing the airway's luminal size and structure as shown by histology. We were certain that the proximal airways were proximal to the distal ones. In addition, in the monkey, we knew that the proximal airways were histologically bronchi or bronchioles, whereas the distal airways were almost entirely respiratory bronchioles. In the rat, this histological verification was not possible, because the proximal and distal airways in the rat appear similar until the terminal bronchiole. Although airway luminal size does not correlate well with airway generation, in general, the distal airways were smaller than the proximal airways, as would be expected. With regard to the lobes, we picked the lobe from each species that would provide the straightest axial airway. In the monkey, the accessory lobe axial airway curves sharply at first and then straightens out and divides in a monopodial fashion until, in the final one-fourth of the lobe, it begins to divide dichotomously. In the rat, the infracardiac lobe airway has a less sharp curve proximally and remains monopodial throughout the lobe. A large axial airway throughout the lobe probably explains why the distal airways of the rats were larger than those of the monkey, despite coming from the same relative section of the lobe.

Other potential methodological concerns in the between-species comparisons are differences in drugs used at necropsy (heparin in the rat and ketamine in the monkey) and viability. With regard to the differences in drugs at necropsy, the vasculature was cleared of drug by buffer perfusing the rats and exsanguinating the monkeys. The slices were then washed eight times for a total of at least 40 min before the study. We do not believe that residual drug remained in the tissues with this procedure. Finally, we believe all airways were viable. The proximal airways of both species and the distal airways of the rat all closed by at least 40% to methacholine, suggesting that they were viable. Although some of the distal airways of monkey did not show as much response to methacholine, these slices were handled identically. Further evidence of viability of the tissue is that on histological evaluation the epithelium was intact, without blebbing, vacuolization, or sloughing.

Videomicrometry is a useful tool to study differences in airway physiology. Recently, Martin et al. (16) used videomicrometry elegantly to show that endothelin-1 affects small and large airways in the rat to the same extent, whereas a thromboxane agonist was 10-fold more potent in contracting small airways compared with large airways. Our present work demonstrates that this technique of studying the responsiveness of a distinct airway can be coupled with evaluating the wall structure of that same airway. This attribute of our videomicrometry method will also make it useful in studying toxicological, allergic, and infectious influences that differ by airway generation. Such site-specific differences among airways have been shown after exposure to ozone on airway epithelial cell injury (11), as well as glutathione levels (6). Furthermore, within a generation, there may be focal injury (20). Thus combining airway physiology with histology in the same slice will allow for studying such airway changes.

In conclusion, we have shown that videomicrometry is a powerful tool for evaluating responsiveness of individual intrapulmonary airways. This is especially true for the smaller distal airways, which are difficult to study by other techniques. Furthermore, this technique allows for selective study and comparison of proximal and distal airways. More specifically, in the present study, we have shown that the respiratory bronchioles of the monkey were less responsive, and the distal bronchioles of the rat were more responsive, than the proximal airways of either species. In the monkey, both proximal and distal airways were more responsive in younger than in 3-yr-old animals. We have shown that heterogeneity in airways is less than previously thought except for maximum closure of respiratory bronchioles. Most importantly, we have shown that respiratory bronchioles can be studied as distinct airways, that they are responsive to methacholine, and that they differ in their responsiveness from that of the most distal airway of the rat. This adds to the evidence that the monkey is a better experimental model for human airway responsiveness. Use of videomicrometry methods in combination with histological evaluation will greatly facilitate our understanding of the constitutive function of the airways and allow for better investigation of effects of toxins and allergens on specific airway generations.