CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709
Received May 1, 2001; accepted August 15, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Long-Evans rats; deposition efficiency; nasal airway; impaction; in vivo deposition.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Deposition of ultrafine particles, or particles with geometric diameters less than 0.1 µm, in the upper respiratory tracts of rats has been experimentally studied in vivo (Chen et al., 1989; Gerde et al., 1991
; Wolff et al., 1984
) and in nasal molds (Cheng et al., 1990
). Deposition of fine and coarse particles, or particles with geometric diameters ranging from about 0.5 to 5 µm, in rat upper respiratory tracts has been studied in vivo (Raabe et al., 1977
, 1988
) and in nasal molds (Kelly et al., 2001
, Schreider, 1983
).
The results of studies by Raabe et al. (1977, 1988), as discussed in Kelly et al. (2001), warrant further research into nasal deposition of fine and coarse particles in the rat. The nasal molds used in the studies by Kelly et al. (2001) and by Schreider (1983) were made from postmortem casts of rat nasal airways. The cross-sectional area of a postmortem airway cast has been indicated as being up to twice that of a live subject (Guilmette et al., 1989; Yeh et al., 1997
). Tissue shrinkage has been cited as a possible reason for the observed dimensional differences (Guilmette et al., 1989
). Differences in morphology between the nasal airways of the live rat and the nasal mold may result in differences in deposition. Therefore, further in vivo study of nasal deposition of fine and coarse particles in the rat is warranted.
The objective of this study was to determine aerosol deposition efficiencies, or fractions of particles entering the nose and depositing in the nasal airways of Long-Evans rats, for particles with diameters ranging from 0.5 to 4 µm.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aerosol generation and characterization.
The experimental apparatus is illustrated in Figure 1A and 1B. Polystyrene latex (PSL) microspheres (density of about 1 g/cm3) were suspended in distilled water and nebulized using a Lovelace nebulizer (In-Tox Products, Albuquerque, NM). PSL microspheres of 10 monodisperse particle diameters ranging from 0.5 to 4.2 µm (0.5, 0.8, 1.1, 2.1, and 2.8 µm from Polysciences, Warrington, PA; 1.8, 2.8, 3.2, 3.6, and 4.2 µm from Spherotech, Inc., Libertyville, IL) were used. Particle sizes were supplied by the manufacturers and verified with an aerodynamic particle sizer (APS) (TSI Model 3320, St. Paul, MN). A dry aerosol with charge Boltzman equilibrium was obtained by passing nebulized droplets through a silica gel diffusion dryer and a 2-mCi 85Kr neutralizer.
|
Rats were anesthetized prior to exposure with a 0.3-ml intraperitoneal injection of Rompun (xylazine, 20 mg/ml injectable; Bayer Corp., Agriculture Division, Animal Health, Shawnee Mission, KS) in 1:1 combination with Ketaset (ketamine HCL, 100 mg/ml; Fort Dodge Laboratories, Inc., Fort Dodge, IA). Anesthesia was subsequently maintained with small subcutaneous doses (e.g., 0.15 ml) given as required, approximately every 1530 min, to ensure that the animal remained anesthetized during the entire exposure. A high-intensity lamp was used to maintain the body temperature of anesthetized animals. Animals were killed by exsanguination following exposure.
After anesthetization, the trachea was surgically exposed, and a tracheal incision was made about 2 mm posterior to the larynx and parallel to the tracheal rings. One end of a 14-gauge needle, used as a breathing catheter, was immediately inserted caudally through the incision (Fig. 2). The end of the breathing catheter that was external to the animal was angled so that it was clear of obstructions, thereby allowing the animal to spontaneously breathe room air. A second catheter, made from an infant feeding tube (Size 8 French, Cutter Laboratories, Berkeley, CA), was inserted through the same incision in the direction of the larynx. The function of this catheter was to conduct aerosol from the outlet of the nasal airways to the experimental apparatus for measurement during inspiration experiments, and to deliver aerosol to the nasal passages during expiration experiments. Great care was taken to place the aerosol-conducting catheter such that its introduction to the airway caused no flow obstructions; increased pressure drop as a result of flow obstructions unrelated to normal anatomy would compromise the accuracy of the deposition measurements.
|
To ensure proper placement of the aerosol-conducting catheter, air was passed through the nasal airways and the rise of a soap bubble in a bubble flow meter connected in series with the animal was observed. The position of the catheter was delicately manipulated to yield the highest flow rate as indicated by the flow meter. If the flow rate achieved was within a few percentage points of the unobstructed flow rate, flow through the nasal passages was considered to be unobstructed. The flow meter was then placed upstream and downstream of the animal in series with the animal, and the flow rates of air traveling through the nasal passages were measured. The positions of the catheter and mask were adjusted until the flow rates were nearly identical upstream and downstream of the animal, to insure no or minimal air leakage. The catheter was secured in the optimal position with surgical thread, and petroleum jelly was applied to the incision area to eliminate air leakage from the catheter and airway interface.
The surgical procedure described above was used effectively for 5 experiments. However, because of difficulty in consistently achieving an unobstructed flow path using the surgical procedure as described, the procedure was modified for subsequent experiments. The primary cause of flow obstruction was the larynx. Various steps were taken to eliminate flow blockage by the larynx, including varying the position of the animal's head, body, and tongue; manipulating the placement of the catheter; reducing the flow rates; and gavaging the rat. None of these manipulations proved to be a consistent solution. Consequently, in subsequent experiments, the catheter was inserted anterior (instead of posterior) to the larynx, in the nasopharyngeal meatus. In this configuration, aerosol flow from the nasal passages bypassed the larynx, thereby eliminating larynx-related flow obstructions. This modification required the application of ophthalmic solution (Proparacaine Hydrochloride Opthalmic Solution USP, 0.5%; Bausch & Lomb, Tampa, FL) to the sensitive tissue surrounding the incision.
Deposition measurements.
Deposition measurements were recorded using the experimental apparatus employed by Kelly et al. (2001) with a live rat in place of the previously used nasal mold. A brief description follows.
The flow from the nebulizer was split into 2 branches: an exposure line and a bypass line (Fig. 1A). The exposure line included the animal, and the bypass line contained a constriction that equalized the pressure drops between the 2 branches. Since the branches otherwise consisted of identical components, the line losses in the 2 branches were equal. By reversing the configuration of the two 3-way valves located at the ends of the 2 branches, the flow from the exposure line was directed to a particle-detection line, while flow from the bypass line was discarded through a vacuum line and vice-versa. The particle detection line used an APS to measure particle size and concentration from the exposure and bypass lines. A mass flow controller (MKS Instruments, Model 246B, Andover, MA) and a diluter (In-Tox Products, Albuquerque, NM) were used to maintain the flow rate required by the APS. The vacuum line was used to discard flow from one of the branches while maintaining balanced flow conditions between the 2 branches; it included a filter, vacuum pump (Model 4144, Emerson, St Louis, MO), and pressure gauge (Magnehelic, Dwyer Instruments, Inc., Michigan City, IN).
The pulsatile apparatus (Fig. 1B) was assembled by inserting a rodent ventilator (Model 683, Harvard Apparatus, South Natick, MA) into the steady-state apparatus (Fig. 1A
) downstream of the 85Kr neutralizer. Two passive air lines were also added to the apparatus to convert pulsed flows to steady flows upstream of the APS and vacuum pump.
To simulate inspiration, the animal was oriented in the system so that aerosols passed from the exposure mask through the nasal airways and into the catheter. The orientation of the animal was reversed to simulate expiration.
The number of animals used for the surgical and flow conditions studied are listed in Table 1. At least five animals were exposed per flow condition to account for possible intersubject variability. Typically, one animal was exposed to multiple particle sizes at one or more flow rates for a given flow condition (i.e., steady state or pulsatile) for either inspiration or expiration. A total of 27 exposures were performed using 22 animals. For 5 of the 16 steady-state cases, both inspiration and expiration were studied with the same animal. Since the animal's position could not be rotated without altering the catheter placement, an extra length of tubing was added to the exposure line to reverse the flow direction without moving the animal. The influence of this extra length of tubing on deposition was expected to be minimal for all except the largest particle sizes, where deposition may have been artificially increased due to gravitational sedimentation of particles in the exposure line.
|
Particle concentration measurements taken from the bypass line reflected exposure concentrations at the external nares of the rat, while those taken from the exposure line represented conditions at the outlet of the nasal airways. Nasal deposition efficiency () was determined as follows (Gerde et al., 1991
):
![]() | ((1)) |
Occasionally, the flow rate changed during measurements because of disruption of the catheter placement. Changes in flow rate were detected by conducting frequent flow rate measurements, observing changes in the pressure gauge reading, and occasionally by observing large changes in particle concentration. When the flow rate was observed to change during measurements, the measurements were discarded and repeated after the flow had been reset.
Slightly negative deposition efficiencies were occasionally measured as a result of minor fluctuations in nebulizer output concentration. These measurements, which occurred infrequently for the smallest particle sizes only, were discarded because of their physical impossibility. Blockage of the nebulizer jet, in addition to settling of particles in the nebulizer cup, occasionally resulted in a reduction of nebulizer output concentration to near background levels. In these instances, measurements were discarded, the problem was resolved, and measurements were repeated.
Data analysis.
Deposition efficiency curves were generated from experimental measurements by fitting the following model (Zhang and Yu, 1993) to the data:
![]() | ((2)) |
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The curve-fits for the steady-state cases depicted in Figure 3 indicate that the deposition efficiencies for the inspiratory and expiratory cases of steady-state flow were similar at low values of
d2Q (i.e., approximately 1.510 g-µm2/s). For larger values of
d2Q, expiratory deposition efficiency appeared to be somewhat higher for a given value of
d2Q (Fig. 3
). For the pulsatile cases, expiratory deposition efficiency also appeared to be similar to inspiratory deposition efficiency for low values of
d2Q (i.e., approximately 1.5 g-µm2/s) and higher than inspiratory deposition efficiency for larger values of
d2Q (Fig. 4
).
Best-fit curves comparing deposition efficiencies for the pulsatile and steady-state cases are shown in Figure 5. At low values of particle inertia (i.e., 1.5 g/µm2/s), the deposition efficiency for the pulsatile and steady-state cases were similar. For mid-range values of particle inertia (i.e., 1570 g/µm2/s), the pulsatile cases had significantly higher deposition efficiencies than the corresponding steady-state cases. At very large values of inertia (i.e., 100 g/µm2/s), deposition efficiencies tended toward total deposition for both flow conditions.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Some difference in deposition between inspiratory and expiratory scenarios was expected. The flow profile in the nasal airways is different for inspiratory and expiratory flows, because the shape of the airway is asymmetrical. This difference in flow profile resulted in higher deposition for expiratory flows than inspiratory flows.
The result that particle deposition for mid-range values of particle inertia was significantly greater for pulsatile flows than steady-state flows is physically reasonable and has important implications for the study of airway deposition. For small values of particle inertia, particles closely track flow streamlines in both pulsatile and steady-state flows under conditions relevant to the study, because fluid drag forces are much larger than inertial and other forces. Therefore deposition for low values of particle inertia (i.e., 1.5 g/µm2/s) was minimal for both flow conditions. The tracking fidelity of particles for mid-range values of particle inertia (i.e., 1570 g/µm2/s) is worse than for smaller values for conditions relevant to the study, because inertia hinders a particle's ability to quickly adapt its motion to fluid accelerations. Because fluid accelerations are greater in pulsatile flows than steady-state flows, particle trajectories will deviate more from flow streamlines in pulsatile flows, resulting in greater impaction deposition. Therefore particle deposition was higher in pulsatile flows than steady-state flows for mid-range values of particle inertia. For large values of particle inertia (i.e., 100 g-µm2/s), particles do not follow flow streamlines for either flow condition, and so there was nearly total deposition in both cases. Normal breathing inherently involves fluid acceleration, and therefore, the pulsatile flow condition used in the study is probably more physiologically realistic than the steady-state condition. Thus fluid accelerations associated with normal breathing should be considered when determining deposition that is governed by inertial impaction.
A consistent relationship between deposition from scenarios that included the larynx in the flow domain and those that did not was not observed (Fig. 3). Previous work (Raabe et al., 1977
, 1988
) has shown deposition in the larynx to be minimal compared with deposition in the nasal passages. Considering these findings, the influence of the larynx on total head deposition was probably masked by the variability in the data.
Previous in Vivo Studies
Raabe et al. (1977, 1988) studied deposition of aerosols in live rat nasal airways under normal breathing conditions. The deposition data from the Raabe et al. studies (1977, 1988) are plotted with deposition efficiencies from the pulsatile cases of the present study in Figure 4. The Raabe et al. studies reported deposition in terms of deposition fraction, or fraction of the total number of particles in the exposure atmosphere that deposit in a given airway. Deposition is presented here in terms of deposition efficiency, or the fraction of the number of particles entering an airway that deposit. To facilitate comparison, reported deposition fractions (Raabe et al., 1977
, 1988
) were converted to deposition efficiencies. The conversion was made by assuming that inspiratory and expiratory deposition efficiencies were equal and accounting for aerosol deposition in the lower respiratory tract.
Flow rates were calculated for the Raabe et al. studies (1977, 1988) to determine d2Q. For the Raabe et al. study (1977), the reported average male and female minute volumes were averaged, and the result was doubled, yielding a flow rate of 318 ml/min (i.e., flow rate = 2 x average min volume). For the Raabe et al. study (1988), the flow rate of 228 ml/min was calculated from average animal weight (205 g) using Guyton's formula (Guyton, 1947
). Because of uncertainty regarding individual minute volumes for corresponding deposition measurements, the calculated flow rates are not ideal representations of the actual flow rates.
Data from the Raabe et al. study (1988) agree well with deposition efficiencies from the pulsatile cases in the present study (Fig. 4). Although there are limited data available for comparison, deposition from the Raabe et al. (1988) study closely matches the data from the inspiratory case of pulsatile flow. Deposition from the Raabe et al. study of 1977 is somewhat lower than the data from the pulsatile cases used in the present study (Fig. 4
). Making a thorough comparison between the Raabe et al. study (1977) and the present one is difficult because of the limited data available. Differences between previous work and the present study are probably the result of different study methods as well as inaccuracy in estimating the flow rates from previous studies.
Variability
Due to morphological differences among the nasal airways of the rats studied, some difference in airflow patterns, and consequently in deposition, was expected among subjects. Intersubject variability was examined by comparing the deposition data acquired from different animals. Differences in the weights of animals were small and did not have a consistent relationship with deposition. The variability of the data depicted in Figures 3 and 4 was in excess of expected intersubject variability.
The surgical preparation was probably the primary source of variability. Placement of the catheter posterior to the larynx resulted in flow obstructions of varying degree, and placement of the catheter anterior to the larynx, while eliminating flow obstructions, proved difficult. Because of the limited area available for catheter placement anterior to the larynx and the soft nature of the tissue in this area, manipulation of the catheter position, which promised to yield a smoother, more physiologically realistic flow path was severely limited. Differences in flow as a result of differences in catheter placement and other factors such as animal breathing (which could influence the catheter if rapid or deep) occurred among animals.
Summary
In vivo measurements of fine and coarse aerosol deposition in the nasal airways of female Long-Evans rats were conducted. Deposition increased sharply with increasing particle inertia for all exposure scenarios. Expiratory deposition efficiency appeared to be somewhat higher than inspiratory deposition efficiency for both steady-state and pulsatile flow conditions. Pulsatile flow yielded significantly higher deposition than steady-state flow. This result emphasizes the importance of considering fluid accelerations that are inherent in normal breathing when determining aerosol deposition that is dominated by inertial impaction. Variability in the data, which was suspected to result primarily from the difficult surgical procedure, was in excess of expected intersubject variability. Deposition results of the present work are timely, valuable data to be incorporated into extrapolation modeling and risk assessment activities for inhaled pollutants.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed. Fax: (919) 558-1213. E-mail: asgharian{at}ciit.org.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cheng, Y. S., Hansen, G. K., Su, F. Y., Yeh, H. C., and Morgan, K. T. (1990). Deposition of ultrafine aerosols in rat nasal molds. Toxicol. Appl. Pharmacol. 106, 222233.[ISI][Medline]
Gerde, P., Cheng, Y. S., and Medinsky, M. A. (1991). In vivo deposition of ultrafine aerosols in the nasal airways of the rat. Fundam. Appl. Toxicol. 16, 330336.[ISI][Medline]
Guilmette, R. A., Wicks, J. D., and Wolff, R. K. (1989). Morphometry of human nasal airways in vivo using magnetic resonance imaging. J. Aerosol Med. 2, 365377.
Guyton, A. C. (1947). Measurement of the respiratory volumes of laboratory animals. Am. J. Physiol. 150, 7077.[ISI]
Heyder, J., and Rudolf, G. (1977). Deposition of aerosol particles in the human nose. In Inhaled Particles IV (W. H. Walton, Ed.), pp. 107126. Pergamon Press, Oxford.
Kelly, J. T., Kimbell, J. S., and Asgharian, B. (2001). Deposition of fine and coarse aerosols in a rat nasal mold. Inhal. Toxicol. 13, 577588.[ISI][Medline]
Lippmann, M., Albert, R. E., and Peterson, H. T., Jr. (1977). The regional deposition of inhaled aerosols in man. In Inhaled Particles III (W. H. Walton, Ed.), pp. 105122. Pergamon Press, Oxford.
Ménache, M. G., Miller, F. J., and Raabe, O. G. (1995). Particle inhalability curves for humans and small laboratory animals. Ann. Occup. Hyg. 39, 317328.[ISI][Medline]
Raabe, O. G., Al-Bayati, M. A., Teague, S. V., and Rasolt, A. (1988). Regional deposition of inhaled, monodisperse, coarse and fine aerosol particles in small laboratory animals. In Inhaled Particles VI (J. Dogson, R. I. McCallum, M. R. Bailey, and D. R. Fischer, Eds.), pp. 5363. Pergamon Press, Oxford.
Raabe, O. G., Yeh, H. C., Newton, G. J., Phalen, R. F., and Velasquez, D. J. (1977). Deposition of inhaled monodisperse aerosols in small rodents. In Inhaled Particles IV (Walton, W. H., Ed.), pp. 320. Pergamon Press, Oxford.
Schreider, J. P. (1983). Nasal airway anatomy and inhalation deposition in experimental animals and people. In Nasal Tumors in Animals and Man (G. Reznik, and S.F. Stinson, Eds.), Vol. 3, pp. 126. CRC Press, Boca Raton, FL.
Wolff, R. K., Kanapilly, G. M., Gray, R. H., and McClellan, R. O. (1984). Deposition and retention of inhaled aggregate 67Ga2O3 particles in beagle dogs, Fischer-344 rats, and CD-1 mice. Am. Ind. Hyg. Assoc. J. 45, 377381.[ISI][Medline]
Yeh, H. C., Brinker, R. M., Harkema, J. R., and Muggenburg, B. A. (1997). A comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts. J. Aerosol Med. 10, 319329.[ISI][Medline]
Zhang, L., and Yu, C. P. (1993). Empirical equations for nasal deposition of inhaled particles in small laboratory animals and humans. Aerosol Sci. Technol.19 5156.