Figure shows micrographs from various imaging methods, which demonstrate the distribution of MWCNTs in the lungs 1 day after the termination of the 12 day inhalation exposure period. Figure A is a light micrograph of a Sirius Red stained section showing MWCNTs in a bronchiole, alveolar macrophage, and a case of a fiber midway in penetration between the alveolar airspace and alveolar epithelial surface. Granulomatous responses surrounding airspace MWCNT structures were not observed after inhalation of the well dispersed MWCNT aerosol. Figure B shows a MWCNT-loaded macrophage in a bronchiole of the lungs. An FESEM image of a MWCNT-loaded alveolar macrophage is shown in Figure C. No large, airspace clumps of MWCNTs or granulomatous lesions surrounding MWCNT masses were observed. Penetrations of the visceral pleura by MWCNT fibers (Figure D, arrow) were also observed in inhalation-exposed lungs similar to that previously reported for aspiration exposure [13
] and acute inhalation exposure [3
Figure 1 Micrographs of illustrating distribution of MWCNTs in the lungs 1 day after termination of inhalation exposure. Examples of MWCNTs (arrows) in the three highest initial sites of deposition, alveolar macrophage, alveolar interstitium and bronchiole, (more ...)
At 14 days and later times post-exposure, MWCNT-loaded alveolar macrophages, similar to Figure C and singlet or small clusters of MWCNTs in the alveolar tissue were the most prominent compartments in which MWCNTs were observed. The enhanced darkfield image of Figure shows singlet MWCNTs and small clusters of MWCNTs within alveolar septa of the transition region between a terminal bronchiole and first alveolar duct bifurcation at 168 days after exposure to MWCNTs. As was typically observed at 14 days and later times, a cluster of MWCNTs are present within the ridge of the first alveolar duct bifurcation of Figure . Inhaled particles on the ridge of the first alveolar duct bifurcation are the principal site of deposition following inhalation exposures to particles [14
] and asbestos fibers [15
]. Typically in these inhalation studies, particles or fibers are densely focused on the ridge of the first alveolar duct bifurcation, are proportionately lower in subsequent alveolar duct bifurcations, and are only sparsely found in alveolar septa adjacent to alveolar duct bifurcation ridges. As shown in Figure , inhaled MWCNTs also produced a high concentration of fibers on the bifurcation ridge. In addition, singlet or small MWCNT structures were consistently observed in adjacent alveolar septa (small arrows) and throughout the more distal alveolar septa of the lungs. The occurrence of MWCNTs throughout alveolar tissues of the lungs is unlike that observed for other particles.
Figure 2 Enhanced darkfield image of the transition region between terminal bronchiole and first alveolar duct bifurcation at 168 days after exposure to MWCNT. The large arrow in this micrograph indicates a cluster of MWCNTs (white fibers) in the ridge (more ...)
The lung burden of MWCNTs in airway and alveolar region components of the lungs at 1, 14, 84, 168 and 336 days post-exposure are given in Figure . Initially, MWCNTs in the airways accounted for 16 percent of the initial distribution with 84 percent in the alveolar region (including 1.2 percent of MWCNTs in the subpleural tissue region). At 336 days, 4.2 percent of the initial lung burden was found in the airways and 95.8 percent of the initial lung burden remained in the alveolar region (including 4.8 percent in the subpleural tissue region). Alveolar macrophages initially contained nearly 3 fold the burden present in the alveolar tissue and also had the greatest clearance, declining from 15.8 ug at 1 day post-exposure to 10.2 ug at 168 days post-exposure. Over the same period, the content of MWCNTs in the alveolar tissue increased from 5.8 ug at 1 day post-exposure to 9.5 ug at 168 days post-exposure. The airway region, after an initial rapid decline between 1 and 14 days post-exposure, contained a nearly constant, low level of burden between day 14 and 168 days post-exposure. Total lung burdens of MWCNTs were 28.1
1.2, and 18.3
1.1 ug (Mean
8) at 1, 14, 84, 168 and 336 days, respectively (Mean
8). A highly significant (P
0.0001) decrease in MWCNT lung burden with post-exposure time was determined.
Figure 3 Changes in the distribution of MWCNTs in the lungs after inhalation exposure to MWCNTs. Changes in the lung burden are given for total lung burden, the airway region and the alveolar region. Burden in the alveolar region is further subdivided into that (more ...)
Examples of MWCNT structures containing 1, 2, 3, 4 and >4 fibers per MWCNT structure are shown in the enhanced darkfield image of Figure (upper image) taken from a 1 day post-exposure lung. Counting of these cases was based on using serial sections to avoid bias of selecting larger fibers and to accurately count the number of fibers per structure and thus determine how the distribution of MWCNT fibers changed once deposited in the lungs. MWCNT fibers and most nanoparticles in general, are efficient at scattering light over a broad wavelength. When the scattered light is imaged by an enhanced darkfield microscope, the fibers appear bright white due to scattering of light, while tissue is dark and airspaces are black. This contrast significantly enhances the ability to detect isolated or rare nanoparticles. The right hand, FESEM image of Figure is an example of a singlet MWCNT penetration of the alveolar epithelium at 168 days post-exposure. Partial shells of the MWCNT structure are visible at the airspace end (arrow).
Figure 4 Examples of varying fiber numbers for MWCNT structures. Left, enhanced darkfield image (1 day post-exposure) shows examples of MWCNT structures containing 1, 2, 3, 4 and >4 fibers per structure. MWCNT fibers (and most nanoparticles in (more ...)
Measurements of the fiber number per MWCNT structure at 1, 14 and 168 days after the termination of inhalation exposure are given in Figure . The results given in Figure demonstrate that clearance depends on the number of fibers in the MWCNT structure. Large agglomerates (>4 fibers/ MWCNT structure) account for the majority of MWCNT clearance from the lungs. From day 1 to 168 days, 28.3 percent of the initial lung burden was cleared from the lungs by removal of large agglomerate (>4 fibers/MWCNT structure). The initial lung burden present as singlet MWCNTs was essentially unchanged over the 168 day post-exposure period. Intermediate percentages of initial lung burden decreasing in the lungs in the cases of 2, 3 and 4 fibers per MWCNT structure. Total MWCNT fiber number of the initial lung burden was 1,321 million based on an initial lung burden of 28.1 ug/lung and a conversion of 47 million MWCNT fibers per ug [16
Figure 5 Changes in the distribution of MWCNT lung burden between 1 day and 168 days post-exposure. The majority of MWCNT structures initially in the lungs contained 5 or more fibers and were reduced from 53.6±2.3 at 1 day (more ...)
MWCNT-induced cytotoxicity in the lung was assessed by determining BAL fluid LDH activities (Figure A) and albumin while pulmonary inflammation was assessed by determining BAL polymorphonuclear leukocytes (PMNs) (Figure B). The time course of albumin in first BAL fluid followed the same pattern as that of LDH activity (data not shown). MWCNT-exposed mice had significantly higher BAL fluid LDH activities, albumin and BAL PMN levels in comparison to the corresponding air-exposed mice at all post-exposure times. For mice exposed to MWCNTs, BAL fluid LDH activities and BAL PMNs decreased significantly with post-exposure time, but statistically significantly different from controls out to 168 days. At 336 days post-exposure BAL fluid LDH activities, albumin and BAL PMN levels still elevated above controls but no longer statistically different.
Figure 6 BAL analysis of pulmonary injury and inflammation. Mice were exposed by inhalation exposure to air (vehicle) or MWCNTs (5 mg/m3) for 12 days. BAL LDH and BAL PMNs studies were conducted at 1, 14, 28, 84, 168 and 336 days post-exposure. (more ...)
Figure shows representative light micrographs of Sirius Red stained sections from a clean-air control lung and a lung at 168 days after MWCNT inhalation. As illustrated in Figure , areas of the lung where MWCNT fibers were observed in the interstitial space were found to develop dense bands of fibrillar collagen (arrows). In our prior study using aspiration exposure to MWCNTs, sites with concentrations of interstitial MWCNTs were also found to be foci for a fibrotic response [7
Figure 7 Light microscopy images of collagen fibers in clean-air control and MWCNT-exposed lungs. Left micrograph shows normal clean-air control section of the alveolar region. Right micrograph is from fibrotic region of a MWCNT-exposed lung at 168 days (more ...)
Morphometric assessment of the fibrillar collagen response, based on Sirius Red binding to fibrillar collagen, at 1, 14, 84, 168 and 336 day post-exposure is given in Figure . The average thickness of fibrillar collagen for the clean-air controls was 0.16
0.02 um. In a prior morphometric study, where high resolution transmission electron microscopy imaging was used to identify and quantify fibrillar collagen, at a resolution of individual fibrils, a value of 0.15
0.02 um was obtained in comparable body weight of CD-1 mice [17
]. Morphometric measurements of that study were shown to be comparable, albeit approximately 30 percent lower, than to those obtained by biochemical measurements. Biochemical measurement of whole lung or lobe fibrillar collagen also include the connective tissue (fibrillar collagen and elastin) of the airway, blood vessels and their associated bronchovascular cuff. Biochemical measurements significantly over-estimate collagen in the alveolar region and are relatively insensitive to changes in the alveolar region as these non-alveolar areas are rich in fibrillar collagen and contain 68% of the total tissue mass of the lungs and approximately 70% of total lung connective tissue [18
Figure 8 Morphometric determination of fibrillar collagen thickness response to inhaled MWCNT exposure. The average thickness of fibrillar collagen in the alveolar septa was increased by approximately 70 percent by 336 days post-exposure. Asterisks significantly (more ...)
A trend toward increased fibrillar collagen within alveolar septa is apparent at 14 days post-exposure, which is consistent with our earlier observations of a rapid fibrotic response in acute inhalation exposure to MWCNTs [3
]. The increase in fibrillar collagen was significantly elevated over control at 84, 168 and 336 days post-exposure and increases by 62, 56 and 70% above 1 day post-exposure, respectively. Despite the increased thickness of the alveolar fibrillar collagen, mean linear intercept length, a measure of the average size of airspaces in the alveolar region of the lungs was 29.5
.0.2 and 29.1
.0.2 and 29.5
.2 microns for clean-air control, 1 day, 14 day, 84 day and 168 day post-exposure MWCNT-exposed groups. Mean linear intercept length were not significantly different between groups.
A comparison of the fibrillar collagen response between our earlier study [11
] using aspiration delivery of MWCNTs to the present inhalation exposure is given in Figure . The earlier study, Figure in Mercer et al
], used the same morphometric methods to determine the alveolar interstitial collagen fibers response to MWCNT, expressed as the average thickness of connective tissue in the alveolar interstitium, from 1 to 56 days after bolus aspiration.
Figure 9 Comparison of fibrillar collagen response to inhaled versus bolus aspiration of MWCNTs. Although the total lung burden of these two routes differs by a factor of approximately 3, the time course and magnitude of response to bolus aspiration and inhalation (more ...)
The time course of response of bolus aspiration and inhalation exposure of MWCNTs to the lungs is similar in spite of the differences in the rate of exposure, bolus versus 12 day exposure period. The magnitudes of alveolar interstitial fibrosis for aspiration vs. inhalation of MWCNTs appear similar in Figure . However, it should be noted that lung burden from the aspiration study was approximately 3 fold greater than with inhalation, i.e., 80 and 28 ug, respectively. Therefore, on an equivalent lung burden basis, inhaled MWCNTs are 3 fold more fibrogenic than MWCNTs delivered by bulk aspiration. Although some may find this surprising, the potency difference may reflect greater dispersion of the dry aerosol vs. MWCNTs in a biologically compatible fluid, resulting in delivery of a greater number of small MWCNT structures per equal mass.
Table provides a comparative breakdown of the distributions of lung burden between aspiration and inhalation of MWCNTs at 1 day post-exposure and their respective fibrillar collagen responses. Although the bolus aspiration dose (80 ug) was nearly 3-fold higher than the lung burden achieved by inhalation exposure (28 ug), only 8 percent of initial lung burden, versus 21 percent for inhalation, was found in the alveolar interstitium. Both exposures had comparable percentage distributions to airway macrophages and the subpleural alveolar tissue. In spite of the 3-fold difference in lung burden between the two exposures, the higher lung burden for aspiration was counterbalanced by higher efficiency of delivery to the alveolar interstitium for inhalation exposure. This is reflective of the greater number of small MWCNT structures in the dry aerosol compared to the liquid aerosol. The net result was to produce similar initial burdens to the alveolar interstitium at one day post-exposure. This effect was not limited to just the 1 day post-exposure as the inhalation exposure lungs at later time points continued to develop higher percentages of the lung burden delivered to the alveolar interstitium. Thus the alveolar interstitial lung burden of inhalation-exposed mice increased even as clearance from the lungs progressed.
Comparison of Initial Alveolar Interstitial Burden between Bolus aspiration and inhalation of MWCNT
The comparable fibrotic responses between bolus aspiration and inhalation of MWCNT in spite of the significant differences in lung burden appear to be in large part to differences in effective delivery of the lung burden to the alveolar interstitium. Although relatively well dispersed on administration, the bolus nature of the aspiration is likely to produce a greater degree of MWCNT agglomeration on the lungs surface as higher concentrations of MWCNTs on the alveolar surface produced by the short duration of exposure are more likely to agglomerate.