Length-dependent retention of carbon nanotubes in the pleural space of mice initiates sustained inflammation and progressive fibrosis on the parietal pleura

Abstract

The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.

Figure 1

CNT panel. A: Transmission electron microscopy (different magnifications used). B: Light micrographs of each member of the CNT panel. For light micrographs, CNTs and fibers were dispersed by ultrasonication in 0.5% BSA-saline at a concentration of 50 μg/mL to the standard degree of dispersion used for intrapleural injection. NPCB indicates nanoparticle carbon black.

Figure 2

Acute inflammatory response to intrapleural injection of CNT. Total cell number (A), total granulocytes (B), and total protein (C) were measured in the lavage fluid of mice injected with 5 μg of CNT and controls at 24 hours after injection. Total granulocytes (D) were measured in lavage fluid of mice treated with either NT_tang1 or NT_long2 up to 168 days (24 weeks) after injection. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle (Veh) control. Data represent mean ± SEM (n = 5 mice per treatment group). E: Histological examination of chest wall samples from mice injected with NT_tang1 and NT_long2 at 1 and 7 days after injection. Aggregates of inflammatory cells are present in both NT_tang1 and NT_long2 samples at 1 day but only in NT_long2 samples at 7 days. The arrowhead indicates long CNT aggregates in ×100 magnification of NT_long2 (7-day sample). Scale bar = 20 μm. NPCB indicates nanoparticle carbon black.

Figure 3

Fibrotic lesion development. A: Parietal pleura of the chest wall of mice treated with NT_tang1 and NT_long2 were examined histologically at 4, 12, and 24 weeks after injection. Sections were stained with both H&E for gross morphological features and picrosirius red for collagen. Arrows indicate level of original mesothelium. Call outs to a high-power view show aggregates of CNT present within the fibrous lesion at each point. Scale bar = 20 μm. B: Lesion size and collagen content were quantified for the NT_long2 samples and expressed as area per length of chest wall section (mm²/mm). Data are represented as the mean ± SEM (n = 3). C: High-power views of the lesion at 24 weeks show the lesion is vascularized (V) and covered with a layer of mesothelial cells (M). Scale bar = 20 μm.

Figure 4

SEM analysis of lesion development over time. The surface of the chest wall parietal pleura was examined by SEM at 1, 4, 12, and 24 weeks after injection. A–L: Low-magnification images (×30) show continuous normal mesothelium (M) in vehicle control–treated mice. L indicates the inflammatory lesions (also outlined in red), which are mild and resolve quickly in NT_tang1-treated mice or are extensive and persistent in NT_long2-treated mice. P indicates papillae, tonguelike extensions from the mesothelium, in NT_long2-treated samples. Scale bar = 500 μm. M–P: High-power view of the lesions present in NT_long2-treated mice shows the changing nature of the lesion over time from fibrinous leukocyte aggregates at early points to the progressive resurfacing of the lesion by mesothelial cells. The papillae present at 24 weeks appear to contain and be covered by mesothelial cells, as identified by the presence of microvilli. Scale bar = 10 μm.

Figure 5

Cellular proliferation along the mesothelium of CNT-treated mice. Cellular proliferation along the parietal pleural of the chest wall and diaphragm was examined in mice treated with vehicle control, NT_tang1, and NT_long2 at 24 weeks after injection. Sections were stained with anti–Ki-67 antibody, a marker for actively proliferating cells. Ki-67–positive staining was only detected along the mesothelium in mice treated with NT_long2. Scale bar = 20 μm.

Figure 6

Size-dependent inflammatory response to compact particles in the pleural space. A: SEM image of a lymphatic stoma (diameter, 10 μm) found on the parietal pleura of the internal chest wall of a mouse. L indicates a leukocyte passing through the stoma. B: Mean size of particles as measured by dynamic light scattering. Data are represented as mean ± SEM. C: Total granulocytes measured in pleural lavage fluid of mice 24 hours after injection of “inflammatory” particles: quartz (DQ12) and coal mine dust or 3- and 10-μm beads. Only the 10-μm beads caused a significant increase in granulocytes. Data are represented by mean ± SEM. ***P < 0.001 versus vehicle (Veh) control.

Figure 7

Clearance of CNTs from the pleural space. Whole-body Nano-SPECT/CT imaging of NT_short-DTPA[¹¹¹In]. Imaging was performed immediately (0 to 1 hour) and 1 day (23 to 24 hours) after intrapleural administration of 5 μg of NT_short-DTPA[¹¹¹In] (A and B) or [¹¹¹In]DTPA (C and D). The scanning duration was 60 minutes. SPECT/CT-fused images of the whole body (anterior view) and sagittal planes are shown. Arrows indicate the cranial mediastinal LNs (A and B) and bladder (C and D) in the case of NT_short-DTPA[¹¹¹In] and [¹¹¹In]DTPA, respectively. E: Cranial mediastinal LNs removed from mice intrapleurally injected with nonradiolabeled long and short CNTs sectioned and stained with H&E.

Figure 8

Clearance of short NiNWs to the cranial mediastinal LNs. A: Transmission electron microscopy images of short and long NiNWs. B: Total granulocytes in the pleural lavage fluid 24 hours after injection with vehicle (Veh) control or short or long NiNWs. ***P < 0.001 versus Veh control. C: Histological sections of cranial mediastinal LNs excised from mice 24 hours after intrapleural injection of short or long NiNWs (n = 3) and stained with picrosirius red to make the NiNWs more visible. Arrows indicate NiNWs. Scale bar = 10 μm. D: Fiber number per square millimeter of LN tissue was determined by counting the total number of fibers from six sequential sections of each cranial mediastinal LN and measuring the area of tissue in each section. Data are presented as the mean ± SEM (n = 3 mice per treatment group).

Figure 9

Diagrammatic representation of length-dependent clearance from the pleural space. A: Short fibers and small CNT tangles that deposit in alveoli that are situated subpleurally and migrate to the pleural space and exit in the flow of pleural fluid through the stomata, where they follow the lymphatic drainage to the mediastinal LNs. B: Long fibers and long CNTs also reach the pleural space from subpleural alveoli but they cannot negotiate the stomata and are retained, where they cause inflammation and potentially long-term disease.