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* From the Servei de Pneumologia (Drs. Ferrer and Villarino), Hospital General Vall d’Hebron, Barcelona; the Departament de Biologia Cellular (Drs. Montes and García-Valero), Facultat de Biologia, Universitat de Barcelona, Spain; and Department of Medicine (Dr. Light), Saint Thomas Hospital and the Center for Lung Research, Vanderbilt University, Nashville, TN.
Correspondence to: Jaume Ferrer, MD, Servei de Pneumologia Hospital General Vall d’Hebron Passeig Vall d’Hebron, 119-129 08035 Barcelona, Spain; e-mail: jjferrer{at}hg.vhebron.es
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Abstract |
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Study objectives: To test the hypothesis that particle size influences extrapleural talc deposition and pleural inflammation after talc slurry pleurodesis.
Design: Thirty rabbits underwent pleurodesis as follows: 10 rabbits received 200 mg/kg of the talc used for human pleurodesis, normal talc (NT); 10 rabbits received 200 mg/kg of talc with particles of larger size, large talc (LT); and 10 rabbits received saline solution. Samples from the ipsilateral lung, chest wall, diaphragm, mediastinal pleura, heart, liver, spleen, and right kidney were obtained at 24 h and 7 days and processed for optic and electron microscopy and energy-dispersive x-ray analysis.
Results: Visceral pleural thickening was greater with NT than with LT, but no differences were observed in the macroscopic score of adhesions. There was more talc in the lungs of the rabbits that received NT than in those that received LT. Talc particles were detected in mediastinum (100%) and pericardium (20%), irrespective of the talc used. Three animals, all receiving NT, had talc particles in the liver.
Conclusions: Our study shows that while both talcs were equally effective in achieving pleurodesis, the intrapleural injection of NT elicits greater pulmonary and systemic talc particle deposition than LT. Moreover, pleural inflammation was greater with NT than with LT.
Key Words: particle size • pleural • pleurodesis • talc
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Talc is a hydrated magnesium silicate, Mg3Si4O10 (OH)2. It is one of the most commonly used agents for pleurodesis. The reason for its high popularity is its high effectiveness2 and low cost. Moreover, a possible therapeutic effect of talc on mesothelioma cells has been suggested.3
However, concerns persist as to the development of the ARDS after the intrapleural administration of talc. ARDS has been reported with either insufflated talc powder4 or talc slurry,5 and with different talc doses.6 7 The frequency of ARDS after talc pleurodesis was 3 to 9% in three different series,6 8 9 and in some cases this complication was lethal.10 Evidence exists supporting the hypothesis that talc particles instilled into the pleural cavity can escape and migrate to extrapleural organs, thus provoking an inflammatory reaction and acute lung failure. In two studies, talc particles could be detected in the BAL of patients who presented with acute pneumonitis after talc pleurodesis.5 7 In addition, the extrapleural dissemination of talc particles after pleurodesis has been demonstrated in the experimental model. In a study11 in rabbits, after pleurodesis with talc slurry, talc particles were detected by optic microscopy in 17 to 40% of different extrapleural organs. In a more recent study12 performed in rats, birefringent particles were found in 100% of extrapleural organs after talc pleurodesis. Talc dissemination can be significant, since lung and hepatic granulomas have been detected after talc was administered by inhalation or IV.13
Talc particle size can be a key factor in explaining the extrapleural dissemination of talc from the pleural cavity. Mean particle size among the sterile talcs used for pleurodesis in several countries ranges from 10 to 33 µm, but the lowest mean sizes correspond to the talcs used in the United States.14 This is remarkable, since most patients who had acute lung disease develop after talc pleurodesis had been treated in the United States.6 8 9 These facts suggest that particle size can influence the extrapleural dissemination of talc after pleurodesis and may be related to development of acute lung injury.
The aim of the present study was to analyze the extrapleural inorganic deposition and its corresponding histologic lesions after pleurodesis with two talcs of different size distributions. We tested the hypothesis that the smaller the size of the talc particle, the higher the extrapleural deposition of talc and the greater the tissue damage.
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Materials and Methods |
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General Strategy
Approval for animal experimentation was obtained from the Ethic Committee on Animal Experimentation of the University of Barcelona. Thirty white male New Zealand rabbits weighing 1.5 to 2.0 kg were randomly assigned to the following three experimental groups: NT, LT, and control. Animals from the NT and LT groups received 200 mg/kg of the corresponding talc suspended in 2 mL of saline solution. Control rabbits received only the saline solution. Half of the animals in each group were killed at 24 h, and half were killed 7 days after instillation.
Experimental Procedure
Rabbits were anesthetized with ketamine hydrochloride, 35 mg/kg, and xylazine hydrochloride, 5 mg/kg, administered IM. Under direct view of the parietal pleura, after aseptic surgery talc slurry was instilled into the right pleural cavity with a 27-gauge needle. After suture, animals were turned over to ensure a homogeneous distribution.
The animals were killed with 40 mg/kg of pentobarbital solution injected into the marginal ear vein. The thoracic and abdominal cavities were immediately examined macroscopically. The degree of pleurodesis was graded according to the scheme of Light et al.15 Samples from lung, chest wall, diaphragm, mediastinal pleura, interpleural adhesions, heart, liver, spleen and right kidney were resected, fixed in 2% paraformaldehyde in 0.1 mol/L phosphate-buffered saline solution (pH 7.4), and processed for optic and electron microscope examination.
Sample Processing
Optical Microscopy:
After being fixed by immersion, samples from lung, chest wall, diaphragm, mediastinal pleura, interpleural adhesions, heart, liver, spleen and kidney were dehydrated in solutions with an increasing percentage of ethanol and embedded in paraffin. Samples were then cut with a microtome (model 0325; Anglia Scientific Instruments; Cambridge, UK) to obtain slices at a nominal thickness of 6 µm, which were placed on previously gelatinized slices. Sections were then dewaxed with xylol, hydrated, and stained with Harri’s hematoxylin-eosin. Examination was carried out by light field and polarizing microscopy in an optic microscope (Polyvar 2; Reichert-Jung; Vienna, Austria).
Scanning Electron Microscopy: Samples from the lung lower lobe, chest wall, diaphragm, mediastinal pleura, spleen and kidney were cryoprotected by infusion with 30% sucrose in phosphate-buffered saline solution, embedded in optimum-cutting temperature compound (OCT; Miles Laboratories; Naperville, IL), quickly frozen in dry ice and stored at - 35°C. Sections 10 µm thick were cut using a cryostat 2800 Frigocut-E (Reichert-Jung) and spread on stubs covered with poly-l-lysine (Sigma; St Louis, MO). Sections were then washed with double-distilled water, dehydrated, and freeze-dried by the critic point technique and finally recovered with coal. Five slices (lung, chest wall, diaphragm and mediastinal pleura) and 20 slices (spleen and kidney) were prepared for backscattering observation.
Scanning Electron Microscopy and Backscattering Observation and EDXA
Observation based on the retrodispersed electrons, which give a high discrimination power between tissue and talc particles, was carried out with a Cambridge Stereoscan S-120 scanning electron microscope (Cambridge Instruments; Cambridge, UK). Scanning electron microscopy was performed at 20 kV, distance of 21 mm, and angle of 90°. Backscattering observation was carried out with an intensity of 0.9 nanoamperes. The elemental composition of the particles was determined by means of an energy-dispersive x-ray analyzer (Kevex PCXA; LINK; High Wecombe, UK). Elemental silicon (Si) and magnesium (Mg) peaks corresponded to talc.
Visceral Pleural Thickening Estimation
The distance between the pulmonary surface and the underlying parenchyma was measured on hematoxylin-eosin–stained lung sections to quantify the thickening of the visceral pleura. In talc-treated groups, determinations were carried out at a minimum of 5 mm from the focal talc depositions identified in six random selected sections of each rabbit. In the control group, three measurements were obtained in four randomly selected sections of each animal.
Stereologic Estimations
A systematic random strategy was applied as a general rule to sampling blocks, sections, and recounting areas. Quantitative analysis included stereologic parameters as the volume density (Vv) to estimate fractional volumes, mean average volume number-weighted (Vn), and surface density (Sv) to characterize particle populations.16
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All light and electron microscopic measurements were carried out by investigators blinded to the category of the rabbit.
Statistical Analysis
All data were expressed as mean ± SEM. Data from estimations were analyzed using a one-way analysis of variance followed by the Fisher exact test. Moreover, specific pairs of estimations were analyzed using two-sample t test. Data were considered statistically significant at p < 0.05.
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Results |
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By light microscopy, 24 h after talc slurry instillation, NT and LT groups showed focal inflammatory reactions that expanded centrifugally from those points on the pleural surface where talc masses were located (Fig 1 , top left, A). This inflammatory process included both denudement of mesothelium and regression of the basal lamina and underlying connective tissue. Likewise, submesothelial capillary vasodilatation and local endothelial necrosis with extravasation of leukocytes and erythrocytes were observed. In the lung, the inflammation also affected the underlying pulmonary parenchyma, which showed capillary vasodilatation, leukocyte infiltration, and edematous areas (Fig 1 , top right, B, and center right, C). The extent of inflammation (Table 3 ) was significantly greater in visceral pleura and subpleural lung of rabbits that underwent pleurodesis with NT (Fig 1 , top right, B) than those treated with LT (Fig 1 , center right, C).
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Between 24 h and 7 days after talc slurry instillation, neovascularization stemming from marginal vessels occurred (Fig 2 , bottom left, E), and fibroblasts, initially located in inflamed submesothelial areas, were progressively incorporated into fibrin matrixes. At 7 days, fibroblasts were enlarged with strongly stained cytoplasm and lax chromatin, indicating a high protein synthesis rate, with their major axis oriented parallel to collagen fibers (Fig 2 , bottom right, F). These collagen fibers were oriented perpendicularly with reference to the pleural surfaces, and were continuous with the visceral and parietal submesothelial connective tissues. This continuity and the progressive inversion of the cellular fraction to a noncellular component favored the stabilization of the adhesion. Macrophages in the early fibrin matrix persisted in this new scarring tissue, forming both epithelioid and multinucleated giant cells associated with talc particles.
From the first day following talc administration, mesothelial cells located at the margins of denuded areas underwent an active proliferation that re-epithelized all the previously inflamed areas, including fibrotic thickening (Fig 2 , top left, A, and top right, B), granulomas (Fig 2 , center left, C) and adhesions (Fig 2 , bottom right, F). This newly formed mesothelium was composed of poorly differentiated cells without microvilli. Its basal domain was initially associated with the fibrin matrix, and at 7 days a thin basal lamina was observed underlying the mesothelial cells.
Lung
Several rabbits undergoing talc pleurodesis exhibited talc in the lung. Substantial differences were observed between the experimental groups.
EDXA revealed that 60% (6 of 10 animals) from the NT group showed talc in lung parenchyma. Particle diameter covered the entire spectrum of the sample. Talc was usually distributed as massive accumulations of particles located mainly in the peripheral parenchyma. The subsequent inflammation of adjacent lung parenchyma resulted in edema and some degree of necrosis. Some contiguous airways, particularly alveoli, alveolar ducts and bronchioles, were disorganized and contained aggregates of particles. No fibroblasts or fibrotic changes were observed related to these particle aggregates. Occasionally, talc followed the bronchovascular spaces and reached small blood and lymphatic vessels, forming small thrombi with blood cells and fibrin (Fig 3 ).
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We defined the following score to semiquantitate the talc deposition in the pleura and lung: I, talc particles incorporated into the pleura but no parenchymal deposition (Fig 3 , top left, A, and top right, B); II, individual or small aggregates of talc particles randomly distributed in parenchyma (Fig 3 , center left, C, and center right, D); and III, diffuse deposition of particles affecting a variable percentage of pulmonary parenchyma (Fig 3 , bottom left, E, and bottom right, F). According to this score, the intensity of talc particle deposition was greater with NT than with LT, irrespective of the experimental time (Table 4 ). With NT, five of the six animals showing a pulmonary deposition were classified as stage III, whereas with LT both rabbits with pulmonary deposition were classified only as stage II.
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Liver
At 7 days, macroscopic examination of the abdominal cavity revealed that three of the five rabbits treated with NT, but none of those receiving LT, showed talc deposition on the liver surface (Table 5)
. In one of these animals, a firm symphysis > 2 cm2 containing a high collection of talc particles was established between the diaphragmatic connective tissue and the Glisson capsule, which became fibrotic and disorganized (Fig 4
, bottom, C). Hepatocytes underlying these talc aggregates became dysplastic. In addition, polarized light microscopy revealed that one rabbit from the NT group, which exhibited the greatest macroscopic deposition of talc, presented small particles (Dmax < 10 µm) inside macrophages located in portal spaces.
Spleen
By EDXA, only two rabbits belonging to the early experimental time showed talc particles in the spleen (Table 5)
. Whereas one animal from the NT group showed six particles (Fig 5
, top left, A, and bottom left, B), one rabbit treated with LT had one particle (Fig 5
, top right, C, and bottom right, D). In both cases, particles were associated with the white periarteriolar substance, and their Dmax were always < 10 µm. Statistically significant (p < 0.001) hyperplasia of the white periarteriolar substance was observed at 24 h in all talc-treated animals, but not in control animals (Table 6
). At 7 days, the relative volume of the splenic white substance had diminished, but the difference between animals undergoing talc pleurodesis and control animals remained significant (p < 0.001).
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Discussion |
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We found that pleurodesis with NT produced greater talc particle deposition in the ipsilateral lung than pleurodesis with LT; more animals were affected with NT, and those that were affected had more talc particles. Our observations suggest that talc reaches the lung parenchyma by breaking the mesothelial and elastic layer. Other proposed escape routes, such as cellular engulfment or intercellular junctions,18 were not supported by the observations in this study. The fact that the size distribution of talc particles instilled in the pleural cavity and that of NT particles deposited in the lung was the same further supports this mechanism of dissemination.
The results of several studies7 12 suggest that ARDS after talc pleurodesis is due to pulmonary deposition of talc. According to the present results, talc could be found in the lung of any patient undergoing talc pleurodesis, and not only in those who have ARDS develop. However, the fact that only animals treated with NT have diffuse or massive depositions of talc in the lung suggests that the deposition of talc is critically dependent on the size of the particle. If ARDS is due to talc deposition, this provides an explanation for why most patients reported to have this complication received American talc, the particle size of which is the smallest.14
A systemic inflammatory reaction seems to develop after talc pleurodesis, as suggested by the fact that all animals undergoing talc pleurodesis showed hyperplasia of the white periarteriolar substance of the spleen. The finding of Mitchem et al,19 that rabbits undergoing talc slurry pleurodesis have elevated angiotensin-converting enzyme levels in serum and lung, supports this possibility. However, lung parenchymal changes after talc pleurodesis are basically a granulomatous reaction.
Many talc particles of varying sizes were found in mediastinum in all animals. Some were located in milky spots, thus suggesting that talc, like other particles, is drained from the pleural cavity by the lymphatic system, probably in the parietal pleura.20 The presence of talc particles in epicardium and pericardium is also remarkable. Particles had different sizes and their number slightly increased between 1 day and 7 days. Talc can reach these structures directly by mechanical progression passing across the mediastinal space. After breaking the pleural mesothelium, talc could progress mechanically and directly penetrate into the pericardial space, which is an alternative pathway to lymphatic dissemination.18
Strikingly, three animals undergoing pleurodesis with NT had macroscopically visible talc masses on the liver surface, and one of them had a thick adhesion between the diaphragm and the liver. Since NT produces greater inflammation than LT and a wide range of particle sizes was found in this hepatic adhesion, it can be hypothesized that talc massively leaked from the pleural cavity and reached the diaphragm by mechanical progression through necrotic openings of the damaged mesothelium.
In previous studies, talc particles were detected in abdominal organs after experimental animal pleurodesis. In a study11 in the rabbit, birefringent bodies were found in abdominal organs in 15 to 40% of the animals studied, but were surprisingly absent in the ipsilateral lung. In a second study12 in the rat, all extrathoracic organs studied contained birefringent bodies. In the present study, only a few talc particles, all < 10 µm, were observed in the spleen (affecting 2 of 20 animals) and the right kidney (affecting 1 of 20 animals). This was a low-probability phenomenon, probably as a consequence of a passive dissemination via the bloodstream. Furthermore, the low number of affected animals and the rapidity of the process (< 1 day) suggest that particles could have leaked from the pleural cavity into the bloodstream by erosions of the pleural or pulmonary tissue during talc instillation.
There is no doubt that the inflammatory changes observed in the pleura were due to talc, since control animals treated with saline solution showed no such alterations. The main difference between NT and LT was that the intensity of the inflammation was greater with NT, while qualitative aspects were similar. A direct effect of the talc particles on the mesothelium seems to have been the initial mechanism triggering the pleural inflammatory reaction, as suggested by the coincidence of inflammation and talc particle deposition in time and space. Since the pleural cavity is a virtual space, the instillation of talc slurry may place talc particles in rough contact with the mesothelial layer, thus facilitating its direct damage, as proposed for asbestos fibers.21
What is the reason for the greater inflammatory power of NT? Since both talcs were asbestos-free and similarly sterilized, the physical characteristics of the particles may be responsible for the differences observed. First, NT, with a lower mean particle size, has a greater number of particles per mass unit. Furthermore, the higher specific surface of NT particles could result in greater cellular damage by particle-dependent mechanisms such as direct injury and oxidative mechanisms.22 However, cytokine-mediated inflammation may also be related to physical particle characteristics.
High levels of interleukin 8 and monocyte chemoattractant protein 1 are detected in pleural fluid in the first 24 h after human talc pleurodesis,23 and these cytokines are released by human pleural mesothelial cells after in vitro talc stimulation.24 Several of the changes observed in the present study, such as loss of cellular adhesion, necrosis, and talc phagocytosis, are known to be stimuli for the production of cytokines.25 The first two are dependent on the particle size and are presumably higher with NT than with LT, as previously discussed. Regarding phagocytosis, we found that only talc particles with a maximal diameter < 10 µm are phagocytized by macrophages, which therefore perhaps explains why NT, with 300% more particles < 10 µm than LT, provokes more phagocytosis-related cytokine release and tissue injury.
The main purpose of pleurodesis is to achieve pleural symphysis and thus prevent further fluid accumulations. In the present study, the constitution of interpleural adhesions was qualitatively similar with both talcs. NT caused greater pleural thickness than LT, but no differences were found in the number of macroscopically visible adhesions or in their microscopic appearance. Thus, the effectiveness of NT and LT as a pleural sclerosing agent appears to be similar.
Some considerations should be made for the results of this study to be extrapolated to clinical practice. First, the talcs used in this study fulfil the requirements for clinical use, and their size is comparable to those used in human pleurodesis. Although the difference in median particle size between NT and LT was not great, a clear difference existed in size distribution and the percentage of particles < 10 µm. Since the median particle size of talcs from different countries ranges from 10 to 33 µm,14 differences in extrapleural talc dissemination in human patients could be higher than those observed in this study.
Second, the dose of talc used in this study equaled 12 g in a human patient weighing 60 kg. The reason for administering this high dose was that it is necessary to produce pleural symphysis in rabbits.15 We do not believe that this high dose was responsible for the extrapleural talc dissemination, since similar or greater talc disseminations were observed after low doses as 60 to 70 mg/kg.11 12
Extrapleural talc dissemination has been demonstrated in the present and other animal studies11 12 and in man.7 Thus, it seems preferable not to treat patients with benign pleural diseases with intrapleural talc, since the long-term effects of this mineral have not been definitively established. Patients with pleural malignancies are also at risk for ARDS. According to the results of the present study, if talc is chosen as the agent for pleurodesis for those patients, it is probably better to use talc with large particles, since it appears to be equally effective and causes less extrapleural dissemination. The optimum size of talc particles cannot be established from the present data, but it seems advisable to eliminate particles < 10 µm to avoid phagocytosis-related inflammation. Additional studies assessing the safety and effectiveness of pleurodesis with large-particle talc are required to confirm the present data.
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Acknowledgements |
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Footnotes |
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Dr. Ferrer and Dr. Montes contributed equally to the design of the study and writing the article.
Supported by grants from the Fundació Catalana de Pneumologia (Beca Maria Ravà; 1996 FUCAP), Fundación Española de Patología Respiratoria (FEPAR 1997) y Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS-1998).
Received for publication July 27, 2001. Accepted for publication April 3, 2002.
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