INTRODUCTION

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Chemical pleurodesis is important in the management of recurrent pleural effusions and pneumothorax. The ideal pleurodesing agent should produce pleurodesis effectively, safely, and in the shortest possible time.

Although talc is effective, there is increasing concern of its safety. Systemic embolization of talc after its intrapleural administration has been shown in several animal studies (1, 2) as well as in humans (3). More importantly, acute respiratory distress syndrome (ARDS) occurs in as many as 9% of patients who receive talc intrapleurally (4). The speed at which talc produces pleurodesis in humans is unknown. However, various animal studies have shown that talc, even in high doses, is slow in inducing pleural fibrosisrequiring as long as 4 wk to produce a satisfactory pleurodesis (5).

We recently demonstrated that transforming growth factor- ₂ (TGF-₂) is effective in producing pleurodesis in rabbits (6). Conventional pleurodesing agents, including talc, act indirectly by inducing pleural injury, which results in acute inflammation and subsequent fibrosis (2). TGF- is a potent profibrotic cytokine. It increases extracellular matrix by stimulating collagen and fibronectin synthesis as well as by inhibiting matrix degradation (7, 8). The direct fibrogenic action of TGF- may allow it to produce pleurodesis faster than talc by bypassing the pleural injury and subsequent inflammatory processes.

TGF- also carries important immune-modulatory properties. In situations of excess inflammation, TGF- can down-regulate lymphocytic functions and the expression of proinflammatory cytokines, including tumor necrosis factor (TNF)- and interleukin (IL)-1 (7, 8).

The purpose of the present study was to compare the temporal evolution of TGF-- and talc-induced pleurodesis. We hypothesized that the intrapleural administration of TGF-₂ would (1) produce an effective pleurodesis faster than the intrapleural administration of talc, (2) stimulate more collagen deposition than talc, and (3) induce less inflammation than talc.

	METHODS

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TGF-₂

A recombinant human TGF-₂ (Genzyme Corp., Framingham, MA) produced in Chinese hamster ovary cells was used. TGF-₂ was formulated in a vehicle consisting of 20 mM sodium phosphate, 130 mM sodium chloride, 15% (wt/wt) propylene glycol, and 20% (wt/wt) polyethylene glycol 400. The pH of the solution was 7.2. The vehicle was prepared using USP/NF grade reagents in water for injection and sterile-filtered through a 0.2 micron filter. The TGF-₂ concentration was determined by a sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies that cross-react with both TGF-₂ and TGF-₃. The activity of TGF-₂ was determined using a mink lung cell (Mv1Lu) antiproliferation assay, modified from the method described by Ogawa and Seyedin (9).

Animal Experiment

The study protocol was approved by the Vanderbilt University Institutional Animal Care and Use Committee. The method used was similar to that described in our previous study (6). New Zealand white rabbits weighing 1.5 to 2.0 kg were anesthetized with an intramuscular injection of 35 mg/kg of ketamine hydrochloride (Fort Dodge Animal Health Laboratories, Fort Dodge, IA) and 5 mg/kg of xylazine hydrochloride (Fermenta, Kansas City, MO). The chest was shaved and the skin was sterilized with 10% povidone iodine (Baxter, Deerfield, IL). The rabbit was placed in the lateral decubitus position and a small (< 3 cm) skin incision was made midway between the tip of the scapula and the sternum approximately 2 cm above the costal margin. Chest tubes were made from intravenous solution set tubes (Baxter) with three extra openings near the distal end of the tube to enhance drainage. The chest tube was inserted by blunt dissection into the right pleural cavity. The left pleural cavity was used for control. The chest tube was secured at the muscle layers with purse-string sutures. The proximal end of the chest tube was then tunneled underneath the skin and drawn out through the skin posteriorly and superiorly between the two scapulae. The exterior end of the chest tube was sealed with a one-way valve with cap (Medexinc, Hilliard, OH) via an adapter and sutured to the skin. A three-way stopcock was attached to the end of the chest tube through which any aspirated air was evacuated from the pleural space.

Thirty rabbits were divided into two groups of 15 each. Rabbits in the TGF-₂ group received an intrapleural injection of 1.7 µg of TGF-₂ (Genzyme Corp.), in 2.5 ml via the chest tube, and rabbits in the talc group received 400 mg/kg of sterilized talc slurry (Sigma, St. Louis, MO) in 2.5 ml via the chest tube. In both groups, injection of the pleurodesing agent was followed with an injection of 1.0 mL of 0.9% sodium chloride solution to clear the dead space of the chest tube.

Talc at 400 mg/kg was shown to be the most effective dosage in a previous dose-response study of talc pleurodesis in rabbits (10). It is the most common dosage of talc used in rabbit pleurodesis studies (11, 12). The dose of TGF-₂ used was based on the results of our recent study on TGF-₂-induced pleurodesis (6).

After the intrapleural injection, the chest tube was aspirated at 24-h intervals for any pleural fluid. The chest tube was removed under light sedation when the pleural fluid drainage was less than 5 ml over the preceding 24 h. The volume of the pleural fluid collected was recorded. The protein, glucose, and lactate dehydrogenase (LDH) levels were determined with an automated analyzer (Johnson & Johnson, Rochester, NY).

Five rabbits in each group were killed at each of the following time points: Days 1, 4, and 7. At the time of death, the rabbits were sedated and euthanized with carbon dioxide. The thorax was removed en bloc. The lungs were expanded by the injection of 50 ml of 10% neutral-buffered formalin into the exposed trachea via a plastic catheter (6 mm diameter). The trachea was then ligated and the entire thorax submerged into 10% neutral-buffered formalin solution for at least 48 h.

Pleurodesis Scoring Scheme

The pleural cavity was carefully exposed after methodology previously described (13). A consensus grading was reached by two blinded investigators (KBL and RWL) on the degree of macroscopic pleurodesis using a semiquantitative scheme (as below). Any evidence of hemothorax, infection, or empyema was recorded if present.

Grading of Pleurodesis Score

The degree of pleurodesis was graded on a scale of 1 to 8. Adhesions were defined as fibrous connections between the visceral and parietal pleura. Symphysis was present if the visceral and parietal pleura were difficult to separate as a result of adhesions.

1 = No adhesions between the visceral and parietal pleura.

2 = Rare adhesions between the visceral and parietal pleura with no symphysis.

3 = A few scattered adhesions between the visceral and parietal pleura with no symphysis.

4 = Many adhesions between the visceral and parietal pleura with no symphysis.

5 = Many adhesions between the visceral and parietal pleura with symphysis involving less than 5% of the hemithorax.

6 = Many adhesions between the visceral and parietal pleura with symphysis involving 5 to 25% of the hemithorax.

7 = Many adhesions between the visceral and parietal pleura with symphysis involving 25 to 50% of the hemithorax.

8 = Many adhesions between the visceral and parietal pleura with symphysis involving greater than 50% of the hemithorax.

Microscopic Examination of the Pleura

At the time the pleura was evaluated grossly, samples of the visceral pleura and lung from each hemithorax were obtained and placed in 10% neutral buffered formalin. The tissue samples were stained with hematoxylin-eosin (H&E) and picrosirius stains for histologic examination. The degree of microscopic inflammation and fibrosis were graded from the H&E slide by an experienced examiner (LRT) blinded to the treatment agent, as previously described (12, 14, 15). The pleural inflammation and fibrosis were graded as none (0), equivocal (1), mild (2), moderate (3), or severe (4). The thickness of the pleura was measured using the Leica Q500IW Imaging Workstation, Image Processing and Analysis System (Leica Imaging Systems Ltd., Cambridge, UK). With this system, the image obtained was transformed from pixels to microns. Measurements were obtained at 10 different points on each sample, and the mean result was reported.

Collagen fibers were subdivided into mature (thick) and immature (thin) fibers using picrosirius staining as reported by Andrade and colleagues (16). With this method the tissue blocks were sectioned at 5 µm and stained for 1 h in a 0.2% solution of Sirius Red, Direct Red 80 (Aldrich Chemical Co., Milwaukee, WI) dissolved in aqueous saturated picric acid (17). The enhancement of collagen birefringence elicited by picrosirius staining is specific for collagen and discloses its distinct patterns of physical aggregation. Immature (thin) fibers, as those present in early granulation tissue, are shown as weakly birefringent green structures, whereas mature (thick) fibers, characteristic of mature fibrotic lesions, are identified by their strong birefringence and their yellow or red color. The areas covered by mature and immature collagen fibers were measured by the Leica Q500IW Imaging Workstation, Image Processing and Analysis System (Leica Imaging Systems). This system detects all pixels in the image that are equivalent to, or nearly equivalent to, the color levels of the select area. The total area of collagen deposition was the sum of the areas covered by mature and immature fibers in the same field and is expressed as the percentage of the total area of the pleural surface. For each sample, readings were taken from six representative fields and the mean result used for analysis.

Statistical Analysis

Student's t test (for parametric data) and the Mann-Whitney Rank Sum test (for nonparametric data) were used to compare the values between subgroups. Two-way analysis of variance (ANOVA) and the Tukey test were used to compare the values between talc and TGF-₂ groups, using the pleurodesing agent and the days after injection as the two factors for analysis. If the values were not normally distributed, the data were log-transformed before analysis with the two-way ANOVA. A p value of less than 0.05 was considered significant. Data were presented as mean ± SD (standard deviation), unless otherwise stated. All data were analyzed with Sigma Stat V2.03 statistic software program (SPSS; San Rafael, CA).

	RESULTS

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Pleurodesis Scores

The intrapleural injection of TGF-₂ produced a more rapid pleurodesis than did the intrapleural injection of talc slurry. The mean pleurodesis scores were significantly higher in rabbits that received TGF-₂ than those injected with talc slurry at all three time points studied (Figure 1). By Day 7, rabbits in the TGF-₂ group had achieved excellent pleurodesis, whereas those rabbits in the talc group had developed significantly less pleurodesis (pleurodesis scores, 6.2 ± 2.2 versus 2.4 ± 1.8; p < 0.02).

View larger version (11K): [in this window] [in a new window]   Figure 1.   Pleurodesis scores of the TGF-2 (closed triangles) and the talc (open triangles) groups at different time points. The differences between the pleurodesis scores of the TGF-2 and the talc groups were significant at all time points: Day 1, p = 0.01; Day 4, p < 0.001; Day 7, p < 0.02.

One rabbit in the TGF-₂ group developed an empyema 48 h after the chest tube injection and was killed and replaced. No other rabbits died before the time of sacrifice. One rabbit in the TGF-₂ group (killed at Day 7) had a mild hemothorax occupying < 15% of the hemithorax.

Microscopic Pleural Histology

The pleural fibrosis score, pleural thickness, and degree of inflammation are presented in Table 1. The pleural fibrosis and pleural thickening scores increased with time in both groups. In keeping with the macroscopic pleurodesis score, the TGF-₂ group had higher levels of pleural fibrosis and pleural thickening at all three time points than did the talc group. When analyzed as a group, rabbits that received TGF-₂ had significantly higher pleural fibrosis score and pleural thickness than did rabbits in the talc group (p = 0.01, p < 0.05, respectively). Although TGF-₂ produced substantially more pleural fibrosis and thickening, the degree of pleural inflammation was similar in the two groups at each of the time points.

The amount of collagen deposition (expressed as a percentage of the total area of the pleura) and the proportion of mature and immature fibers between the two groups at different time points are shown in Figure 2. Significantly more total collagen, as well as mature (thick) and immature (thin) fiber depositions, were seen in the pleura of rabbits that received TGF-₂ when compared with those that received talc (p < 0.01 for all three collagen groups). The amount of collagen deposition was approximately fivefold higher in the TGF-₂ group than in the talc group consistently at all time points studied. As expected, there were more total collagen and mature and immature collagen fiber depositions (p < 0.001; p < 0.05; p < 0.01, respectively) at Day 7 than in the earlier time points. The proportion of mature and immature collagen fibers deposited were comparable in each group.

View larger version (28K): [in this window] [in a new window]   Figure 2.   The amount of mature and immature collagen deposition in the TGF-2 and the talc groups at different time points. (The differences in total collagen, mature collagen, and immature collagen deposition between the TGF-2 and talc groups were all statistically significant, p < 0.01.)

Pleural Fluid Analysis

The pleural fluids collected at 24 h after the injection of TGF-₂ or talc slurry were compared (Table 2). The TGF-₂ group of rabbits produced significantly more fluid than did the talc group at 24 h postinjection. The fluid produced after TGF-₂ injection was characterized by lower LDH levels, though the protein levels were similar in both groups. The amount of fluid produced after TGF-₂ decreased rapidly over the following days. None of the rabbits killed at Day 4 had pleural fluid present at the time of necropsy.

	DISCUSSION

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This present study demonstrated that TGF-₂ produces pleurodesis significantly faster than talc does. Rabbits in the TGF-₂ group achieved excellent pleurodesis within 7 d. Intrapleural administration of TGF-₂ induced more collagen deposition and fibrosis with comparable degrees of pleural inflammation when compared with talc slurry.

An ideal pleurodesing agent is one that produces pleurodesis effectively, safely, and quickly. Although talc is commonly used, there have been increasing reports on serious adverse effects after talc pleurodesis (3, 4). Also it is slow in inducing pleurodesis, as shown in various animal studies. Even in doses more than five times higher than those used in humans, talc does not usually produce significant pleurodesis in rabbits until 4 wk after administration (5). Talc produces pleurodesis by provoking acute pleural injury, inflammation, and hence fibrosis (2). The more intense the insult, the more successful is the pleurodesis. Pain and feversecondary to the pleural inflammationare common side effects with talc pleurodesis (18). Talc-induced ARDS (3, 4) and systemic talc embolization (1, 2) are two major clinical concerns.

We recently demonstrated that TGF-₂ is at least as effective as talc in inducing pleurodesis in rabbits (6). TGF- is a unique cytokine with both potent profibrotic as well as immune-modulatory properties, which make it an attractive agent for pleurodesis.

TGF- is more potent than any other growth factor in stimulating extracellular matrix production (8). It induces matrix deposition by stimulating the transcription of the genes of matrix proteins, thereby increasing the synthesis of collagen, fibronectin, proteoglycan, and other matrix proteins by severalfold (7, 19). It also simultaneously inhibits the synthesis of matrix-degrading proteases and increases the production of protease inhibitors (20). Furthermore, TGF- can inhibit fibrinolysis by increasing the levels of plasminogen activator inhibitor-1 (PAI-1) (21). TGF- is produced by and is an important chemoattractant for fibroblasts (22). Overproduction of TGF- underlies a wide range of fibrotic diseases, including pulmonary (22), renal (7), and liver fibrosis (23). Conversely, the use of TGF- antagonists can reduce or obliterate the fibrotic changes (7, 24).

The potent and direct fibrogenic actions of TGF- can explain the effective and rapid pleurodesis seen after intrapleural injection of TGF-₂ in our study. In keeping with the known fibrogenic effects of TGF-, the rabbits that received intrapleural TGF-₂ had significantly more collagen (both mature and immature fibers) deposition, thicker pleural membranes, and more fibrosis. The rate of pleurodesis induced by TGF-₂ is significantly faster than all other agents previously studied, including talc (5, 11, 25), doxycycline (14), and mitoxantrone (26)all of which required 28 to 60 d to achieve satisfactory pleurodesis in animal models. Our study compared the differences of talc- and TGF-₂-induced pleurodesis in the first 7 d. Future studies are required to determine the long-term differences in pleurodesis induced by these two agents.

The other important advantage of using TGF- is its anti-inflammatory functions. TGF- coordinates and modulates immune responses. It can reduce or prevent excessive inflammation by regulating both T- and B-lymphocyte activities and their production of TNF- and IL-1 (8). TGF-₁ knockout mice have markedly elevated TNF- and IL-1 levels and die rapidly after birth from autoimmune-like diseases (27, 28). TGF-, under suitable circumstances, is also capable of down-regulating IL-6 and IL-8 (29, 30) and modulating cytotoxicity of macrophages by suppressing their superoxide and nitric oxide production (7, 31). There is increasing evidence that the immunosuppressive actions of glucocorticoids (32) and cyclosporine (33) are mediated, at least in part, via TGF-.

This present study did not examine for systemic side effects of the intrapleural injections of TGF-₂ and talc. In our previous study, rabbits injected intrapleurally with low doses of TGF-₂ such as 1.7 µg did not show any obvious systemic abnormality at necropsy. However, rabbits given extreme high doses, e.g., four doses of 20 µg, of TGF-₂ did develop fibrosis in the contralateral pleural space and in the peritoneum (6). In a recent study using a new sheep model for pleurodesis, we found no histologic abnormalities in extrapulmonary organs 14 d after the intrapleural administration of 0.25 µg/kg of TGF-₂ (34).

For conventional pleurodesing agents, the more intense the inflammation they elicit, the more fibrosis and adhesions they produce. In the case of TGF-, however, significantly more fibrosis and pleurodesis were produced, and yet the degree of pleural inflammation was similar to that induced by talc and that the pleural fluid had significantly lower inflammatory indices when compared with talc. This is most likely explained by the fact that TGF-, unlike talc, produces pleurodesis without inducing acute pleural injury as well as the potent anti-inflammatory properties of TGF- described above.

TGF-₂ stimulated the production of significant amount of pleural fluid with low inflammatory indices in the first 24 h after injection, similar to our previous observation (6). In vitro, TGF-₂ can alter the morphology of mesothelial cells, increase the size of the intercellular spaces, and hence the permeability of mesothelial cell monolayers (35). TGF- is also one of the most potent stimulator of the production of vascular endothelial growth factor (VEGF) (36)a cytokine well known for its ability to increase vascular permeability (37). VEGF has been postulated to be an important mediator in the formation of pleural and peritoneal fluid (38). In our study, the amount of pleural fluid produced decreased rapidly after the first 2 d, and no fluid was present by Day 4.

One possible explanation for the differences in the results between the two treatment groups is that the chest tubes remained in place longer in the TGF-₂ group, as there were more pleural fluid induced after TGF-₂ injection. In our protocol, the chest tubes were left in place until the pleural fluid drainage was < 5 ml in the preceding 24 h. However, we believe it is unlikely that the better pleurodesis seen in the TGF-₂ group was due to the longer duration of chest tube placement. Rabbits killed at Day 1 in both groups had chest tubes for the same duration, and the differences between the two groups were already significant at that time point. Also, in previous studies, the presence of chest tubes for as long as 96 h in rabbits or sheep that received injections of buffer or saline did not lead to the development of significant pleural adhesions (6, 34).

In conclusion, our study demonstrated that intrapleural administration of TGF-₂ produced excellent pleurodesis in rabbits at a rate faster than talc slurry and all other pleurodesing agents that had been investigated before. TGF-₂ stimulated more collagen deposition without inducing excess inflammation when compared with talc slurry. If these data can be extrapolated to humans, TGF-₂ will have an advantage over talc slurry in preventing rapid reaccumulation of pleural effusion and in stopping air leak in patients with pneumothorax.