Results

PLEURODESIS

The intrapleural injection of TGFβ₂ was very effective in inducing pleurodesis. Intrapleural TGFβ₂ in doses of 1.0 μg/kg, 0.5 μg/kg, and 0.25 μg/kg (groups A, B and C) produced a maximum pleurodesis score of 8 in all nine sheep. The three sheep given 0.125 μg/kg TGFβ₂ (group D) all had a pleurodesis score of 6. At the time of death the pleurae of the treatment side were grossly thickened in all sheep (fig 1). The lungs were tightly adhered to the chest wall and could only be separated from the chest wall after intense blunt dissection. Nevertheless, after the lung was freed from the chest wall it could be easily inflated with positive pressure.

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Figure 1

The visceral pleura (lifted with forceps) is significantly thickened and can only be separated from the underlying lung after intense blunt dissection.

In the six sheep in groups C and D given a buffer injection into the left (control) pleural space, there was essentially no pleurodesis with scores of 1–2 in all animals. One sheep in group D had evidence of a small loculated empyema in the left pleural space (control side) at the necroscopic examination. There was no haemothorax in any of the sheep.

The volume of effusion produced at 24 hours and in total, the biochemical analysis, and the total leucocyte count of the pleural fluid from the treatment side of the sheep in each dosage group are summarised in table 1. None of the six sheep receiving 0.125 μg/kg or 0.25 μg/kg TGFβ₂ (groups C and D) produced significant amounts of pleural fluid (<5 ml) while sheep receiving higher doses produced a large volume of effusion (mean >700 ml for both groups A and B) after intrapleural administration of TGFβ₂. There was no significant drainage in these sheep 72 hours after the injection. None of the sheep developed more than 2 ml of fluid in the first 72 hours on the control side. The volume was considered insignificant and the fluid was not analysed. The effusions produced were exudative with mean protein concentrations of 3.9 mg/dl in group A and 3.6 mg/dl in group B, and mean LDH levels of 958 IU/l and 896 IU/l, respectively. The white cell counts were relatively low but increased with decreasing doses of TGFβ₂ (1352/mm³ in group A, 1938/mm³ in group B, and 3475/mm³ in group C).

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Table 1

Results of pleurodesis score, volume of effusion produced, and analysis of pleural effusion in sheep after intrapleural TGFβ₂ administration

HISTOLOGICAL EXAMINATION

The pleura of the treated side of all sheep had significant thickening with fibrovascular proliferation and a minimal inflammatory component (fig 2). There were no differences noted in the nature or intensity of the pleural reaction between animals receiving different doses of TGFβ₂. Minor inflammatory changes were noted in the underlying lung including interstitial non-suppurative inflammation, peribronchial lymphoid aggregates, and localised small patchy consolidations. These findings were considered spontaneous and incidental, commonly observed in sheep, and unlikely to be related to the intrapleural administration of TGFβ₂.

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Figure 2

The pleura of the treatment side (A) was markedly thickened with a large amount of collagen and fibrous tissues compared with the pleura of the control side (B) of the sheep. Trichrome stain, magnification ×10.

Specific changes that can occur with TGFβ were examined. In the kidneys tuberlointerstitial chronic inflammation and periglomerular sclerosis were either not present or minimal (grade 1) in all the samples. Likewise, in the adrenal gland, medullary lymphoid infiltration was occasionally seen (median grade 0 or 1 in all dosage groups). Urinary bladder samples showed no abnormality. Two of the sheep were female and both had normal ovaries and uteri. Liver samples showed minimal chronic inflammation (median grade = 1) while splenic congestion was present to a mild degree (median = 2) in all samples. One sheep had evidence of focal necrotising enteritis and four others had very mild coccidiosis; these findings are common in necroscopic examinations of sheep and were considered incidental.

Discussion

This study has shown that TGFβ₂ is effective in producing pleurodesis in sheep, an animal species with a thick pleural membrane similar to that of humans. TGFβ₂ induced pleurodesis in a dose dependent fashion and was effective in creating pleural symphysis even at the low dose of 0.25 μg/kg. At this dosage there was no histological evidence of systemic complications in any of the extrapulmonary organs examined. To our knowledge this is the first study to establish the use of a sheep model to determine the effect of pleurodesing agents.

It is estimated that 20 000 patients require pleurodesis each year in the USA.1 Currently available agents either have a low success rate (for example, bleomycin)2 or have potentially serious (for example, doxycycline)2 or fatal complications (for example, talc).3 ,4 Talc is considered the most effective compound among commonly used agents.2 ,16However, talc induced ARDS is a growing concern and can occur in up to 9% of patients.4 Systemic embolisation of talc has also been identified in patients during necroscopic examination3 and was found in all organs in rats after talc pleurodesis.6 Hence, there is an ongoing search for a safe and effective agent for chemical pleurodesis.

We recently established that TGFβ₂ was effective in producing pleurodesis in rabbits.11 TGFβ₂ is a multifunctional cytokine with diverse functions. In particular, it is one of the most powerful stimulators of extracellular matrix production and fibrosis in tissue repair.9 It also has potent anti-inflammatory properties that include downregulation of interferon induced class II MHC expression, and inhibition of T and B lymphocytes and their production of tumour necrosis factor α and interleukin 1.10

The rabbit is the most common animal model used to study pleurodesis5 ,17-19 although dogs20 ,21 and pigs22 have occasionally been studied. The visceral pleurae of small animals such as rabbits, dogs, and cats are thin and are different in structure from the pleurae of larger animals such as sheep, horses, and cows.12 The pleurae of these larger animals are more similar to the human pleura which is relatively thick. The results of pleurodesis studies using animals with thin pleurae are often different from those in humans. For example, bleomycin was completely ineffective in creating pleurodesis in rabbits18 and a much higher dose (>5 fold) of talc is required to produce satisfactory pleurodesis in rabbits17 than in humans. The sheep model we described can provide a useful method for future studies of pleurodesis.

We found that TGFβ₂ was as effective in generating pleurodesis in sheep as it was in rabbits. All animals receiving 0.25 μg/kg or above reached our maximum pleurodesis score indicating symphysis of over 50% of the chest wall. This minimal effective dose in sheep was only 10% of that needed in rabbits (approximately 2.5 μg/kg).11 Interestingly, intrapleural injection of a higher dose of TGFβ₂ induced the production of a large volume of fluid that was dose related. At lower doses minimal amounts of pleural fluid were produced. The production of fluid neither hindered nor was necessary for the production of pleurodesis as excellent pleurodesis was generated in sheep whether or not they developed any sizeable effusion.

More importantly, the effusion induced was relatively non-inflammatory as reflected by the low leucocyte count, protein and LDH concentrations, in keeping with our observations in rabbits. The pleural biopsy samples taken at day 14 also showed no evidence of pleural inflammation. This may in part be due to the potent anti-inflammatory effects of TGFβ.9 ,10 It supports the idea that TGFβ₂ produces pleural fibrosis through a novel pathway by stimulation of extracellular matrix deposition and inhibition of its degradation. This is in contrast to talc and tetracycline derivatives which produce pleurodesis by inducing acute pleural injury.5

Most currently available pleurodesis agents produce the side effects of fever and pain2 which are probably the result of acute pleural injury and intense inflammation. Fever can occur in up to 62% of patients following talc pleurodesis2 and may last for 72 hours.23 Severe pain was reported in over 50% of patients who were given tetracycline pleurodesis for pneumothorax in the VA cooperative study.24 Recent studies have also shown that levels of pro-inflammatory cytokines in the pleural fluids were significantly raised following chemical pleurodesis with talc or quinacrine.25 ,26 By minimising the inflammatory process, pleurodesis with TGFβ₂ may therefore produce less pain or fever, a distinct advantage as pleurodesis is mainly performed for symptomatic comfort in patients with incurable malignancies.

In sheep and humans the visceral pleurae are thick and the pleural space is drained by the lymphatic system via stroma in the parietal pleura which opens eventually into the systemic circulation.27 The systemic absorption of TGFβ₂ following its intrapleural injection has not been studied. TGFβ is rapidly cleared from the systemic circulation by binding to α₂-macroglobulin and by redistribution to the kidney, lung, liver, and spleen.28 Hence, the serum half life of TGFβ is short (<5 minutes).10 Given the short half life and the lack of information on the time course of systemic absorption (if any) of TGFβ₂ from the pleural space, we did not attempt to measure the changes in serum levels of TGFβ₂. Instead we examined the lungs and extrapulmonary organs for evidence of adverse effects.

Previous studies evaluating the systemic effects of TGFβ have yielded different results. When TGFβ₂ was applied topically to rats in doses up to 800 μg/kg there was no evidence of systemic absorption.28 No acute systemic or local reactions were seen after intravenous TGFβ₂ administration in human or animal studies.29 ,30 Intravenous TGFβ₂ was given three times a week for four weeks to 11 patients with multiple sclerosis in doses of 0.2 μg/kg, 0.6 μg/kg, or 2.0 μg/kg. Reversible reductions in the glomerular filtration rate were observed at 2.0 μg/kg. Mild and reversible increases in hepatocellular enzymes and anaemia were also seen in some of the patients.29

Other animal studies have shown that administration of very high doses of TGFβ could lead to fibrotic changes in the liver and kidneys. Rabbits given intravenous TGFβ₁ in a dose of 1000 μg/kg/day developed periportal fibrosis and marked centrilobular degeneration in the liver.31 Rats treated with 800 μg/kg TGFβ₂ together with volume depletion developed subcutaneous fibrous nodules and thickened vessel walls in the kidneys.30 These changes were not seen in rabbits or rats following chronic systemic administration of lower doses of TGFβ.31 ,32 In our study none of the sheep had any of these changes. There were occasional cases of hepatic or splenic congestion and minimal renal changes in some of the sheep. Although the possibility that these effects are related to TGFβ₂cannot be entirely excluded without a control group for comparison, these findings were mild and are usually considered as incidental findings in farm sheep. Most importantly, the potential side effects of hepatic, renal, and peritoneal fibrosis were not seen in any of the sheep.

In conclusion, this study has established that the intrapleural injection of TGFβ₂ is effective in producing pleurodesis in sheep, a species with a thick visceral pleura similar to that of humans. The intrapleural injection of TGFβ₂ stimulated significant pleural fibrosis but produced minimal pleural inflammation. High doses of TGFβ₂ also induced the production of large amounts of pleural fluid. Low doses of TGFβ₂ produced excellent pleurodesis without inducing pleural fluid formation. We have also shown that the intrapleural administration of TGFβ₂is safe and does not result in any systemic histological changes. A phase I clinical trial is needed to ascertain the effectiveness of TGFβ₂ as a pleurodesing agent in humans.