Background and Objective Pleurodesis is a frequently preferred procedure in thoracic surgery, and many factors may affect the process. We aimed to determine whether the administration of systemic diclofenac sodium diminishes the effectiveness of the pleurodesis induced by intrapleural tetracycline in rabbits.
Methods Twelve male New Zealand rabbits that received tetracycline 35 mg/kg intrapleurally were allocated into two groups. The first group (diclofenac group, n = 6) received 2 mg/kg diclofenac sodium intramuscularly for 10 days, and the second group (control group, n = 6) received acetaminophen 30 mg/kg orally for 10 days after the pleurodesis procedure. The rabbits were sacrificed after 28 days, and the pleural spaces were assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis, inflammation, and collagenization.
Results The mean macroscopic pleurodesis score of the diclofenac group was 2.16 ± 0.40 compared with 2.83 ± 0.40 in the control group (p = .027). The mean microscopic pleurodesis score of the diclofenac group was 2. 3 ± 1.03, whereas it was 3.5 ± 0.54 in the control group (p = .045).
Conclusion The administration of diclofenac sodium for 10 days following tetracycline pleurodesis reduces the effectiveness of pleurodesis in rabbits.
- diclofenac sodium
- nonsteroidal anti-inflammatory drug
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Pleurodesis is a common procedure, especially in malignant pleural effusions and recurrent pneumothoraces. Tetracycline is often referred for chemical pleurodesis in clinical and experimental applications.1-3Most patients who undergo pleurodesis are ordered nonsteroidal anti-inflammatory drugs (NSAIDs) for postoperative pain management.
Because pleurodesis itself is a painful procedure, and a significant percentage of patients have malignant diseases, pain management in these patients is an important issue. Because the aim in pleurodesis is to induce fibrosis by artificial inflammation in the pleural space, the use of anti-inflammatory drugs for analgesia should be questioned.
The objective of this study was to evaluate the effect of diclofenac sodium on the pleurodesis induced by tetracycline in rabbits. We hypothesized that pleurodesis would be less effective in rabbits receiving diclofenac sodium, an NSAID.
MATERIAL AND METHODS
This study was carried out in the animal laboratory of our institution. All animals received humane care in compliance with Principles of Laboratory Animal Care, formulated by the National Society for Medical Research,4and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences.5
Twelve male New Zealand rabbits weighing 1.5 to 2.0 kg were used and were housed at a temperature of 21 to 24°C. All animals were fed on a standard laboratory diet ad libitum. The rabbits were divided into two groups (six animals in each group). The first group (diclofenac group) received 2 mg/kg diclofenac sodium intramuscularly (IM) for 10 days, and the second group (control group) received acetaminophen 30 mg/kg orally for 10 days.
The rabbits were not fed the night before surgery and were anesthetized with ketamine (35 mg/kg) and xylazine (5 mg/kg) by IM injection. The right chest wall was shaved, the animal was placed in a left side down position, and the operative area was sterilized with alcohol. A 1 cm skin incision was made over the right anterolateral chest wall. The muscles in the fifth intercostal spaces were bluntly dissected and exposed. As a standard procedure, a 25-gauge needle connected to a three-way stopcock was inserted into the pleural space. Initially, aspiration was performed for the evaluation of possible accidental air, and 2 mL of tetracycline solution at a dose of 35 mg/kg was administered into the pleural space. Under direct vision, the muscles and skin were closed with silk sutures. The left hemithorax received no injection and was referred to as control evaluation for each rabbit. After the surgery, the rabbits were closely monitored for clinical evidence of pain (vocalization, tachypnea).
The rabbits were randomly assigned to two groups (six rabbits in each group). The study group (diclofenac) received diclofenac sodium 2 mg/kg/daily IM, and the control group received acetaminophen 30 mg/kg/daily orally for 10 days.
The rabbits were sacrificed on the twenty-eighth postprocedural day by an injection of pentobarbital solution, 40 mg/kg intravenously. The thorax was removed en bloc from the rabbit. The lung was filled by the injection of 10% formalin intratracheally. The trachea was then ligated with silk suture, and the thorax specimen was saved in neutral buffered 10% formaldehyde solution at least until pathologic examination.2
Necropsy was performed by a pathologist, who was blinded to the groups. Each pleural cavity was exposed by performing bilateral incisions through the ribs at the midclavicular line. The sternum and the medial portions of the anterior ribs were removed. Then the lungs and pleural cavities were evaluated. The gross pleurodesis and fibrosis were graded according to the following scheme: 0 = normal; 1 = less than three adhesions; 2 = more than three adhesions, 3 = generalized scattered adhesions; 4 = complete obliteration of pleural space.2At the time that the pleura was evaluated grossly, samples of the visceral pleura and lung from each hemithorax were obtained from the anterior lower lobes and placed in neutral buffered 10% formaldehyde solution. These tissue samples were prepared by standard procedure and stained with hematoxylin-eosin and Masson's trichrome stains. The degree of microscopic fibrosis was graded by the same pathologist. The fibrosis was graded as absent = 0, slight = 1, mild = 2, moderate = 3, and severe = 4. The 4-point fibrosis score was used to evaluate the collagen deposition, fibrosis, and inflammation in the visceral pleura:
0 = no inflammation, no collagenization
1 = a few foci of inflammation, slight or no collagenization
2 = more inflammation, a few fibers of collagen
3 = more fibers of collagen but not organized
4 = high density of collagen fibers, partially organized in thick layers
The data were expressed as median ± SD. A nonparametric analysis of variance (Mann-Whitney test, SPSS for Windows, version 6.0, SPSS Inc., Chicago, IL) was performed. A p value of less than .05 was considered significant.
All rabbits were alive, without any complication until the sacrifice. The intrapleural instillation of tetracycline resulted in moderate pleurodesis without well-formed scarring. Although a higher amount of organized collagen fibers was not observed in both groups, partially organized collagen fibers in thick layers were present in the control group. In this connective tissue, the proliferation of capillaries and fibroblast was moderate. The pleurodesis score was 0 in the contralateral hemithorax in all rabbits of both groups. The distribution of the macroscopic and microscopic pleural grades for each rabbit and pleurodesis scores in the two different groups is shown in Table 1The photomicrographs of the different stages of microscopic fibrosis are shown in five panels (Figure 1). The macroscopic and microscopic pleurodesis scores of the diclofenac group were found to be significantly lower than those of the controls (p< .05).
Hemothorax was detected in about 15% of the hemithoraces in the control group and in about 30% in the diclofenac group. Additionally, the amount of alveolar erythrocyte was higher in the diclofenac group than in the acetaminophen group. Hemothorax was not seen in the contralateral left pleural space. Microscopic examination of the lungs did not show any markers of alveolar inflammation, alveolar fibrosis, or atelectasis in either group.
Chemical pleurodesis should be considered in patients with malignant pleural effusions or recurrent spontaneous pneumothorax. The pleurodesis is produced by the intrapleural injection of a sclerosing agent that induces an inflammatory reaction leading to a fibrotic process that fuses the lung and chest wall.
This study showed that the administration of diclofenac sodium for an analgesic purpose (2 mg/kg IM, 10 days) reduced the efficacy of the tetracycline-induced pleurodesis in rabbits. Chest pain was the most common problem associated with tetracycline pleurodesis. Postprocedural analgesia is necessary in both patients and animals. We preferred to study the anti-inflammatory effect of diclofenac sodium on tetracycline pleurodesis because it is a popular agent for pain management. Acetaminophen is known to have no anti-inflammatory effect, so we used it only for analgesic management of control animals.6Pain can influence respiratory mechanics and the level of stress hormones such as steroids. Steroid levels increase immediately after injury, pain, or fever in response to the stimulation of corticotrophin-releasing hormone secretion by various cytokines.7Acetaminophen has antipyretic and analgesic properties but differs from NSAIDs by exhibiting little effect on inflammation.8Because the aim of this study was to show the effect of anti-inflammatory medicine on pleurodesis, we preferred acetaminophen, which provides analgesia with a negligible effect on inflammation, in the control group.
The mechanism of pleurodesis by the intrapleural administration of a sclerosing agent is not completely understood. It is thought that the first event occurring after the intrapleuralinjection of a sclerosing agent is a pleural injury manifestedby the denudation of the mesothelial cells and the developmentof an exudative pleural effusion.3,9-11After this initialinjury, several factors influence the subsequent process thatresults in either the development of a pleurodesis or restorationof the pleural space to its normal state. These include the degreeof injury, the capacity of the mesothelial cells and fibroblasts tosecrete collagen, and the relative balance between metalloproteinases,which degrade collagen, and their inhibitors.12Fibrin has been noted along the pleural surface in many models of acute pleural injury and is responsible for a latticework that is the first lesion in pleural adhesion formation. Fibrin deposition is regulated by the balance between procoagulant activity and fibrinolysis in the tissue milieu. Strange and colleagues indicated that early anticoagulation or fibrinolytic intervention can attenuate subsequent pleural symphysis in tetracycline-induced pleurodesis.13Collagen synthesis by fibroblasts begins at 3 to 5 days after injury and continues for several weeks. Collagen synthesis is enhanced by several factors, including growth factors (platelet-derived growth factor, fibroblast growth factor, and transforming growth factor [TGF]-β) and cytokines (interleukin [IL]-1, IL-4), which are secreted by leukocytes and fibroblasts in healing wounds.14Tetracycline for pleurodesis also stimulates mesothelial cells to release a growth factor-like activity for fibroblasts. This phenomenon may play an important role in tetracycline-induced pleural fibrosis.15
It is reported that the administration of corticosteroids decreases the effectiveness of pleurodesis in rabbits and human studies.9,11,12Corticosteroids are drugs known for their prominent anti-inflammatory properties. Because the formation of adhesions requires an inflammatory process, and NSAIDs counteract inflammatory processes through inhibition of prostaglandin synthesis, the postoperative analgesia by NSAIDs may diminish the development of pleurodesis.
We evaluated the late phase of the fibrosis, which includes fibroblast proliferation and deposition of the extracellular matrix, after the rabbits were sacrificed on the twenty-eighth day. We observed less adhesion and microscopic fibrosis in the diclofenac group. Diclofenac sodium interferes with the inflammatory process that is necessary for the development of fibrosis for successful pleurodesis. Diclofenac is one of the most effective agents in inhibiting fibroblast proliferation and decreasing the chemotaxis induced by substance P and TGF-β.16-18Lardinois and colleagues, using a pig model, demonstrated that postoperative use of NSAIDs adversely affects the quality of pleural adhesions in mechanical pleurodesis.19In our study, we applied tetracycline-induced pleurodesis in a rabbit model, and our results support the previous observation by Lardinois and colleagues. NSAIDs also act as immunomodulating agents by blocking the production of endogenous prostaglandin E. Prostaglandin E is involved in regulating monocyte production of collagenase and mediating inflammation, and collagenase plays an important role in the breakdown and remodeling of collagen.19It has been demonstrated that collagen synthesis is inhibited by diclofenac in some experimental models.20,21In another study, diclofenac prevented lung collagen accumulation after intratracheal bleomycin.22
The intrapleural administration of tetracycline in animals is associated with a high incidence of hemothorax.23This could be due to the rupture of fragile capillaries associated with the neovascularization in the pleura. Wu and colleagues reported in a previous study that the insertion of a chest tube will prevent hemothorax.3In addition, we observed relatively more hemothoraces and alveolar erythrocyte deposition in the diclofenac group with respect to control. This can be explained by the inhibitory role of diclofenac sodium on platelet aggregation.24
In conclusion, this study demonstrates that the administration of diclofenac sodium for 10 days following tetracycline pleurodesis reduces the effectiveness of the pleurodesis in rabbits. Our results may have important clinical implications. Many patients undergoing pleurodesis are receiving NSAIDs for analgesic purposes. If the results of this experimental study could be extrapolated to humans, the quality of pleurodesis might be low in patients receiving diclofenac.
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